Creating stable stem regions for loop elongation in Fcabs — Insights from combining yeast surface display, in silico loop reconstruction and molecular dynamics simulations

PubMed Central

Hasenhindl, Christoph; Lai, Balder; Delgado, Javier; Traxlmayr, Michael W.; Stadlmayr, Gerhard; Rüker, Florian; Serrano, Luis; Oostenbrink, Chris; Obinger, Christian

2014-01-01

Fcabs (Fc antigen binding) are crystallizable fragments of IgG where the C-terminal structural loops of the CH3 domain are engineered for antigen binding. For the design of libraries it is beneficial to know positions that will permit loop elongation to increase the potential interaction surface with antigen. However, the insertion of additional loop residues might impair the immunoglobulin fold. In the present work we have probed whether stabilizing mutations flanking the randomized and elongated loop region improve the quality of Fcab libraries. In detail, 13 libraries were constructed having the C-terminal part of the EF loop randomized and carrying additional residues (1, 2, 3, 5 or 10, respectively) in the absence and presence of two flanking mutations. The latter have been demonstrated to increase the thermal stability of the CH3 domain of the respective solubly expressed proteins. Assessment of the stability of the libraries expressed on the surface of yeast cells by flow cytometry demonstrated that loop elongation was considerably better tolerated in the stabilized libraries. By using in silico loop reconstruction and mimicking randomization together with MD simulations the underlying molecular dynamics were investigated. In the presence of stabilizing stem residues the backbone flexibility of the engineered EF loop as well as the fluctuation between its accessible conformations were decreased. In addition the CD loop (but not the AB loop) and most of the framework regions were rigidified. The obtained data are discussed with respect to the design of Fcabs and available data on the relation between flexibility and affinity of CDR loops in Ig-like molecules. PMID:24792385

Creating stable stem regions for loop elongation in Fcabs - insights from combining yeast surface display, in silico loop reconstruction and molecular dynamics simulations.

PubMed

Hasenhindl, Christoph; Lai, Balder; Delgado, Javier; Traxlmayr, Michael W; Stadlmayr, Gerhard; Rüker, Florian; Serrano, Luis; Oostenbrink, Chris; Obinger, Christian

2014-09-01

Fcabs (Fc antigen binding) are crystallizable fragments of IgG where the C-terminal structural loops of the CH3 domain are engineered for antigen binding. For the design of libraries it is beneficial to know positions that will permit loop elongation to increase the potential interaction surface with antigen. However, the insertion of additional loop residues might impair the immunoglobulin fold. In the present work we have probed whether stabilizing mutations flanking the randomized and elongated loop region improve the quality of Fcab libraries. In detail, 13 libraries were constructed having the C-terminal part of the EF loop randomized and carrying additional residues (1, 2, 3, 5 or 10, respectively) in the absence and presence of two flanking mutations. The latter have been demonstrated to increase the thermal stability of the CH3 domain of the respective solubly expressed proteins. Assessment of the stability of the libraries expressed on the surface of yeast cells by flow cytometry demonstrated that loop elongation was considerably better tolerated in the stabilized libraries. By using in silico loop reconstruction and mimicking randomization together with MD simulations the underlying molecular dynamics were investigated. In the presence of stabilizing stem residues the backbone flexibility of the engineered EF loop as well as the fluctuation between its accessible conformations were decreased. In addition the CD loop (but not the AB loop) and most of the framework regions were rigidified. The obtained data are discussed with respect to the design of Fcabs and available data on the relation between flexibility and affinity of CDR loops in Ig-like molecules. Copyright © 2014. Published by Elsevier B.V.

Designing synthetic RNAs to determine the relevance of structural motifs in picornavirus IRES elements

NASA Astrophysics Data System (ADS)

Fernandez-Chamorro, Javier; Lozano, Gloria; Garcia-Martin, Juan Antonio; Ramajo, Jorge; Dotu, Ivan; Clote, Peter; Martinez-Salas, Encarnacion

2016-04-01

The function of Internal Ribosome Entry Site (IRES) elements is intimately linked to their RNA structure. Viral IRES elements are organized in modular domains consisting of one or more stem-loops that harbor conserved RNA motifs critical for internal initiation of translation. A conserved motif is the pyrimidine-tract located upstream of the functional initiation codon in type I and II picornavirus IRES. By computationally designing synthetic RNAs to fold into a structure that sequesters the polypyrimidine tract in a hairpin, we establish a correlation between predicted inaccessibility of the pyrimidine tract and IRES activity, as determined in both in vitro and in vivo systems. Our data supports the hypothesis that structural sequestration of the pyrimidine-tract within a stable hairpin inactivates IRES activity, since the stronger the stability of the hairpin the higher the inhibition of protein synthesis. Destabilization of the stem-loop immediately upstream of the pyrimidine-tract also decreases IRES activity. Our work introduces a hybrid computational/experimental method to determine the importance of structural motifs for biological function. Specifically, we show the feasibility of using the software RNAiFold to design synthetic RNAs with particular sequence and structural motifs that permit subsequent experimental determination of the importance of such motifs for biological function.

The Role of elF4E Activity in Breast Cancer

DTIC Science & Technology

2011-08-01

protein; ORF, open reading frame; qPCR, quantitative PCR; RACE, rapid amplification of cDNA ends; RT, reverse transcriptase ; uORF, upstream ORF; UTR...Reactions were also performed using template lacking RT ( reverse transcriptase ): products were either undetectable or greatly reduced (>30000-fold less...that a 5’UTR expressed from the human AXIN2 gene contains a sixty nucleotide sequence that is predicted to form a stable stem-loop structure6. This

Generation of PCV2 in PK15 cells transfected with recombinant baculovirus containing a 1.1 copy of the PCV2 genome.

PubMed

Cai, Jie; Xie, Xiaohong; Hu, Yi; Zhan, Yang; Yu, Wanting; Wang, Aibing; Wang, Naidong

2017-06-01

Porcine circovirus associated diseases (PCVAD) caused by PCV2 are responsible for severe economic losses in the swine industry. The mechanism of PCV2 replication has not been fully elucidated yet. PCV2 may be successfully rescued by means of either an infectious DNA clone containing the full length of the viral genomic DNA, or from PCV2-infected clinical tissues in PK15 cell culture. However, viruses harvested by both methods have low titres. In this study, PCV2 was prepared with a higher titre from PK15 cells infected by recombinant baculoviruses containing 1PCV2 (one stem-loop structure) or 1.1PCV2 (two stem-loop structure) genomic DNA copy. In addition, infectious DNA clones containing two stem-loop structures in either plasmid or baculovirus backbones are capable of generating a higher virus titre than the DNA clones with only one copy of stem-loop structure.

The complete mitochondrial genome of the pink stem borer, Sesamia inferens, in comparison with four other Noctuid moths.

PubMed

Chai, Huan-Na; Du, Yu-Zhou

2012-01-01

The complete 15,413-bp mitochondrial genome (mitogenome) of Sesamia inferens (Walker) (Lepidoptera: Noctuidae) was sequenced and compared with those of four other noctuid moths. All of the mitogenomes analyzed displayed similar characteristics with respect to gene content, genome organization, nucleotide comparison, and codon usages. Twelve-one protein-coding genes (PCGs) utilized the standard ATN, but the cox1 gene used CGA as the initiation codon; cox1, cox2, and nad4 genes had the truncated termination codon T in the S. inferens mitogenome. All of the tRNA genes had typical cloverleaf secondary structures except for trnS1(AGN), in which the dihydrouridine (DHU) arm did not form a stable stem-loop structure. Both the secondary structures of rrnL and rrnS genes inferred from the S. inferens mitogenome closely resembled those of other noctuid moths. In the A+T-rich region, the conserved motif "ATAGA" followed by a long T-stretch was observed in all noctuid moths, but other specific tandem-repeat elements were more variable. Additionally, the S. inferens mitogenome contained a potential stem-loop structure, a duplicated 17-bp repeat element, a decuplicated segment, and a microsatellite "(AT)(7)", without a poly-A element upstream of the trnM in the A+T-rich region. Finally, the phylogenetic relationships were reconstructed based on amino acid sequences of mitochondrial 13 PCGs, which support the traditional morphologically based view of relationships within the Noctuidae.

The Complete Mitochondrial Genome of the Pink Stem Borer, Sesamia inferens, in Comparison with Four Other Noctuid Moths

PubMed Central

Chai, Huan-Na; Du, Yu-Zhou

2012-01-01

The complete 15,413-bp mitochondrial genome (mitogenome) of Sesamia inferens (Walker) (Lepidoptera: Noctuidae) was sequenced and compared with those of four other noctuid moths. All of the mitogenomes analyzed displayed similar characteristics with respect to gene content, genome organization, nucleotide comparison, and codon usages. Twelve-one protein-coding genes (PCGs) utilized the standard ATN, but the cox1 gene used CGA as the initiation codon; cox1, cox2, and nad4 genes had the truncated termination codon T in the S. inferens mitogenome. All of the tRNA genes had typical cloverleaf secondary structures except for trnS1(AGN), in which the dihydrouridine (DHU) arm did not form a stable stem-loop structure. Both the secondary structures of rrnL and rrnS genes inferred from the S. inferens mitogenome closely resembled those of other noctuid moths. In the A+T-rich region, the conserved motif “ATAGA” followed by a long T-stretch was observed in all noctuid moths, but other specific tandem-repeat elements were more variable. Additionally, the S. inferens mitogenome contained a potential stem-loop structure, a duplicated 17-bp repeat element, a decuplicated segment, and a microsatellite “(AT)7”, without a poly-A element upstream of the trnM in the A+T-rich region. Finally, the phylogenetic relationships were reconstructed based on amino acid sequences of mitochondrial 13 PCGs, which support the traditional morphologically based view of relationships within the Noctuidae. PMID:22949858

A small modified hammerhead ribozyme and its conformational characteristics determined by mutagenesis and lattice calculation.

PubMed Central

Lustig, B; Lin, N H; Smith, S M; Jernigan, R L; Jeang, K T

1995-01-01

A prototypic hammerhead ribozyme has three helices that surround an asymmetrical central core loop. We have mutagenized a hammerhead type ribozyme. In agreement with previous studies, progressive removal of stem-loop II from a three stemmed ribozyme showed that this region is not absolutely critical for catalysis. However, complete elimination of stem II and its loop did reduce, but did not eliminate, function. In a stem-loop II-deleted ribozyme, activity was best preserved when a purine, preferably a G, was present at position 10.1. This G contributed to catalysis irregardless of its role as either one part of a canonical pair with a C residue at 11.1 or a lone nucleotide with C (11.1) deleted. Computational methods using lattices generated 87 million three-dimensional chain forms for a stem-loop II-deleted RNA complex that preserved one potential G.C base pair at positions 10.1 and 11.1. This exhaustive set of chain forms included one major class of structures with G(10.1) being spatially proximal to the GUCX cleavage site of the substrate strand. Strong correlations were observed between colinear arrangement of stems I and III, constraints of base-pairing in the central core loop, and one particular placement of G(10.1) relative to the cleavage site. Our calculations of a stem-loop II-deleted ribozyme indicate that without needing to invoke any other constraints, the inherent asymmetry in the lengths of the two loop strands (3 nt in one and 7 nt in the other) that compose the core and flank G10.1-C11.1 stipulated strongly this particular G placement. This suggests that the hammerhead ribozyme maintains an asymmetry in its internal loop for a necessary structure/function reason. Images PMID:7567466

HCV IRES domain IIb affects the configuration of coding RNA in the 40S subunit's decoding groove.

PubMed

Filbin, Megan E; Kieft, Jeffrey S

2011-07-01

Hepatitis C virus (HCV) uses a structured internal ribosome entry site (IRES) RNA to recruit the translation machinery to the viral RNA and begin protein synthesis without the ribosomal scanning process required for canonical translation initiation. Different IRES structural domains are used in this process, which begins with direct binding of the 40S ribosomal subunit to the IRES RNA and involves specific manipulation of the translational machinery. We have found that upon initial 40S subunit binding, the stem-loop domain of the IRES that contains the start codon unwinds and adopts a stable configuration within the subunit's decoding groove. This configuration depends on the sequence and structure of a different stem-loop domain (domain IIb) located far from the start codon in sequence, but spatially proximal in the IRES•40S complex. Mutation of domain IIb results in misconfiguration of the HCV RNA in the decoding groove that includes changes in the placement of the AUG start codon, and a substantial decrease in the ability of the IRES to initiate translation. Our results show that two distal regions of the IRES are structurally communicating at the initial step of 40S subunit binding and suggest that this is an important step in driving protein synthesis.

Predicted stem-loop structures and variation in nucleotide sequence of 3' noncoding regions among animal calicivirus genomes.

PubMed

Seal, B S; Neill, J D; Ridpath, J F

1994-07-01

Caliciviruses are nonenveloped with a polyadenylated genome of approximately 7.6 kb and a single capsid protein. The "RNA Fold" computer program was used to analyze 3'-terminal noncoding sequences of five feline calicivirus (FCV), rabbit hemorrhagic disease virus (RHDV), and two San Miguel sea lion virus (SMSV) isolates. The FCV 3'-terminal sequences are 40-46 nucleotides in length and 72-91% similar. The FCV sequences were predicted to contain two possible duplex structures and one stem-loop structure with free energies of -2.1 to -18.2 kcal/mole. The RHDV genomic 3'-terminal RNA sequences are 54 nucleotides in length and share 49% sequence similarity to homologous regions of the FCV genome. The RHDV sequence was predicted to form two duplex structures in the 3'-terminal noncoding region with a single stem-loop structure, resembling that of FCV. In contrast, the SMSV 1 and 4 genomic 3'-terminal noncoding sequences were 185 and 182 nucleotides in length, respectively. Ten possible duplex structures were predicted with an average structural free energy of -35 kcal/mole. Sequence similarity between the two SMSV isolates was 75%. Furthermore, extensive cloverleaflike structures are predicted in the 3' noncoding region of the SMSV genome, in contrast to the predicted single stem-loop structures of FCV or RHDV.

Minor groove RNA triplex in the crystal structure of a ribosomal frameshifting viral pseudoknot

NASA Technical Reports Server (NTRS)

Su, L.; Chen, L.; Egli, M.; Berger, J. M.; Rich, A.

1999-01-01

Many viruses regulate translation of polycistronic mRNA using a -1 ribosomal frameshift induced by an RNA pseudoknot. A pseudoknot has two stems that form a quasi-continuous helix and two connecting loops. A 1.6 A crystal structure of the beet western yellow virus (BWYV) pseudoknot reveals rotation and a bend at the junction of the two stems. A loop base is inserted in the major groove of one stem with quadruple-base interactions. The second loop forms a new minor-groove triplex motif with the other stem, involving 2'-OH and triple-base interactions, as well as sodium ion coordination. Overall, the number of hydrogen bonds stabilizing the tertiary interactions exceeds the number involved in Watson-Crick base pairs. This structure will aid mechanistic analyses of ribosomal frameshifting.

Helix-length compensation studies reveal the adaptability of the VS ribozyme architecture.

PubMed

Lacroix-Labonté, Julie; Girard, Nicolas; Lemieux, Sébastien; Legault, Pascale

2012-03-01

Compensatory mutations in RNA are generally regarded as those that maintain base pairing, and their identification forms the basis of phylogenetic predictions of RNA secondary structure. However, other types of compensatory mutations can provide higher-order structural and evolutionary information. Here, we present a helix-length compensation study for investigating structure-function relationships in RNA. The approach is demonstrated for stem-loop I and stem-loop V of the Neurospora VS ribozyme, which form a kissing-loop interaction important for substrate recognition. To rapidly characterize the substrate specificity (k(cat)/K(M)) of several substrate/ribozyme pairs, a procedure was established for simultaneous kinetic characterization of multiple substrates. Several active substrate/ribozyme pairs were identified, indicating the presence of limited substrate promiscuity for stem Ib variants and helix-length compensation between stems Ib and V. 3D models of the I/V interaction were generated that are compatible with the kinetic data. These models further illustrate the adaptability of the VS ribozyme architecture for substrate cleavage and provide global structural information on the I/V kissing-loop interaction. By exploring higher-order compensatory mutations in RNA our approach brings a deeper understanding of the adaptability of RNA structure, while opening new avenues for RNA research.

An efficient algorithm for planar drawing of RNA structures with pseudoknots of any type.

PubMed

Byun, Yanga; Han, Kyungsook

2016-06-01

An RNA pseudoknot is a tertiary structural element in which bases of a loop pair with complementary bases are outside the loop. A drawing of RNA secondary structures is a tree, but a drawing of RNA pseudoknots is a graph that has an inner cycle within a pseudoknot and possibly outer cycles formed between the pseudoknot and other structural elements. Visualizing a large-scale RNA structure with pseudoknots as a planar drawing is challenging because a planar drawing of an RNA structure requires both pseudoknots and an entire structure enclosing the pseudoknots to be embedded into a plane without overlapping or crossing. This paper presents an efficient heuristic algorithm for visualizing a pseudoknotted RNA structure as a planar drawing. The algorithm consists of several parts for finding crossing stems and page mapping the stems, for the layout of stem-loops and pseudoknots, and for overlap detection between structural elements and resolving it. Unlike previous algorithms, our algorithm generates a planar drawing for a large RNA structure with pseudoknots of any type and provides a bracket view of the structure. It generates a compact and aesthetic structure graph for a large pseudoknotted RNA structure in O([Formula: see text]) time, where n is the number of stems of the RNA structure.

Rational engineering of the Neurospora VS ribozyme to allow substrate recognition via different kissing-loop interactions.

PubMed

Lacroix-Labonté, Julie; Girard, Nicolas; Dagenais, Pierre; Legault, Pascale

2016-08-19

The Neurospora VS ribozyme is a catalytic RNA that has the unique ability to specifically recognize and cleave a stem-loop substrate through formation of a highly stable kissing-loop interaction (KLI). In order to explore the engineering potential of the VS ribozyme to cleave alternate substrates, we substituted the wild-type KLI by other known KLIs using an innovative engineering method that combines rational and combinatorial approaches. A bioinformatic search of the protein data bank was initially performed to identify KLIs that are structurally similar to the one found in the VS ribozyme. Next, substrate/ribozyme (S/R) pairs that incorporate these alternative KLIs were kinetically and structurally characterized. Interestingly, several of the resulting S/R pairs allowed substrate cleavage with substantial catalytic efficiency, although with reduced activity compared to the reference S/R pair. Overall, this study describes an innovative approach for RNA engineering and establishes that the KLI of the trans VS ribozyme can be adapted to cleave other folded RNA substrates. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Structure-Function Model for Kissing Loop Interactions That Initiate Dimerization of Ty1 RNA

PubMed Central

Gamache, Eric R.; Doh, Jung H.; Ritz, Justin; Laederach, Alain; Bellaousov, Stanislav; Mathews, David H.; Curcio, M. Joan

2017-01-01

The genomic RNA of the retrotransposon Ty1 is packaged as a dimer into virus-like particles. The 5′ terminus of Ty1 RNA harbors cis-acting sequences required for translation initiation, packaging and initiation of reverse transcription (TIPIRT). To identify RNA motifs involved in dimerization and packaging, a structural model of the TIPIRT domain in vitro was developed from single-nucleotide resolution RNA structural data. In general agreement with previous models, the first 326 nucleotides of Ty1 RNA form a pseudoknot with a 7-bp stem (S1), a 1-nucleotide interhelical loop and an 8-bp stem (S2) that delineate two long, structured loops. Nucleotide substitutions that disrupt either pseudoknot stem greatly reduced helper-Ty1-mediated retrotransposition of a mini-Ty1, but only mutations in S2 destabilized mini-Ty1 RNA in cis and helper-Ty1 RNA in trans. Nested in different loops of the pseudoknot are two hairpins with complementary 7-nucleotide motifs at their apices. Nucleotide substitutions in either motif also reduced retrotransposition and destabilized mini- and helper-Ty1 RNA. Compensatory mutations that restore base-pairing in the S2 stem or between the hairpins rescued retrotransposition and RNA stability in cis and trans. These data inform a model whereby a Ty1 RNA kissing complex with two intermolecular kissing-loop interactions initiates dimerization and packaging. PMID:28445416

Stability of a stem-loop involving the initiator AUG controls the efficiency of internal initiation of translation on hepatitis C virus RNA.

PubMed Central

Honda, M; Brown, E A; Lemon, S M

1996-01-01

The initiation of translation on the positive-sense RNA genome of hepatitis C virus (HCV) is directed by an internal ribosomal entry site (IRES) that occupies most of the 341-nt 5' nontranslated RNA (5'NTR). Previous studies indicate that this IRES differs from picornaviral IRESs in that its activity is dependent upon RNA sequence downstream of the initiator AUG. Here, we demonstrate that the initiator AUG of HCV is located within a stem-loop (stem-loop IV) involving nt -12 to +12 (with reference to the AUG). This structure is conserved among HCV strains, and is present in the 5'NTR of the phylogenetically distant GB virus B. Mutant, nearly genome-length RNAs containing nucleotide substitutions predicted to enhance the stability of stem-loop IV were generally deficient in cap-independent translation both in vitro and in vivo. Additional mutations that destabilize the stem-loop restored translation to normal. Thus, the stability of the stem-loop is strongly but inversely correlated with the efficiency of internal initiation of translation. In contrast, mutations that stabilize this stem-loop had comparatively little effect on translation of 5' truncated RNAs by scanning ribosomes, suggesting that internal initiation of translation follows binding of the 40S ribosome directly at the site of stem-loop IV. Because stem-loop IV is not required for internal entry of ribosomes but is able to regulate this process, we speculate that it may be stabilized by interactions with a viral protein, providing a mechanism for feedback regulation of translation, which may be important for viral persistence. PMID:8849773

The structure of the L3 loop from the hepatitis delta virus ribozyme: a syn cytidine.

PubMed Central

Lynch, S R; Tinoco, I

1998-01-01

The structure of the L3 central hairpin loop isolated from the antigenomic sequence of the hepatitis delta virus ribozyme with the P2 and P3 stems from the ribozyme stacked on top of the loop has been determined by NMR spectroscopy. The 26 nt stem-loop structure contains nine base pairs and a 7 nt loop (5'-UCCUCGC-3'). This hairpin loop is critical for efficient catalysis in the intact ribozyme. The structure was determined using homonuclear and heteronuclear NMR techniques on non-labeled and15N-labeled RNA oligonucleotides. The overall root mean square deviation for the structure was 1.15 A (+/- 0.28 A) for the loop and the closing C.G base pair and 0.90 A (+/- 0.18 A) for the loop and the closing C.G base pair but without the lone purine in the loop, which is not well defined in the structure. The structure indicates a U.C base pair between the nucleotides on the 5'- and 3'-ends of the loop. This base pair is formed with a single hydrogen bond involving the cytosine exocyclic amino proton and the carbonyl O4 of the uracil. The most unexpected finding in the loop is a syn cytidine. While not unprecedented, syn pyrimidines are highly unusual. This one can be confidently established by intranucleotide distances between the ribose and the base determined by NMR spectroscopy. A similar study of the structure of this loop showed a somewhat different three-dimensional structure. A discussion of differences in the two structures, as well as possible sites of interaction with the cleavage site, will be presented. PMID:9461457

Water-soluble mercury ion sensing based on the thymine-Hg2+-thymine base pair using retroreflective Janus particle as an optical signaling probe.

PubMed

Chun, Hyeong Jin; Kim, Saemi; Han, Yong Duk; Kim, Dong Woo; Kim, Ka Ram; Kim, Hyo-Sop; Kim, Jae-Ho; Yoon, Hyun C

2018-05-01

Herein, we report an optical sensing platform for mercury ions (Hg 2+ ) in water based on the integration of Hg 2+ -mediated thymine-thymine (T-T) stabilization, a biotinylated stem-loop DNA probe, and a streptavidin-modified retroreflective Janus particle (SA-RJP). Two oligonucleotide probes, including a stem-loop DNA probe and an assistant DNA probe, were utilized. In the absence of Hg 2+ , the assistant DNA probe does not hybridize with the stem-loop probe due to their T-T mismatch, so the surface-immobilized stem-loop DNA probe remains a closed hairpin structure. In the presence of Hg 2+ , the DNA forms a double-stranded structure with the loop region via Hg 2+ -mediated T-T stabilization. This DNA hybridization induces stretching of the stem-loop DNA probe, exposing biotin. To translate these Hg 2+ -mediated structural changes in DNA probe into measurable signal, SA-RJP, an optical signaling label, is applied to recognize the exposed biotin. The number of biospecifically bound SA-RJPs is proportional to the concentration of Hg 2+ , so that the concentration of Hg 2+ can be quantitatively analyzed by counting the number of RJPs. Using the system, a highly selective and sensitive measurement of Hg 2+ was accomplished with a limit of detection of 0.027nM. Considering the simplified optical instrumentation required for retroreflection-based RJP counting, RJP-assisted Hg 2+ measurement can be accomplished in a much easier and inexpensive manner. Moreover, the detection of Hg 2+ in real drinking water samples including tap and commercial bottled water was successfully carried out. Copyright © 2018 Elsevier B.V. All rights reserved.

The icosahedral RNA virus as a grotto: organizing the genome into stalagmites and stalactites.

PubMed

Harvey, Stephen C; Zeng, Yingying; Heitsch, Christine E

2013-03-01

There are two important problems in the assembly of small, icosahedral RNA viruses. First, how does the capsid protein select the viral RNA for packaging, when there are so many other candidate RNA molecules available? Second, what is the mechanism of assembly? With regard to the first question, there are a number of cases where a particular RNA sequence or structure--often one or more stem-loops--either promotes assembly or is required for assembly, but there are others where specific packaging signals are apparently not required. With regard to the assembly pathway, in those cases where stem-loops are involved, the first step is generally believed to be binding of the capsid proteins to these "fingers" of the RNA secondary structure. In the mature virus, the core of the RNA would then occupy the center of the viral particle, and the stem-loops would reach outward, towards the capsid, like stalagmites reaching up from the floor of a grotto towards the ceiling. Those viruses whose assembly does not depend on protein binding to stem-loops could have a different structure, with the core of the RNA lying just under the capsid, and the fingers reaching down into the interior of the virus, like stalactites. We review the literature on these alternative structures, focusing on RNA selectivity and the assembly mechanism, and we propose experiments aimed at determining, in a given virus, which of the two structures actually occurs.

Model for an RNA tertiary interaction from the structure of an intermolecular complex between a GAAA tetraloop and an RNA helix.

PubMed

Pley, H W; Flaherty, K M; McKay, D B

1994-11-03

In large structured RNAs, RNA hairpins in which the strands of the duplex stem are connected by a tetraloop of the consensus sequence 5'-GNRA (where N is any nucleotide, and R is either G or A) are unusually frequent. In group I introns there is a covariation in sequence between nucleotides in the third and fourth positions of the loop with specific distant base pairs in putative RNA duplex stems: GNAA loops correlate with successive 5'-C-C.G-C base pairs in stems, whereas GNGA loops correlate with 5'-C-U.G-A. This has led to the suggestion that GNRA tetraloops may be involved in specific long-range tertiary interactions, with each A in position 3 or 4 of the loop interacting with a C-G base pair in the duplex, and G in position 3 interacting with a U-A base pair. This idea is supported experimentally for the GAAA loop of the P5b extension of the group I intron of Tetrahymena thermophila and the L9 GUGA terminal loop of the td intron of bacteriophage T4 (ref. 4). NMR has revealed the overall structure of the tetraloop for 12-nucleotide hairpins with GCAA and GAAA loops and models have been proposed for the interaction of GNRA tetraloops with base pairs in the minor groove of A-form RNA. Here we describe the crystal structure of an intermolecular complex between a GAAA tetraloop and an RNA helix. The interactions we observe correlate with the specificity of GNRA tetraloops inferred from phylogenetic studies, suggesting that this complex is a legitimate model for intramolecular tertiary interactions mediated by GNRA tetraloops in large structured RNAs.

Negative feedback regulation of wild-type p53 biosynthesis.

PubMed Central

Mosner, J; Mummenbrauer, T; Bauer, C; Sczakiel, G; Grosse, F; Deppert, W

1995-01-01

When growth-arrested mouse fibroblasts re-entered the cell-cycle, the rise in tumour suppressor p53 mRNA level markedly preceded the rise in expression of the p53 protein. Furthermore, gamma-irradiation of such cells led to a rapid increase in p53 protein biosynthesis even in the presence of the transcription inhibitor actinomycin D. Both findings strongly suggest that p53 biosynthesis in these cells is regulated at the translational level. We present evidence for an autoregulatory control of p53 expression by a negative feed-back loop: p53 mRNA has a predicted tendency to form a stable stem-loop structure that involves the 5'-untranslated region (5'-UTR) plus some 280 nucleotides of the coding sequence. p53 binds tightly to the 5'-UTR region and inhibits the translation of its own mRNA, most likely mediated by the p53-intrinsic RNA re-annealing activity. The inhibition of p53 biosynthesis requires wild-type p53, as it is not observed with MethA mutant p53, p53-catalysed translational inhibition is selective; it might be restricted to p53 mRNA and a few other mRNAs that are able to form extensive stem-loop structures. Release from negative feed-back regulation of p53 biosynthesis, e.g. after damage-induced nuclear transport of p53, might provide a means for rapidly increasing p53 protein levels when p53 is required to act as a cell-cycle checkpoint determinant after DNA damage. Images PMID:7556087

Chimeric RNase H–Competent Oligonucleotides Directed to the HIV-1 Rev Response Element

PubMed Central

Prater, Chrissy E.; Saleh, Anthony D.; Wear, Maggie P.; Miller, Paul S.

2007-01-01

Chimeric oligo-2′-O-methylribonucleotides containing centrally located patches of contiguous 2′-deoxyribonucleotides and terminating in a nuclease resistant 3′-methylphosphonate internucleotide linkage were prepared. The oligonucleotides were targeted to the 3′-side of HIV Rev response element (RRE) stem-loop IIB RNA, which is adjacent to the high affinity Rev protein binding site and is critical to virus function. Thermal denaturation experiments showed that chimeric oligonucleotides form very stable duplexes with a complementary single-stranded RNA, and gel electrophoretic mobility shift assays (EMSA) showed that they bind with high affinity and specificity to RRE stem-loop II RNA (KD approximately 200 nM). The chimeric oligonucleotides promote RNase H-mediated hydrolysis of RRE stem-loop II RNA and have half lives exceeding 24 h when incubated in cell culture medium containing 10% fetal calf serum. One of the chimeric oligonucleotides inhibited RRE mediated expression of chloramphenicol acetyl transferase (CAT) approximately 60% at a concentration of 300 nM in HEK 293T cells co-transfected with p-RRE/CAT and p-Rev mammalian expression vectors. PMID:17566743

Complete Mitochondrial Genome Sequence of Acrida cinerea (Acrididae: Orthoptera) and Comparative Analysis of Mitochondrial Genomes in Orthoptera

PubMed Central

Liu, Nian; Huang, Yuan

2010-01-01

The complete 15,599-bp mitogenome of Acrida cinerea was determined and compared with that of the other 20 orthopterans. It displays characteristic gene content, genome organization, nucleotide composition, and codon usage found in other Caelifera mitogenomes. Comparison of 21 orthopteran sequences revealed that the tRNAs encoded by the H-strand appear more conserved than those by the L-stand. All tRNAs form the typical clover-leaf structure except trnS (agn), and most of the size variation among tRNAs stemmed from the length variation in the arm and loop of TΨC and the loop of DHU. The derived secondary structure models of the rrnS and rrnL from 21 orthoptera species closely resemble those from other insects on CRW except a considerably enlarged loop of helix 1399 of rrnS in Caelifera, which is a potentially autapomorphy of Caelifera. In the A+T-rich region, tandem repeats are not only conserved in the closely related mitogenome but also share some conserved motifs in the same subfamily. A stem-loop structure, 16 bp or longer, is likely to be involved in replication initiation in Caelifera and Grylloidea. A long T-stretch (>17 bp) with conserved stem-loop structure next to rrnS on the H-strand, bounded by a purine at either end, exists in the three species from Tettigoniidae. PMID:21197069

Degenerate RNA packaging signals in the genome of Satellite Tobacco Necrosis Virus: implications for the assembly of a T=1 capsid.

PubMed

Bunka, David H J; Lane, Stephen W; Lane, Claire L; Dykeman, Eric C; Ford, Robert J; Barker, Amy M; Twarock, Reidun; Phillips, Simon E V; Stockley, Peter G

2011-10-14

Using a recombinant, T=1 Satellite Tobacco Necrosis Virus (STNV)-like particle expressed in Escherichia coli, we have established conditions for in vitro disassembly and reassembly of the viral capsid. In vivo assembly is dependent on the presence of the coat protein (CP) N-terminal region, and in vitro assembly requires RNA. Using immobilised CP monomers under reassembly conditions with "free" CP subunits, we have prepared a range of partially assembled CP species for RNA aptamer selection. SELEX directed against the RNA-binding face of the STNV CP resulted in the isolation of several clones, one of which (B3) matches the STNV-1 genome in 16 out of 25 nucleotide positions, including across a statistically significant 10/10 stretch. This 10-base region folds into a stem-loop displaying the motif ACAA and has been shown to bind to STNV CP. Analysis of the other aptamer sequences reveals that the majority can be folded into stem-loops displaying versions of this motif. Using a sequence and secondary structure search motif to analyse the genomic sequence of STNV-1, we identified 30 stem-loops displaying the sequence motif AxxA. The implication is that there are many stem-loops in the genome carrying essential recognition features for binding STNV CP. Secondary structure predictions of the genomic RNA using Mfold showed that only 8 out of 30 of these stem-loops would be formed in the lowest-energy structure. These results are consistent with an assembly mechanism based on kinetically driven folding of the RNA. Copyright © 2011 Elsevier Ltd. All rights reserved.

The role of RNA structure in the interaction of U1A protein with U1 hairpin II RNA

PubMed Central

Law, Michael J.; Rice, Andrew J.; Lin, Patti; Laird-Offringa, Ite A.

2006-01-01

The N-terminal RNA Recognition Motif (RRM1) of the spliceosomal protein U1A interacting with its target U1 hairpin II (U1hpII) has been used as a paradigm for RRM-containing proteins interacting with their RNA targets. U1A binds to U1hpII via direct interactions with a 7-nucleotide (nt) consensus binding sequence at the 5′ end of a 10-nt loop, and via hydrogen bonds with the closing C–G base pair at the top of the RNA stem. Using surface plasmon resonance (Biacore), we have examined the role of structural features of U1hpII in binding to U1A RRM1. Mutational analysis of the closing base pair suggests it plays a minor role in binding and mainly prevents “breathing” of the loop. Lengthening the stem and nontarget part of the loop suggests that the increased negative charge of the RNA might slightly aid association. However, this is offset by an increase in dissociation, which may be caused by attraction of the RRM to nontarget parts of the RNA. Studies of a single stranded target and RNAs with untethered loops indicate that structure is not very relevant for association but is important for complex stability. In particular, breaking the link between the stem and the 5′ side of the loop greatly increases complex dissociation, presumably by hindering simultaneous contacts between the RRM and stem and loop nucleotides. While binding of U1A to a single stranded target is much weaker than to U1hpII, it occurs with nanomolar affinity, supporting recent evidence that binding of unstructured RNA by U1A has physiological significance. PMID:16738410

The role of RNA structure in the interaction of U1A protein with U1 hairpin II RNA.

PubMed

Law, Michael J; Rice, Andrew J; Lin, Patti; Laird-Offringa, Ite A

2006-07-01

The N-terminal RNA Recognition Motif (RRM1) of the spliceosomal protein U1A interacting with its target U1 hairpin II (U1hpII) has been used as a paradigm for RRM-containing proteins interacting with their RNA targets. U1A binds to U1hpII via direct interactions with a 7-nucleotide (nt) consensus binding sequence at the 5' end of a 10-nt loop, and via hydrogen bonds with the closing C-G base pair at the top of the RNA stem. Using surface plasmon resonance (Biacore), we have examined the role of structural features of U1hpII in binding to U1A RRM1. Mutational analysis of the closing base pair suggests it plays a minor role in binding and mainly prevents "breathing" of the loop. Lengthening the stem and nontarget part of the loop suggests that the increased negative charge of the RNA might slightly aid association. However, this is offset by an increase in dissociation, which may be caused by attraction of the RRM to nontarget parts of the RNA. Studies of a single stranded target and RNAs with untethered loops indicate that structure is not very relevant for association but is important for complex stability. In particular, breaking the link between the stem and the 5' side of the loop greatly increases complex dissociation, presumably by hindering simultaneous contacts between the RRM and stem and loop nucleotides. While binding of U1A to a single stranded target is much weaker than to U1hpII, it occurs with nanomolar affinity, supporting recent evidence that binding of unstructured RNA by U1A has physiological significance.

Autologous serum improves bone formation in a primary stable silica-embedded nanohydroxyapatite bone substitute in combination with mesenchymal stem cells and rhBMP-2 in the sheep model

PubMed Central

Boos, Anja M; Weigand, Annika; Deschler, Gloria; Gerber, Thomas; Arkudas, Andreas; Kneser, Ulrich; Horch, Raymund E; Beier, Justus P

2014-01-01

New therapeutic strategies are required for critical size bone defects, because the gold standard of transplanting autologous bone from an unharmed area of the body often leads to several severe side effects and disadvantages for the patient. For years, tissue engineering approaches have been seeking a stable, axially vascularized transplantable bone replacement suitable for transplantation into the recipient bed with pre-existing insufficient conditions. For this reason, the arteriovenous loop model was developed and various bone substitutes have been vascularized. However, it has not been possible thus far to engineer a primary stable and axially vascularized transplantable bone substitute. For that purpose, a primary stable silica-embedded nanohydroxyapatite (HA) bone substitute in combination with blood, bone marrow, expanded, or directly retransplanted mesenchymal stem cells, recombinant human bone morphogenetic protein 2 (rhBMP-2), and different carrier materials (fibrin, cell culture medium, autologous serum) was tested subcutaneously for 4 or 12 weeks in the sheep model. Autologous serum lead to an early matrix change during degradation of the bone substitute and formation of new bone tissue. The best results were achieved in the group combining mesenchymal stem cells expanded with 60 μg/mL rhBMP-2 in autologous serum. Better ingrowth of fibrovascular tissue could be detected in the autologous serum group compared with the control (fibrin). Osteoclastic activity indicating an active bone remodeling process was observed after 4 weeks, particularly in the group with autologous serum and after 12 weeks in every experimental group. This study clearly demonstrates the positive effects of autologous serum in combination with mesenchymal stem cells and rhBMP-2 on bone formation in a primary stable silica-embedded nano-HA bone grafting material in the sheep model. In further experiments, the results will be transferred to the sheep arteriovenous loop model in order to engineer an axially vascularized primary stable bone replacement in clinically relevant size for free transplantation. PMID:25429218

Structural features of the DNA hairpin d(ATCCTA-GTTA-TAGGAT): formation of a G-A base pair in the loop.

PubMed Central

van Dongen, M J; Mooren, M M; Willems, E F; van der Marel, G A; van Boom, J H; Wijmenga, S S; Hilbers, C W

1997-01-01

The three-dimensional structure of the hairpin formed by d(ATCCTA-GTTA-TAGGAT) has been determined by means of two-dimensional NMR studies, distance geometry and molecular dynamics calculations. The first and the last residues of the tetraloop of this hairpin form a sheared G-A base pair on top of the six Watson-Crick base pairs in the stem. The glycosidic torsion angles of the guanine and adenine residues in the G-A base pair reside in the anti and high- anti domain ( approximately -60 degrees ) respectively. Several dihedral angles in the loop adopt non-standard values to accommodate this base pair. The first and second residue in the loop are stacked in a more or less normal helical fashion; the fourth loop residue also stacks upon the stem, while the third residue is directed away from the loop region. The loop structure can be classified as a so-called type-I loop, in which the bases at the 5'-end of the loop stack in a continuous fashion. In this situation, loop stability is unlikely to depend heavily on the nature of the unpaired bases in the loop. Moreover, the present study indicates that the influence of the polarity of a closing A.T pair is much less significant than that of a closing C.G base pair. PMID:9092659

The cellular RNA-binding protein EAP recognizes a conserved stem-loop in the Epstein-Barr virus small RNA EBER 1.

PubMed Central

Toczyski, D P; Steitz, J A

1993-01-01

EAP (EBER-associated protein) is an abundant, 15-kDa cellular RNA-binding protein which associates with certain herpesvirus small RNAs. We have raised polyclonal anti-EAP antibodies against a glutathione S-transferase-EAP fusion protein. Analysis of the RNA precipitated by these antibodies from Epstein-Barr virus (EBV)- or herpesvirus papio (HVP)-infected cells shows that > 95% of EBER 1 (EBV-encoded RNA 1) and the majority of HVP 1 (an HVP small RNA homologous to EBER 1) are associated with EAP. RNase protection experiments performed on native EBER 1 particles with affinity-purified anti-EAP antibodies demonstrate that EAP binds a stem-loop structure (stem-loop 3) of EBER 1. Since bacterially expressed glutathione S-transferase-EAP fusion protein binds EBER 1, we conclude that EAP binding is independent of any other cellular or viral protein. Detailed mutational analyses of stem-loop 3 suggest that EAP recognizes the majority of the nucleotides in this hairpin, interacting with both single-stranded and double-stranded regions in a sequence-specific manner. Binding studies utilizing EBER 1 deletion mutants suggest that there may also be a second, weaker EAP-binding site on stem-loop 4 of EBER 1. These data and the fact that stem-loop 3 represents the most highly conserved region between EBER 1 and HVP 1 suggest that EAP binding is a critical aspect of EBER 1 and HVP 1 function. Images PMID:8380232

Stem loop recognition by DDX17 facilitates miRNA processing and antiviral defense

PubMed Central

Moy, Ryan H.; Cole, Brian S.; Yasunaga, Ari; Gold, Beth; Shankarling, Ganesh; Varble, Andrew; Molleston, Jerome M.; tenOever, Benjamin R.; Lynch, Kristen W.; Cherry, Sara

2014-01-01

DEAD-box helicases play essential roles in RNA metabolism across species, but emerging data suggest that they have additional functions in immunity. Through RNAi screening we identify an evolutionarily conserved and interferon-independent role for the DEAD-box helicase DDX17 in restricting Rift Valley fever virus (RVFV), a mosquito-transmitted virus in the bunyavirus family that causes severe morbidity and mortality in humans and livestock. Loss of Drosophila DDX17 (Rm62) in cells and flies enhanced RVFV infection. Similarly, depletion of DDX17 but not the related helicase DDX5 increased RVFV replication in human cells. Using cross-linking immunoprecipitation high-throughput sequencing (CLIP-seq), we show that DDX17 binds the stem loops of host pri-miRNA to facilitate their processing, and also an essential stem loop in bunyaviral RNA to restrict infection. Thus, DDX17 has dual roles in the recognition of stem loops: in the nucleus for endogenous miRNA biogenesis and in the cytoplasm for surveillance against structured non-self elements. PMID:25126784

Alternative Mode of E-Site tRNA Binding in the Presence of a Downstream mRNA Stem Loop at the Entrance Channel.

PubMed

Zhang, Yan; Hong, Samuel; Ruangprasert, Ajchareeya; Skiniotis, Georgios; Dunham, Christine M

2018-03-06

Structured mRNAs positioned downstream of the ribosomal decoding center alter gene expression by slowing protein synthesis. Here, we solved the cryo-EM structure of the bacterial ribosome bound to an mRNA containing a 3' stem loop that regulates translation. Unexpectedly, the E-site tRNA adopts two distinct orientations. In the first structure, normal interactions with the 50S and 30S E site are observed. However, in the second structure, although the E-site tRNA makes normal interactions with the 50S E site, its anticodon stem loop moves ∼54 Å away from the 30S E site to interact with the 30S head domain and 50S uL5. This position of the E-site tRNA causes the uL1 stalk to adopt a more open conformation that likely represents an intermediate state during E-site tRNA dissociation. These results suggest that structured mRNAs at the entrance channel restrict 30S subunit movement required during translation to slow E-site tRNA dissociation. Copyright © 2018 Elsevier Ltd. All rights reserved.

Biomolecular and structural analyses of cauliflower-like DNAs by ultraviolet, circular dichroism, and fluorescence spectroscopies in comparison with natural DNA.

PubMed

Gill, Pooria; Ranjbar, Bijan; Saber, Reza; Khajeh, Khosro; Mohammadian, Mehdi

2011-07-01

Cauliflower-like DNAs are stem-loop DNAs that are fabricated periodically in inverted repetitions from deoxyribonucleic acid phosphates (dNTPs) by loop-mediated isothermal amplification (LAMP). Cauliflower-like DNAs have ladder-shape behaviors on gel electrophoresis, and increasing the time of LAMP leads to multiplying the repetitions, stem-loops, and electrophoretic bands. Cauliflower-like DNAs were fabricated via LAMP using two loop primers, two bumper primers, dNTPs, a λ-phage DNA template, and a Bst DNA polymerase in 75- and 90-min periods. These times led to manufacturing two types of cauliflower-like DNAs with different contents of inverted repetitions and stem-loops, which were clearly indicated by two comparable electrophoresis patterns in agarose gel. LAMP-fabricated DNAs and natural dsB-DNA (salmon genomic DNA) were dialyzed in Gomori phosphate buffer (10 mM, pH 7.4) to be isolated from salts, nucleotides, and primers. Dialyzed DNAs were studied using UV spectroscopy, circular dichroism spectropolarimetry, and fluorescence spectrophotometry. Structural analyses indicated reduction of the molecular ellipticity and extinction coefficients in comparison with B-DNA. Also, cauliflower-like DNAs demonstrated less intrinsic and more extrinsic fluorescence in comparison with natural DNA. The overwinding and lengthening of the cauliflower-like configurations of LAMP DNAs led to changes in physical parameters of this type of DNA in comparison with natural DNA. The results obtained introduced new biomolecular characteristics of DNA macromolecules fabricated within a LAMP process and show the effects of more inverted repeats and stem-loops, which are manufactured by lengthening the process.

Atomistic Free Energy Model for Nucleic Acids: Simulations of Single-Stranded DNA and the Entropy Landscape of RNA Stem-Loop Structures.

PubMed

Mak, Chi H

2015-11-25

While single-stranded (ss) segments of DNAs and RNAs are ubiquitous in biology, details about their structures have only recently begun to emerge. To study ssDNA and RNAs, we have developed a new Monte Carlo (MC) simulation using a free energy model for nucleic acids that has the atomisitic accuracy to capture fine molecular details of the sugar-phosphate backbone. Formulated on the basis of a first-principle calculation of the conformational entropy of the nucleic acid chain, this free energy model correctly reproduced both the long and short length-scale structural properties of ssDNA and RNAs in a rigorous comparison against recent data from fluorescence resonance energy transfer, small-angle X-ray scattering, force spectroscopy and fluorescence correlation transport measurements on sequences up to ∼100 nucleotides long. With this new MC algorithm, we conducted a comprehensive investigation of the entropy landscape of small RNA stem-loop structures. From a simulated ensemble of ∼10(6) equilibrium conformations, the entropy for the initiation of different size RNA hairpin loops was computed and compared against thermodynamic measurements. Starting from seeded hairpin loops, constrained MC simulations were then used to estimate the entropic costs associated with propagation of the stem. The numerical results provide new direct molecular insights into thermodynaimc measurement from macroscopic calorimetry and melting experiments.

Cross-Species Functionality of Pararetroviral Elements Driving Ribosome Shunting

PubMed Central

Pooggin, Mikhail M.; Fütterer, Johannes; Hohn, Thomas

2008-01-01

Background Cauliflower mosaic virus (CaMV) and Rice tungro bacilliform virus (RTBV) belong to distinct genera of pararetroviruses infecting dicot and monocot plants, respectively. In both viruses, polycistronic translation of pregenomic (pg) RNA is initiated by shunting ribosomes that bypass a large region of the pgRNA leader with several short (s)ORFs and a stable stem-loop structure. The shunt requires translation of a 5′-proximal sORF terminating near the stem. In CaMV, mutations knocking out this sORF nearly abolish shunting and virus viability. Methodology/Principal Findings Here we show that two distant regions of the CaMV leader that form a minimal shunt configuration comprising the sORF, a bottom part of the stem, and a shunt landing sequence can be replaced by heterologous sequences that form a structurally similar configuration in RTBV without any dramatic effect on shunt-mediated translation and CaMV infectivity. The CaMV-RTBV chimeric leader sequence was largely stable over five viral passages in turnip plants: a few alterations that did eventually occur in the virus progenies are indicative of fine tuning of the chimeric sequence during adaptation to a new host. Conclusions/Significance Our findings demonstrate cross-species functionality of pararetroviral cis-elements driving ribosome shunting and evolutionary conservation of the shunt mechanism. We are grateful to Matthias Müller and Sandra Pauli for technical assistance. This work was initiated at Friedrich Miescher Institute (Basel, Switzerland). We thank Prof. Thomas Boller for hosting the group at the Institute of Botany. PMID:18286203

RNA-dependent RNA polymerase of hepatitis C virus binds to its coding region RNA stem-loop structure, 5BSL3.2, and its negative strand.

PubMed

Kanamori, Hiroshi; Yuhashi, Kazuhito; Ohnishi, Shin; Koike, Kazuhiko; Kodama, Tatsuhiko

2010-05-01

The hepatitis C virus NS5B RNA-dependent RNA polymerase (RdRp) is a key enzyme involved in viral replication. Interaction between NS5B RdRp and the viral RNA sequence is likely to be an important step in viral RNA replication. The C-terminal half of the NS5B-coding sequence, which contains the important cis-acting replication element, has been identified as an NS5B-binding sequence. In the present study, we confirm the specific binding of NS5B to one of the RNA stem-loop structures in the region, 5BSL3.2. In addition, we show that NS5B binds to the complementary strand of 5BSL3.2 (5BSL3.2N). The bulge structure of 5BSL3.2N was shown to be indispensable for tight binding to NS5B. In vitro RdRp activity was inhibited by 5BSL3.2N, indicating the importance of the RNA element in the polymerization by RdRp. These results suggest the involvement of the RNA stem-loop structure of the negative strand in the replication process.

Hepatitis B virus nuclear export elements: RNA stem-loop α and β, key parts of the HBV post-transcriptional regulatory element.

PubMed

Lim, Chun Shen; Brown, Chris M

2016-09-01

Many viruses contain RNA elements that modulate splicing and/or promote nuclear export of their RNAs. The RNAs of the major human pathogen, hepatitis B virus (HBV) contain a large (~600 bases) composite cis-acting 'post-transcriptional regulatory element' (PRE). This element promotes expression from these naturally intronless transcripts. Indeed, the related woodchuck hepadnavirus PRE (WPRE) is used to enhance expression in gene therapy and other expression vectors. These PRE are likely to act through a combination of mechanisms, including promotion of RNA nuclear export. Functional components of both the HBV PRE and WPRE are 2 conserved RNA cis-acting stem-loop (SL) structures, SLα and SLβ. They are within the coding regions of polymerase (P) gene, and both P and X genes, respectively. Based on previous studies using mutagenesis and/or nuclear magnetic resonance (NMR), here we propose 2 covariance models for SLα and SLβ. The model for the 30-nucleotide SLα contains a G-bulge and a CNGG(U) apical loop of which the first and the fourth loop residues form a CG pair and the fifth loop residue is bulged out, as observed in the NMR structure. The model for the 23-nucleotide SLβ contains a 7-base-pair stem and a 9-nucleotide loop. Comparison of the models with other RNA structural elements, as well as similarity searches of human transcriptome and viral genomes demonstrate that SLα and SLβ are specific to HBV transcripts. However, they are well conserved among the hepadnaviruses of non-human primates, the woodchuck and ground squirrel.

Hepatitis B virus nuclear export elements: RNA stem-loop α and β, key parts of the HBV post-transcriptional regulatory element

PubMed Central

Lim, Chun Shen; Brown, Chris M.

2016-01-01

ABSTRACT Many viruses contain RNA elements that modulate splicing and/or promote nuclear export of their RNAs. The RNAs of the major human pathogen, hepatitis B virus (HBV) contain a large (~600 bases) composite cis-acting 'post-transcriptional regulatory element' (PRE). This element promotes expression from these naturally intronless transcripts. Indeed, the related woodchuck hepadnavirus PRE (WPRE) is used to enhance expression in gene therapy and other expression vectors. These PRE are likely to act through a combination of mechanisms, including promotion of RNA nuclear export. Functional components of both the HBV PRE and WPRE are 2 conserved RNA cis-acting stem-loop (SL) structures, SLα and SLβ. They are within the coding regions of polymerase (P) gene, and both P and X genes, respectively. Based on previous studies using mutagenesis and/or nuclear magnetic resonance (NMR), here we propose 2 covariance models for SLα and SLβ. The model for the 30-nucleotide SLα contains a G-bulge and a CNGG(U) apical loop of which the first and the fourth loop residues form a CG pair and the fifth loop residue is bulged out, as observed in the NMR structure. The model for the 23-nucleotide SLβ contains a 7-base-pair stem and a 9-nucleotide loop. Comparison of the models with other RNA structural elements, as well as similarity searches of human transcriptome and viral genomes demonstrate that SLα and SLβ are specific to HBV transcripts. However, they are well conserved among the hepadnaviruses of non-human primates, the woodchuck and ground squirrel. PMID:27031749

R-loops: targets for nuclease cleavage and repeat instability.

PubMed

Freudenreich, Catherine H

2018-01-11

R-loops form when transcribed RNA remains bound to its DNA template to form a stable RNA:DNA hybrid. Stable R-loops form when the RNA is purine-rich, and are further stabilized by DNA secondary structures on the non-template strand. Interestingly, many expandable and disease-causing repeat sequences form stable R-loops, and R-loops can contribute to repeat instability. Repeat expansions are responsible for multiple neurodegenerative diseases, including Huntington's disease, myotonic dystrophy, and several types of ataxias. Recently, it was found that R-loops at an expanded CAG/CTG repeat tract cause DNA breaks as well as repeat instability (Su and Freudenreich, Proc Natl Acad Sci USA 114, E8392-E8401, 2017). Two factors were identified as causing R-loop-dependent breaks at CAG/CTG tracts: deamination of cytosines and the MutLγ (Mlh1-Mlh3) endonuclease, defining two new mechanisms for how R-loops can generate DNA breaks (Su and Freudenreich, Proc Natl Acad Sci USA 114, E8392-E8401, 2017). Following R-loop-dependent nicking, base excision repair resulted in repeat instability. These results have implications for human repeat expansion diseases and provide a paradigm for how RNA:DNA hybrids can cause genome instability at structure-forming DNA sequences. This perspective summarizes mechanisms of R-loop-induced fragility at G-rich repeats and new links between DNA breaks and repeat instability.

RNA-Mediated cis Regulation in Acinetobacter baumannii Modulates Stress-Induced Phenotypic Variation

PubMed Central

Ching, Carly; Gozzi, Kevin; Heinemann, Björn; Chai, Yunrong

2017-01-01

ABSTRACT In the nosocomial opportunistic pathogen Acinetobacter baumannii, RecA-dependent mutagenesis, which causes antibiotic resistance acquisition, is linked to the DNA damage response (DDR). Notably, unlike the Escherichia coli paradigm, recA and DDR gene expression in A. baumannii is bimodal. Namely, there is phenotypic variation upon DNA damage, which may provide a bet-hedging strategy for survival. Thus, understanding recA gene regulation is key to elucidate the yet unknown DDR regulation in A. baumannii. Here, we identify a structured 5′ untranslated region (UTR) in the recA transcript which serves as a cis-regulatory element. We show that a predicted stem-loop structure in this 5′ UTR affects mRNA half-life and underlies bimodal gene expression and thus phenotypic variation in response to ciprofloxacin treatment. We furthermore show that the stem-loop structure of the recA 5′ UTR influences intracellular RecA protein levels and, in vivo, impairing the formation of the stem-loop structure of the recA 5′ UTR lowers cell survival of UV treatment and decreases rifampin resistance acquisition from DNA damage-induced mutagenesis. We hypothesize that the 5′ UTR allows for stable recA transcripts during stress, including antibiotic treatment, enabling cells to maintain suitable RecA levels for survival. This innovative strategy to regulate the DDR in A. baumannii may contribute to its success as a pathogen. IMPORTANCE Acinetobacter baumannii is an opportunistic pathogen quickly gaining antibiotic resistances. Mutagenesis and antibiotic resistance acquisition are linked to the DNA damage response (DDR). However, how the DDR is regulated in A. baumannii remains unknown, since unlike most bacteria, A. baumannii does not follow the regulation of the Escherichia coli paradigm. In this study, we have started to uncover the mechanisms regulating the novel A. baumannii DDR. We have found that a cis-acting 5′ UTR regulates recA transcript stability, RecA protein levels, and DNA damage-induced phenotypic variation. Though 5′ UTRs are known to provide stability to transcripts in bacteria, this is the first example in which it regulates a bimodal DDR response through recA transcript stabilization, potentially enabling cells to have enough RecA for survival and genetic variability. PMID:28320880

Optimization of a reusable, DNA pseudoknot-based electrochemical sensor for sequence-specific DNA detection in blood serum.

PubMed

Cash, Kevin J; Heeger, Alan J; Plaxco, Kevin W; Xiao, Yi

2009-01-15

We describe in detail a new electrochemical DNA (E-DNA) sensing platform based on target-induced conformation changes in an electrode-bound DNA pseudoknot. The pseudoknot, a DNA structure containing two stem-loops in which the first stem's loop forms part of the second stem, is modified with a methylene blue redox tag at its 3' terminus and covalently attached to a gold electrode via the 5' terminus. In the absence of a target, the structure of the pseudoknot probe minimizes collisions between the redox tag and the electrode, thus reducing faradaic current. Target binding disrupts the pseudoknot structure, liberating a flexible, single-stranded element that can strike the electrode and efficiently transfer electrons. In this article we report further characterization and optimization of this new E-DNA architecture. We find that optimal signaling is obtained at an intermediate probe density ( approximately 1.8 x 10(13) molecules/cm(2) apparent density), which presumably represents a balance between steric and electrostatic blocking at high probe densities and increased background currents arising from transfer from the pseudoknot probe at lower densities. We also find that optimal 3' stem length, which appears to be 7 base pairs, represents a balance between pseudoknot structural stability and target affinity. Finally, a 3' loop comprised of poly(A) exhibits better mismatch discrimination than the equivalent poly(T) loop, but at the cost of decreased gain. Optimization over this parameter space significantly improves the signaling of the pseudoknot-based E-DNA architecture, leading to the ability to sensitively and specifically detect DNA targets even when challenged in complex, multicomponent samples such as blood serum.

Productive mRNA stem loop-mediated transcriptional slippage: Crucial features in common with intrinsic terminators.

PubMed

Penno, Christophe; Sharma, Virag; Coakley, Arthur; O'Connell Motherway, Mary; van Sinderen, Douwe; Lubkowska, Lucyna; Kireeva, Maria L; Kashlev, Mikhail; Baranov, Pavel V; Atkins, John F

2015-04-21

Escherichia coli and yeast DNA-dependent RNA polymerases are shown to mediate efficient nascent transcript stem loop formation-dependent RNA-DNA hybrid realignment. The realignment was discovered on the heteropolymeric sequence T5C5 and yields transcripts lacking a C residue within a corresponding U5C4. The sequence studied is derived from a Roseiflexus insertion sequence (IS) element where the resulting transcriptional slippage is required for transposase synthesis. The stability of the RNA structure, the proximity of the stem loop to the slippage site, the length and composition of the slippage site motif, and the identity of its 3' adjacent nucleotides (nt) are crucial for transcripts lacking a single C. In many respects, the RNA structure requirements for this slippage resemble those for hairpin-dependent transcription termination. In a purified in vitro system, the slippage efficiency ranges from 5% to 75% depending on the concentration ratios of the nucleotides specified by the slippage sequence and the 3' nt context. The only previous proposal of stem loop mediated slippage, which was in Ebola virus expression, was based on incorrect data interpretation. We propose a mechanical slippage model involving the RNAP translocation state as the main motor in slippage directionality and efficiency. It is distinct from previously described models, including the one proposed for paramyxovirus, where following random movement efficiency is mainly dependent on the stability of the new realigned hybrid. In broadening the scope for utilization of transcription slippage for gene expression, the stimulatory structure provides parallels with programmed ribosomal frameshifting at the translation level.

Arsenic Induces Polyadenylation of Canonical Histone mRNA by Down-regulating Stem-Loop-binding Protein Gene Expression*

PubMed Central

Brocato, Jason; Fang, Lei; Chervona, Yana; Chen, Danqi; Kiok, Kathrin; Sun, Hong; Tseng, Hsiang-Chi; Xu, Dazhong; Shamy, Magdy; Jin, Chunyuan; Costa, Max

2014-01-01

The replication-dependent histone genes are the only metazoan genes whose messenger RNA (mRNA) does not terminate with a poly(A) tail at the 3′-end. Instead, the histone mRNAs display a stem-loop structure at their 3′-end. Stem-loop-binding protein (SLBP) binds the stem-loop and regulates canonical histone mRNA metabolism. Here we report that exposure to arsenic, a carcinogenic metal, decreased cellular levels of SLBP by inducing its proteasomal degradation and inhibiting SLBP transcription via epigenetic mechanisms. Notably, arsenic exposure dramatically increased polyadenylation of canonical histone H3.1 mRNA possibly through down-regulation of SLBP expression. The polyadenylated H3.1 mRNA induced by arsenic was not susceptible to normal degradation that occurs at the end of S phase, resulting in continued presence into mitosis, increased total H3.1 mRNA, and increased H3 protein levels. Excess expression of canonical histones have been shown to increase sensitivity to DNA damage as well as increase the frequency of missing chromosomes and induce genomic instability. Thus, polyadenylation of canonical histone mRNA following arsenic exposure may contribute to arsenic-induced carcinogenesis. PMID:25266719

Sequence and structure determinants of Drosophila Hsp70 mRNA translation: 5'UTR secondary structure specifically inhibits heat shock protein mRNA translation.

PubMed Central

Hess, M A; Duncan, R F

1996-01-01

Preferential translation of Drosophila heat shock protein 70 (Hsp70) mRNA requires only the 5'-untranslated region (5'-UTR). The sequence of this region suggests that it has relatively little secondary structure, which may facilitate efficient protein synthesis initiation. To determine whether minimal 5'-UTR secondary structure is required for preferential translation during heat shock, the effect of introducing stem-loops into the Hsp70 mRNA 5'-UTR was measured. Stem-loops of -11 kcal/mol abolished translation during heat shock, but did not reduce translation in non-heat shocked cells. A -22 kcal/mol stem-loop was required to comparably inhibit translation during growth at normal temperatures. To investigate whether specific sequence elements are also required for efficient preferential translation, deletion and mutation analyses were conducted in a truncated Hsp70 5'-UTR containing only the cap-proximal and AUG-proximal segments. Linker-scanner mutations in the cap-proximal segment (+1 to +37) did not impair translation. Re-ordering the segments reduced mRNA translational efficiency by 50%. Deleting the AUG-proximal segment severely inhibited translation. A 5-extension of the full-length leader specifically impaired heat shock translation. These results indicate that heat shock reduces the capacity to unwind 5-UTR secondary structure, allowing only mRNAs with minimal 5'-UTR secondary structure to be efficiently translated. A function for specific sequences is also suggested. PMID:8710519

Identification of a high affinity nucleocapsid protein binding element within the Moloney murine leukemia virus Psi-RNA packaging signal: implications for genome recognition.

PubMed

D'Souza, V; Melamed, J; Habib, D; Pullen, K; Wallace, K; Summers, M F

2001-11-23

Murine leukemia virus (MLV) is currently the most widely used gene delivery system in gene therapy trials. The simple retrovirus packages two copies of its RNA genome by a mechanism that involves interactions between the nucleocapsid (NC) domain of a virally-encoded Gag polyprotein and a segment of the RNA genome located just upstream of the Gag initiation codon, known as the Psi-site. Previous studies indicated that the MLV Psi-site contains three stem loops (SLB-SLD), and that stem loops SLC and SLD play prominent roles in packaging. We have developed a method for the preparation and purification of large quantities of recombinant Moloney MLV NC protein, and have studied its interactions with a series of oligoribonucleotides that contain one or more of the Psi-RNA stem loops. At RNA concentrations above approximately 0.3 mM, isolated stem loop SLB forms a duplex and stem loops SL-C and SL-D form kissing complexes, as expected from previous studies. However, neither the monomeric nor the dimeric forms of these isolated stem loops binds NC with significant affinity. Longer constructs containing two stem loops (SL-BC and SL-CD) also exhibit low affinities for NC. However, NC binds with high affinity and stoichiometrically to both the monomeric and dimeric forms of an RNA construct that contains all three stem loops (SL-BCD; K(d)=132(+/-55) nM). Titration of SL-BCD with NC also shifts monomer-dimer equilibrium toward the dimer. Mutagenesis experiments demonstrate that the conserved GACG tetraloops of stem loops C and D do not influence the monomer-dimer equilibrium of SL-BCD, that the tetraloop of stem loop B does not participate directly in NC binding, and that the tetraloops of stem loops C and D probably also do not bind to NC. These surprising results differ considerably from those observed for HIV-1, where NC binds to individual stem loops with high affinity via interactions with exposed residues of the tetraloops. The present results indicate that MLV NC binds to a pocket or surface that only exists in the presence of all three stem loops. Copyright 2001 Academic Press.

Solution structure of conserved AGNN tetraloops: insights into Rnt1p RNA processing

PubMed Central

Lebars, Isabelle; Lamontagne, Bruno; Yoshizawa, Satoko; Abou Elela, Sherif; Fourmy, Dominique

2001-01-01

Rnt1p, the yeast orthologue of RNase III, cleaves rRNAs, snRNAs and snoRNAs at a stem capped with conserved AGNN tetraloop. Here we show that 9 bp long stems ending with AGAA or AGUC tetraloops bind to Rnt1p and direct specific but sequence-independent RNA cleavage when provided with stems longer than 13 bp. The solution structures of these two tetraloops reveal a common fold for the terminal loop stabilized by non-canonical A–A or A–C pairs and extensive base stacking. The conserved nucleotides are stacked at the 5′ side of the loop, exposing their Watson–Crick and Hoogsteen faces for recognition by Rnt1p. These results indicate that yeast RNase III recognizes the fold of a conserved single-stranded tetraloop to direct specific dsRNA cleavage. PMID:11743001

A small stem-loop structure of the Ebola virus trailer is essential for replication and interacts with heat-shock protein A8

PubMed Central

Sztuba-Solinska, Joanna; Diaz, Larissa; Kumar, Mia R.; Kolb, Gaëlle; Wiley, Michael R.; Jozwick, Lucas; Kuhn, Jens H.; Palacios, Gustavo; Radoshitzky, Sheli R.; J. Le Grice, Stuart F.; Johnson, Reed F.

2016-01-01

Ebola virus (EBOV) is a single-stranded negative-sense RNA virus belonging to the Filoviridae family. The leader and trailer non-coding regions of the EBOV genome likely regulate its transcription, replication, and progeny genome packaging. We investigated the cis-acting RNA signals involved in RNA–RNA and RNA–protein interactions that regulate replication of eGFP-encoding EBOV minigenomic RNA and identified heat shock cognate protein family A (HSC70) member 8 (HSPA8) as an EBOV trailer-interacting host protein. Mutational analysis of the trailer HSPA8 binding motif revealed that this interaction is essential for EBOV minigenome replication. Selective 2′-hydroxyl acylation analyzed by primer extension analysis of the secondary structure of the EBOV minigenomic RNA indicates formation of a small stem-loop composed of the HSPA8 motif, a 3′ stem-loop (nucleotides 1868–1890) that is similar to a previously identified structure in the replicative intermediate (RI) RNA and a panhandle domain involving a trailer-to-leader interaction. Results of minigenome assays and an EBOV reverse genetic system rescue support a role for both the panhandle domain and HSPA8 motif 1 in virus replication. PMID:27651462

Novel cis-acting element within the capsid-coding region enhances flavivirus viral-RNA replication by regulating genome cyclization.

PubMed

Liu, Zhong-Yu; Li, Xiao-Feng; Jiang, Tao; Deng, Yong-Qiang; Zhao, Hui; Wang, Hong-Jiang; Ye, Qing; Zhu, Shun-Ya; Qiu, Yang; Zhou, Xi; Qin, E-De; Qin, Cheng-Feng

2013-06-01

cis-Acting elements in the viral genome RNA (vRNA) are essential for the translation, replication, and/or encapsidation of RNA viruses. In this study, a novel conserved cis-acting element was identified in the capsid-coding region of mosquito-borne flavivirus. The downstream of 5' cyclization sequence (5'CS) pseudoknot (DCS-PK) element has a three-stem pseudoknot structure, as demonstrated by structure prediction and biochemical analysis. Using dengue virus as a model, we show that DCS-PK enhances vRNA replication and that its function depends on its secondary structure and specific primary sequence. Mutagenesis revealed that the highly conserved stem 1 and loop 2, which are involved in potential loop-helix interactions, are crucial for DCS-PK function. A predicted loop 1-stem 3 base triple interaction is important for the structural stability and function of DCS-PK. Moreover, the function of DCS-PK depends on its position relative to the 5'CS, and the presence of DCS-PK facilitates the formation of 5'-3' RNA complexes. Taken together, our results reveal that the cis-acting element DCS-PK enhances vRNA replication by regulating genome cyclization, and DCS-PK might interplay with other cis-acting elements to form a functional vRNA cyclization domain, thus playing critical roles during the flavivirus life cycle and evolution.

Novel cis-Acting Element within the Capsid-Coding Region Enhances Flavivirus Viral-RNA Replication by Regulating Genome Cyclization

PubMed Central

Liu, Zhong-Yu; Li, Xiao-Feng; Jiang, Tao; Deng, Yong-Qiang; Zhao, Hui; Wang, Hong-Jiang; Ye, Qing; Zhu, Shun-Ya; Qiu, Yang; Zhou, Xi; Qin, E-De

2013-01-01

cis-Acting elements in the viral genome RNA (vRNA) are essential for the translation, replication, and/or encapsidation of RNA viruses. In this study, a novel conserved cis-acting element was identified in the capsid-coding region of mosquito-borne flavivirus. The downstream of 5′ cyclization sequence (5′CS) pseudoknot (DCS-PK) element has a three-stem pseudoknot structure, as demonstrated by structure prediction and biochemical analysis. Using dengue virus as a model, we show that DCS-PK enhances vRNA replication and that its function depends on its secondary structure and specific primary sequence. Mutagenesis revealed that the highly conserved stem 1 and loop 2, which are involved in potential loop-helix interactions, are crucial for DCS-PK function. A predicted loop 1-stem 3 base triple interaction is important for the structural stability and function of DCS-PK. Moreover, the function of DCS-PK depends on its position relative to the 5′CS, and the presence of DCS-PK facilitates the formation of 5′-3′ RNA complexes. Taken together, our results reveal that the cis-acting element DCS-PK enhances vRNA replication by regulating genome cyclization, and DCS-PK might interplay with other cis-acting elements to form a functional vRNA cyclization domain, thus playing critical roles during the flavivirus life cycle and evolution. PMID:23576500

Crystal structures of apo wild-type M. jannaschii tyrosyl-tRNA synthetase (TyrRS) and an engineered TyrRS specific for O-methyl-L-tyrosine

PubMed Central

Zhang, Yan; Wang, Lei; Schultz, Peter G.; Wilson, Ian A.

2005-01-01

The Methanococcus jannaschii tRNATyr/TyrRS pair has been engineered to incorporate unnatural amino acids into proteins in E. coli. To reveal the structural basis for the altered specificity of mutant TyrRS for O-methyl-l-tyrosine (OMeTyr), the crystal structures for the apo wild-type and mutant M. jannaschii TyrRS were determined at 2.66 and 3.0 Å, respectively, for comparison with the published structure of TyrRS complexed with tRNATyr and substrate tyrosine. A large conformational change was found for the anticodon recognition loop 257–263 of wild-type TyrRS upon tRNA binding in order to facilitate recognition of G34 of the anticodon loop through π-stacking and hydrogen bonding interactions. Loop 133–143, which is close to the tRNA acceptor stem-binding site, also appears to be stabilized by interaction with the tRNATyr. Binding of the substrate tyrosine results in subtle and cooperative movements of the side chains within the tyrosine-binding pocket. In the OMeTyr-specific mutant synthetase structure, the signature motif KMSKS loop and acceptor stem-binding loop 133–143 were surprisingly ordered in the absence of bound ATP and tRNA. The active-site mutations result in altered hydrogen bonding and steric interactions which favor binding of OMeTyr over l-tyrosine. The structure of the mutant and wild-type TyrRS now provide a basis for generating new active-site libraries to evolve synthetases specific for other unnatural amino acids. PMID:15840835

NMR structure of the 101-nucleotide core encapsidation signal of the Moloney murine leukemia virus.

PubMed

D'Souza, Victoria; Dey, Anwesha; Habib, Dina; Summers, Michael F

2004-03-19

The full length, positive-strand genome of the Moloney Murine Leukemia Virus contains a "core encapsidation signal" that is essential for efficient genome packaging during virus assembly. We have determined the structure of a 101-nucleotide RNA that contains this signal (called mPsi) using a novel isotope-edited NMR approach. The method is robust and should be generally applicable to larger RNAs. mPsi folds into three stem loops, two of which (SL-C and SL-D) co-stack to form an extended helix. The third stem loop (SL-B) is connected to SL-C by a flexible, four-nucleotide linker. The structure contains five mismatched base-pairs, an unusual C.CG base-triple platform, and a novel "A-minor K-turn," in which unpaired adenosine bases A340 and A341 of a GGAA bulge pack in the minor groove of a proximal stem, and a bulged distal uridine (U319) forms a hydrogen bond with the phosphodiester of A341. Phylogenetic analyses indicate that these essential structural elements are conserved among the murine C-type retroviruses.

Thermal stability, structural features, and B-to-Z transition in DNA tetraloop hairpins as determined by optical spectroscopy in d(CG)(3)T(4)(CG)(3) and d(CG)(3)A(4)(CG)(3) oligodeoxynucleotides.

PubMed

Hernández, Belén; Baumruk, Vladimir; Gouyette, Catherine; Ghomi, Mahmoud

2005-05-01

NMR and CD data have previously shown the formation of the T(4) tetraloop hairpin in aqueous solutions, as well as the possibility of the B-to-Z transition in its stem in high salt concentration conditions. It has been shown that the stem B-to-Z transition in T(4) hairpins leads to S (south)- to N (north)-type conformational changes in the loop sugars, as well as anti to syn orientations in the loop bases. In this article, we have compared by means of UV absorption, CD, Raman, and Fourier transform infrared (FTIR), the thermodynamic and structural properties of the T(4) and A(4) tetraloop hairpins formed in 5'-d(CGCGCG-TTTT-CGCGCG)-3' and 5'-d(CGCGCG-AAAA-CGCGCG)-3', respectively. In presence of 5M NaClO(4), a complete B-to-Z transition of the stems is first proved by CD spectra. UV melting profiles are consistent with a higher thermal stability of the T(4) hairpin compared to the A(4) hairpin. Order-to-disorder transition of both hairpins has also been analyzed by means of Raman spectra recorded as a function of temperature. A clear Z-to-B transition of the stem has been confirmed in the T(4) hairpin, and not in the A(4) hairpin. With a right-handed stem, Raman and FTIR spectra have confirmed the C2'-endo/anti conformation for all the T(4) loop nucleosides. With a left-handed stem, a part of the T(4) loop sugars adopt a N-type (C3'-endo) conformation, and the C3'-endo/syn conformation seems to be the preferred one for the dA residues involved in the A(4) tetraloop.

In vitro synthesis of minus-strand RNA by an isolated cereal yellow dwarf virus RNA-dependent RNA polymerase requires VPg and a stem-loop structure at the 3' end of the virus RNA.

PubMed

Osman, Toba A M; Coutts, Robert H A; Buck, Kenneth W

2006-11-01

Cereal yellow dwarf virus (CYDV) RNA has a 5'-terminal genome-linked protein (VPg). We have expressed the VPg region of the CYDV genome in bacteria and used the purified protein (bVPg) to raise an antiserum which was able to detect free VPg in extracts of CYDV-infected oat plants. A template-dependent RNA-dependent RNA polymerase (RdRp) has been produced from a CYDV membrane-bound RNA polymerase by treatment with BAL 31 nuclease. The RdRp was template specific, being able to utilize templates from CYDV plus- and minus-strand RNAs but not those of three unrelated viruses, Red clover necrotic mosaic virus, Cucumber mosaic virus, and Tobacco mosaic virus. RNA synthesis catalyzed by the RdRp required a 3'-terminal GU sequence and the presence of bVPg. Additionally, synthesis of minus-strand RNA on a plus-strand RNA template required the presence of a putative stem-loop structure near the 3' terminus of CYDV RNA. The base-paired stem, a single-nucleotide (A) bulge in the stem, and the sequence of a tetraloop were all required for the template activity. Evidence was produced showing that minus-strand synthesis in vitro was initiated by priming by bVPg at the 3' end of the template. The data are consistent with a model in which the RdRp binds to the stem-loop structure which positions the active site to recognize the 3'-terminal GU sequence for initiation of RNA synthesis by the addition of an A residue to VPg.

In Vitro Synthesis of Minus-Strand RNA by an Isolated Cereal Yellow Dwarf Virus RNA-Dependent RNA Polymerase Requires VPg and a Stem-Loop Structure at the 3′ End of the Virus RNA▿

PubMed Central

Osman, Toba A. M.; Coutts, Robert H. A.; Buck, Kenneth W.

2006-01-01

Cereal yellow dwarf virus (CYDV) RNA has a 5′-terminal genome-linked protein (VPg). We have expressed the VPg region of the CYDV genome in bacteria and used the purified protein (bVPg) to raise an antiserum which was able to detect free VPg in extracts of CYDV-infected oat plants. A template-dependent RNA-dependent RNA polymerase (RdRp) has been produced from a CYDV membrane-bound RNA polymerase by treatment with BAL 31 nuclease. The RdRp was template specific, being able to utilize templates from CYDV plus- and minus-strand RNAs but not those of three unrelated viruses, Red clover necrotic mosaic virus, Cucumber mosaic virus, and Tobacco mosaic virus. RNA synthesis catalyzed by the RdRp required a 3′-terminal GU sequence and the presence of bVPg. Additionally, synthesis of minus-strand RNA on a plus-strand RNA template required the presence of a putative stem-loop structure near the 3′ terminus of CYDV RNA. The base-paired stem, a single-nucleotide (A) bulge in the stem, and the sequence of a tetraloop were all required for the template activity. Evidence was produced showing that minus-strand synthesis in vitro was initiated by priming by bVPg at the 3′ end of the template. The data are consistent with a model in which the RdRp binds to the stem-loop structure which positions the active site to recognize the 3′-terminal GU sequence for initiation of RNA synthesis by the addition of an A residue to VPg. PMID:16928757

Detection of siRNA Mediated Target mRNA Cleavage Activities in Human Cells by a Novel Stem-Loop Array RT-PCR Analysis

DTIC Science & Technology

2016-09-07

sequences of the target mRNA, and a double stranded stem at the 5′ end that forms a stem -loop to function as a forceps to stabilize the secondary...E-mjournal homepage: www.elsevier.com/locate/bbrepDetection of siRNA-mediated target mRNA cleavage activities in human cells by a novel stem -loop...challenges for the accurate and efficient detection and verification of cleavage sites on target mRNAs. Here we used a sensitive stem -loop array reverse

How U38, 39, and 40 of many tRNAs become the targets for pseudouridylation by TruA.

PubMed

Hur, Sun; Stroud, Robert M

2007-04-27

Translational accuracy and efficiency depend upon modification of uridines in the tRNA anticodon stem loop (ASL) by a highly conserved pseudouridine synthase TruA. TruA specifically modifies uridines at positions 38, 39, and/or 40 of tRNAs with highly divergent sequences and structures through a poorly characterized mechanism that differs from previously studied RNA-modifying enzymes. The molecular basis for the site and substrate "promiscuity" was studied by determining the crystal structures of E. coli TruA in complex with two different leucyl tRNAs in conjunction with functional assays and computer simulation. The structures capture three stages of the TruA*tRNA reaction, revealing the mechanism by which TruA selects the target site. We propose that TruA utilizes the intrinsic flexibility of the ASL for site promiscuity and also to select against intrinsically stable tRNAs to avoid their overstabilization through pseudouridylation, thereby maintaining the balance between the flexibility and stability required for its biological function.

Optimization of a Reusable, DNA Pseudoknot-Based Electrochemical Sensor for Sequence-Specific DNA Detection in Blood Serum

PubMed Central

Cash, Kevin J.; Heeger, Alan J.; Plaxco, Kevin W.; Xiao, Yi

2010-01-01

We describe in detail a new electrochemical DNA (E-DNA) sensing platform based on target-induced conformation changes in an electrode-bound DNA pseudoknot. The pseudoknot, a DNA structure containing two stem-loops in which the first stem’s loop forms part of the second stem, is modified with a methylene blue redox tag at its 3′ terminus and covalently attached to a gold electrode via the 5′ terminus. In the absence of a target, the structure of the pseudoknot probe minimizes collisions between the redox tag and the electrode, thus reducing faradaic current. Target binding disrupts the pseudoknot structure, liberating a flexible, single-stranded element that can strike the electrode and efficiently transfer electrons. In this article we report further characterization and optimization of this new E-DNA architecture. We find that optimal signaling is obtained at an intermediate probe density (~1.8 × 1013 molecules/cm2 apparent density), which presumably represents a balance between steric and electrostatic blocking at high probe densities and increased background currents arising from transfer from the pseudoknot probe at lower densities. We also find that optimal 3′ stem length, which appears to be 7 base pairs, represents a balance between pseudoknot structural stability and target affinity. Finally, a 3′ loop comprised of poly(A) exhibits better mismatch discrimination than the equivalent poly(T) loop, but at the cost of decreased gain. Optimization over this parameter space significantly improves the signaling of the pseudoknot-based E-DNA architecture, leading to the ability to sensitively and specifically detect DNA targets even when challenged in complex, multicomponent samples such as blood serum. PMID:19093760

Base Pairing between U3 Small Nucleolar RNA and the 5′ End of 18S rRNA Is Required for Pre-rRNA Processing

PubMed Central

Sharma, Kishor; Tollervey, David

1999-01-01

The loop of a stem structure close to the 5′ end of the 18S rRNA is complementary to the box A region of the U3 small nucleolar RNA (snoRNA). Substitution of the 18S loop nucleotides inhibited pre-rRNA cleavage at site A1, the 5′ end of the 18S rRNA, and at site A2, located 1.9 kb away in internal transcribed spacer 1. This inhibition was largely suppressed by a compensatory mutation in U3, demonstrating functional base pairing. The U3–pre-rRNA base pairing is incompatible with the structure that forms in the mature 18S rRNA and may prevent premature folding of the pre-rRNA. In the Escherichia coli pre-rRNA the homologous region of the 16S rRNA is also sequestered, in that case by base pairing to the 5′ external transcribed spacer (5′ ETS). Cleavage at site A0 in the yeast 5′ ETS strictly requires base pairing between U3 and a sequence within the 5′ ETS. In contrast, the U3-18S interaction is not required for A0 cleavage. U3 therefore carries out at least two functionally distinct base pair interactions with the pre-rRNA. The nucleotide at the site of A1 cleavage was shown to be specified by two distinct signals; one of these is the stem-loop structure within the 18S rRNA. However, in contrast to the efficiency of cleavage, the position of A1 cleavage is not dependent on the U3-loop interaction. We conclude that the 18S stem-loop structure is recognized at least twice during pre-rRNA processing. PMID:10454548

Computational study of stability of an H-H-type pseudoknot motif.

PubMed

Wang, Jun; Zhao, Yunjie; Wang, Jian; Xiao, Yi

2015-12-01

Motifs in RNA tertiary structures are important to their structural organizations and biological functions. Here we consider an H-H-type pseudoknot (HHpk) motif that consists of two hairpins connected by a junction loop and with kissing interactions between the two hairpin loops. Such a tertiary structural motif is recurrently found in RNA tertiary structures, but is difficult to predict computationally. So it is important to understand the mechanism of its formation and stability. Here we investigate the stability of the HHpk tertiary structure by using an all-atom molecular dynamics simulation. The results indicate that the HHpk tertiary structure is stable. However, it is found that this stability is not due to the helix-helix packing, as is usually expected, but is maintained by the combined action of the kissing hairpin loops and junctions, although the former plays the main role. Stable HHpk motifs may form structural platforms for the molecules to realize their biological functions. These results are useful for understanding the construction principle of RNA tertiary structures and structure prediction.

HCV RNA traffic and association with NS5A in living cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fiches, Guillaume N.; Eyre, Nicholas S.; Aloia, Amanda L.

The spatiotemporal dynamics of Hepatitis C Virus (HCV) RNA localisation are poorly understood. To address this we engineered HCV genomes harbouring MS2 bacteriophage RNA stem-loops within the 3′-untranslated region to allow tracking of HCV RNA via specific interaction with a MS2-Coat-mCherry fusion protein. Despite the impact of these insertions on viral fitness, live imaging revealed that replication of tagged-HCV genomes induced specific redistribution of the mCherry-tagged-MS2-Coat protein to motile and static foci. Further analysis showed that HCV RNA was associated with NS5A in both static and motile structures while a subset of motile NS5A structures was devoid of HCV RNA.more » Further investigation of viral RNA traffic with respect to lipid droplets (LDs) revealed HCV RNA-positive structures in close association with LDs. These studies provide new insights into the dynamics of HCV RNA traffic with NS5A and LDs and provide a platform for future investigations of HCV replication and assembly. - Highlights: • HCV can tolerate can bacteriophage MS2 stem-loop insertions within the 3′ UTR. • MS2 stem-loop containing HCV genomes allow for real-time imaging of HCV RNA. • HCV RNA is both static and motile and associates with NS5A and lipid droplets.« less

A small stem-loop structure of the Ebola virus trailer is essential for replication and interacts with heat-shock protein A8.

PubMed

Sztuba-Solinska, Joanna; Diaz, Larissa; Kumar, Mia R; Kolb, Gaëlle; Wiley, Michael R; Jozwick, Lucas; Kuhn, Jens H; Palacios, Gustavo; Radoshitzky, Sheli R; J Le Grice, Stuart F; Johnson, Reed F

2016-11-16

Ebola virus (EBOV) is a single-stranded negative-sense RNA virus belonging to the Filoviridae family. The leader and trailer non-coding regions of the EBOV genome likely regulate its transcription, replication, and progeny genome packaging. We investigated the cis-acting RNA signals involved in RNA-RNA and RNA-protein interactions that regulate replication of eGFP-encoding EBOV minigenomic RNA and identified heat shock cognate protein family A (HSC70) member 8 (HSPA8) as an EBOV trailer-interacting host protein. Mutational analysis of the trailer HSPA8 binding motif revealed that this interaction is essential for EBOV minigenome replication. Selective 2'-hydroxyl acylation analyzed by primer extension analysis of the secondary structure of the EBOV minigenomic RNA indicates formation of a small stem-loop composed of the HSPA8 motif, a 3' stem-loop (nucleotides 1868-1890) that is similar to a previously identified structure in the replicative intermediate (RI) RNA and a panhandle domain involving a trailer-to-leader interaction. Results of minigenome assays and an EBOV reverse genetic system rescue support a role for both the panhandle domain and HSPA8 motif 1 in virus replication. Published by Oxford University Press on behalf of Nucleic Acids Research 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.

Structure of an anti-HIV-1 hammerhead ribozyme complex with a 17-mer DNA substrate analog of HIV-1 gag RNA and a mechanism for the cleavage reaction: 750 MHz NMR and computer experiments

NASA Technical Reports Server (NTRS)

Ojha, R. P.; Dhingra, M. M.; Sarma, M. H.; Myer, Y. P.; Setlik, R. F.; Shibata, M.; Kazim, A. L.; Ornstein, R. L.; Rein, R.; Turner, C. J.;

Mutational analysis of the gag-pol junction of Moloney murine leukemia virus: requirements for expression of the gag-pol fusion protein.

PubMed Central

Felsenstein, K M; Goff, S P

1992-01-01

The gag-pol polyprotein of the murine and feline leukemia viruses is expressed by translational readthrough of a UAG terminator codon at the 3' end of the gag gene. To explore the cis-acting sequence requirements for the readthrough event in vivo, we generated a library of mutants of the Moloney murine leukemia virus with point mutations near the terminator codon and tested the mutant viral DNAs for the ability to direct synthesis of the gag-pol fusion protein and formation of infectious virus. The analysis showed that sequences 3' to the terminator are necessary and sufficient for the process. The results do not support a role for one proposed stem-loop structure that includes the terminator but are consistent with the involvement of another stem-loop 3' to the terminator. One mutant, containing two compensatory changes in this stem structure, was temperature sensitive for replication and for formation of the gag-pol protein. The results suggest that RNA sequence and structure are critical determinants of translational readthrough in vivo. Images PMID:1404606

Mutagenesis Studies of the H5 Influenza Hemagglutinin Stem Loop Region*

PubMed Central

Antanasijevic, Aleksandar; Basu, Arnab; Bowlin, Terry L.; Mishra, Rama K.; Rong, Lijun; Caffrey, Michael

2014-01-01

Influenza outbreaks, particularly the pandemic 1918 H1 and avian H5 strains, are of high concern to public health. The hemagglutinin envelope protein of influenza plays a critical role in viral entry and thus is an attractive target for inhibition of virus entry. The highly conserved stem loop region of hemagglutinin has been shown to undergo critically important conformational changes during the entry process and, moreover, to be a site for inhibition of virus entry by antibodies, small proteins, and small drug-like molecules. In this work we probe the structure-function properties of the H5 hemagglutinin stem loop region by site-directed mutagenesis. We find that most mutations do not disrupt expression, proteolytic processing, incorporation into virus, or receptor binding; however, many of the mutations disrupt the entry process. We further assess the effects of mutations on inhibition of entry by a neutralizing monoclonal antibody (C179) and find examples of increased and decreased sensitivity to the antibody, consistent with the antibody binding site observed by x-ray crystallography. In addition, we tested the sensitivity of the mutants to MBX2329, a small molecule inhibitor of influenza entry. Interestingly, the mutants exhibit increased and decreased sensitivities to MBX2329, which gives further insight into the binding site of the compound on HA and potential mechanisms of escape. Finally, we have modeled the binding site of MBX2329 using molecular dynamics and find that the resulting structure is in good agreement with the mutagenesis results. Together these studies underscore the importance of the stem loop region to HA function and suggest potential sites for therapeutic intervention of influenza entry. PMID:24947513

Mutagenesis studies of the H5 influenza hemagglutinin stem loop region.

PubMed

Antanasijevic, Aleksandar; Basu, Arnab; Bowlin, Terry L; Mishra, Rama K; Rong, Lijun; Caffrey, Michael

2014-08-08

Influenza outbreaks, particularly the pandemic 1918 H1 and avian H5 strains, are of high concern to public health. The hemagglutinin envelope protein of influenza plays a critical role in viral entry and thus is an attractive target for inhibition of virus entry. The highly conserved stem loop region of hemagglutinin has been shown to undergo critically important conformational changes during the entry process and, moreover, to be a site for inhibition of virus entry by antibodies, small proteins, and small drug-like molecules. In this work we probe the structure-function properties of the H5 hemagglutinin stem loop region by site-directed mutagenesis. We find that most mutations do not disrupt expression, proteolytic processing, incorporation into virus, or receptor binding; however, many of the mutations disrupt the entry process. We further assess the effects of mutations on inhibition of entry by a neutralizing monoclonal antibody (C179) and find examples of increased and decreased sensitivity to the antibody, consistent with the antibody binding site observed by x-ray crystallography. In addition, we tested the sensitivity of the mutants to MBX2329, a small molecule inhibitor of influenza entry. Interestingly, the mutants exhibit increased and decreased sensitivities to MBX2329, which gives further insight into the binding site of the compound on HA and potential mechanisms of escape. Finally, we have modeled the binding site of MBX2329 using molecular dynamics and find that the resulting structure is in good agreement with the mutagenesis results. Together these studies underscore the importance of the stem loop region to HA function and suggest potential sites for therapeutic intervention of influenza entry. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Structural Requirement in Clostridium perfringens Collagenase mRNA 5′ Leader Sequence for Translational Induction through Small RNA-mRNA Base Pairing

PubMed Central

Nomura, Nobuhiko; Nakamura, Kouji

2013-01-01

The Gram-positive anaerobic bacterium Clostridium perfringens is pathogenic to humans and animals, and the production of its toxins is strictly regulated during the exponential phase. We recently found that the 5′ leader sequence of the colA transcript encoding collagenase, which is a major toxin of this organism, is processed and stabilized in the presence of the small RNA VR-RNA. The primary colA 5′-untranslated region (5′UTR) forms a long stem-loop structure containing an internal bulge and masks its own ribosomal binding site. Here we found that VR-RNA directly regulates colA expression through base pairing with colA mRNA in vivo. However, when the internal bulge structure was closed by point mutations in colA mRNA, translation ceased despite the presence of VR-RNA. In addition, a mutation disrupting the colA stem-loop structure induced mRNA processing and ColA-FLAG translational activation in the absence of VR-RNA, indicating that the stem-loop and internal bulge structure of the colA 5′ leader sequence is important for regulation by VR-RNA. On the other hand, processing was required for maximal ColA expression but was not essential for VR-RNA-dependent colA regulation. Finally, colA processing and translational activation were induced at a high temperature without VR-RNA. These results suggest that inhibition of the colA 5′ leader structure through base pairing is the primary role of VR-RNA in colA regulation and that the colA 5′ leader structure is a possible thermosensor. PMID:23585542

Domain- and nucleotide-specific Rev response element regulation of feline immunodeficiency virus production

PubMed Central

Na, Hong; Huisman, Willem; Ellestad, Kristofor K.; Phillips, Tom R.; Power, Christopher

2010-01-01

Computational analysis of feline immunodeficiency virus (FIV) RNA sequences indicated that common FIV strains contain a rev response element (RRE) defined by a long unbranched hairpin with 6 stem-loop sub-domains, termed stem-loop A (SLA). To examine the role of the RNA secondary structure of the RRE, mutational analyses were performed in both an infectious FIV molecular clone and a FIV CAT-RRE reporter system. These studies disclosed that the stems within SLA (SA1, 2, 3, 4, and 5) of the RRE were critical but SA6 was not essential for FIV replication and CAT expression. These studies also revealed that the secondary structure rather than an antisense protein (ASP) mediates virus expression and replication in vitro. In addition, a single synonymous mutation within the FIV-RRE, SA3/45, reduced viral reverse transcriptase activity and p24 expression after transfection but in addition also showed a marked reduction in viral expression and production following infection. PMID:20570310

Temperature sensing in Yersinia pestis: translation of the LcrF activator protein is thermally regulated.

PubMed Central

Hoe, N P; Goguen, J D

1993-01-01

The lcrF gene of Yersinia pestis encodes a transcription activator responsible for inducing expression of several virulence-related proteins in response to temperature. The mechanism of this thermoregulation was investigated. An lcrF clone was found to produce much lower levels of LcrF protein at 26 than at 37 degrees C in Y. pestis, although it was transcribed at similar levels at both temperatures. High-level T7 polymerase-directed transcription of the lcrF gene in Escherichia coli also resulted in temperature-dependent production of the LcrF protein. Pulse-chase experiments showed that the LcrF protein was stable at 26 and 37 degrees C, suggesting that translation rate or message degradation is thermally controlled. The lcrF mRNA appears to be highly unstable and could not be reliably detected in Y. pestis. Insertion of the lcrF gene into plasmid pET4a, which produces high levels of plasmid-length RNA, aided detection of lcrF-specific message in E. coli. Comparison of the amount of LcrF protein produced per unit of message at 26 and 37 degrees C indicated that the efficiency of translation of lcrF message increased with temperature. mRNA secondary structure predictions suggest that the lcrF Shine-Dalgarno sequence is sequestered in a stem-loop. A model in which decreased stability of this stem-loop with increasing temperature leads to increased efficiency of translation initiation of lcrF message is presented. Images PMID:7504666

Role of the Pepino mosaic virus 3'-untranslated region elements in negative-strand RNA synthesis in vitro.

PubMed

Osman, Toba A M; Olsthoorn, René C L; Livieratos, Ioannis C

2014-09-22

Pepino mosaic virus (PepMV) is a mechanically-transmitted positive-strand RNA potexvirus, with a 6410 nt long single-stranded (ss) RNA genome flanked by a 5'-methylguanosine cap and a 3' poly-A tail. Computer-assisted folding of the 64 nt long PepMV 3'-untranslated region (UTR) resulted in the prediction of three stem-loop structures (hp1, hp2, and hp3 in the 3'-5' direction). The importance of these structures and/or sequences for promotion of negative-strand RNA synthesis and binding to the RNA dependent RNA polymerase (RdRp) was tested in vitro using a specific RdRp assay. Hp1, which is highly variable among different PepMV isolates, appeared dispensable for negative-strand synthesis. Hp2, which is characterized by a large U-rich loop, tolerated base-pair changes in its stem as long as they maintained the stem integrity but was very sensitive to changes in the U-rich loop. Hp3, which harbours the conserved potexvirus ACUUAA hexamer motif, was essential for template activity. Template-RNA polymerase binding competition experiments showed that the ACUUAA sequence represents a high-affinity RdRp binding element. Copyright © 2014 Elsevier B.V. All rights reserved.

Functional anticodon architecture of human tRNALys3 includes disruption of intraloop hydrogen bonding by the naturally occurring amino acid modification, t6A.

PubMed

Stuart, J W; Gdaniec, Z; Guenther, R; Marszalek, M; Sochacka, E; Malkiewicz, A; Agris, P F

2000-11-07

The structure of the human tRNA(Lys3) anticodon stem and loop domain (ASL(Lys3)) provides evidence of the physicochemical contributions of N6-threonylcarbamoyladenosine (t(6)A(37)) to tRNA(Lys3) functions. The t(6)A(37)-modified anticodon stem and loop domain of tRNA(Lys3)(UUU) (ASL(Lys3)(UUU)- t(6)A(37)) with a UUU anticodon is bound by the appropriately programmed ribosomes, but the unmodified ASL(Lys3)(UUU) is not [Yarian, C., Marszalek, M., Sochacka, E., Malkiewicz, A., Guenther, R., Miskiewicz, A., and Agris, P. F., Biochemistry 39, 13390-13395]. The structure, determined to an average rmsd of 1.57 +/- 0.33 A (relative to the mean structure) by NMR spectroscopy and restrained molecular dynamics, is the first reported of an RNA in which a naturally occurring hypermodified nucleoside was introduced by automated chemical synthesis. The ASL(Lys3)(UUU)-t(6)A(37) loop is significantly different than that of the unmodified ASL(Lys3)(UUU), although the five canonical base pairs of both ASL(Lys3)(UUU) stems are in the standard A-form of helical RNA. t(6)A(37), 3'-adjacent to the anticodon, adopts the form of a tricyclic nucleoside with an intraresidue H-bond and enhances base stacking on the 3'-side of the anticodon loop. Critically important to ribosome binding, incorporation of the modification negates formation of an intraloop U(33).A(37) base pair that is observed in the unmodified ASL(Lys3)(UUU). The anticodon wobble position U(34) nucleobase in ASL(Lys3)(UUU)-t(6)A(37) is significantly displaced from its position in the unmodified ASL and directed away from the codon-binding face of the loop resulting in only two anticodon bases for codon binding. This conformation is one explanation for ASL(Lys3)(UUU) tendency to prematurely terminate translation and -1 frame shift. At the pH 5.6 conditions of our structure determination, A(38) is protonated and positively charged in ASL(Lys3)(UUU)-t(6)A(37) and the unmodified ASL(Lys3)(UUU). The ionized carboxylic acid moiety of t(6)A(37) possibly neutralizes the positive charge of A(+)(38). The protonated A(+)(38) can base pair with C(32), but t(6)A(37) may weaken the interaction through steric interference. From these results, we conclude that ribosome binding cannot simply be an induced fit of the anticodon stem and loop, otherwise the unmodified ASL(Lys3)(UUU) would bind as well as ASL(Lys3)(UUU)-t(6)A(37). t(6)A(37) and other position 37 modifications produce the open, structured loop required for ribosomal binding.

Role for cis-acting RNA sequences in the temperature-dependent expression of the multiadhesive lig proteins in Leptospira interrogans.

PubMed

Matsunaga, James; Schlax, Paula J; Haake, David A

2013-11-01

The spirochete Leptospira interrogans causes a systemic infection that provokes a febrile illness. The putative lipoproteins LigA and LigB promote adhesion of Leptospira to host proteins, interfere with coagulation, and capture complement regulators. In this study, we demonstrate that the expression level of the LigA and LigB proteins was substantially higher when L. interrogans proliferated at 37°C instead of the standard culture temperature of 30°C. The RNA comprising the 175-nucleotide 5' untranslated region (UTR) and first six lig codons, whose sequence is identical in ligA and ligB, is predicted to fold into two distinct stem-loop structures separated by a single-stranded region. The ribosome-binding site is partially sequestered in double-stranded RNA within the second structure. Toeprint analysis revealed that in vitro formation of a 30S-tRNA(fMet)-mRNA ternary complex was inhibited unless a 5' deletion mutation disrupted the second stem-loop structure. To determine whether the lig sequence could mediate temperature-regulated gene expression in vivo, the 5' UTR and the first six codons were inserted between the Escherichia coli l-arabinose promoter and bgaB (β-galactosidase from Bacillus stearothermophilus) to create a translational fusion. The lig fragment successfully conferred thermoregulation upon the β-galactosidase reporter in E. coli. The second stem-loop structure was sufficient to confer thermoregulation on the reporter, while sequences further upstream in the 5' UTR slightly diminished expression at each temperature tested. Finally, the expression level of β-galactosidase was significantly higher when point mutations predicted to disrupt base pairs in the second structure were introduced into the stem. Compensatory mutations that maintained base pairing of the stem without restoring the wild-type sequence reinstated the inhibitory effect of the 5' UTR on expression. These results indicate that ligA and ligB expression is limited by double-stranded RNA that occludes the ribosome-binding site. At elevated temperatures, the ribosome-binding site is exposed to promote translation initiation.

Unified dead-time compensation structure for SISO processes with multiple dead times.

PubMed

Normey-Rico, Julio E; Flesch, Rodolfo C C; Santos, Tito L M

2014-11-01

This paper proposes a dead-time compensation structure for processes with multiple dead times. The controller is based on the filtered Smith predictor (FSP) dead-time compensator structure and it is able to control stable, integrating, and unstable processes with multiple input/output dead times. An equivalent model of the process is first computed in order to define the predictor structure. Using this equivalent model, the primary controller and the predictor filter are tuned to obtain an internally stable closed-loop system which also attempts some closed-loop specifications in terms of set-point tracking, disturbance rejection, and robustness. Some simulation case studies are used to illustrate the good properties of the proposed approach. Copyright © 2014 ISA. Published by Elsevier Ltd. All rights reserved.

Remarkable sequence conservation of the last intron in the PKD1 gene.

PubMed

Rodova, Marianna; Islam, M Rafiq; Peterson, Kenneth R; Calvet, James P

2003-10-01

The last intron of the PKD1 gene (intron 45) was found to have exceptionally high sequence conservation across four mammalian species: human, mouse, rat, and dog. This conservation did not extend to the comparable intron in pufferfish. Pairwise comparisons for intron 45 showed 91% identity (human vs. dog) to 100% identity (mouse vs. rat) for an average for all four species of 94% identity. In contrast, introns 43 and 44 of the PKD1 gene had average pairwise identities of 57% and 54%, and exons 43, 44, and 45 and the coding region of exon 46 had average pairwise identities of 80%, 84%, 82%, and 80%. Intron 45 is 90 to 95 bp in length, with the major region of sequence divergence being in a central 4-bp to 9-bp variable region. RNA secondary structure analysis of intron 45 predicts a branching stem-loop structure in which the central variable region lies in one loop and the putative branch point sequence lies in another loop, suggesting that the intron adopts a specific stem-loop structure that may be important for its removal. Although intron 45 appears to conform to the class of small, G-triplet-containing introns that are spliced by a mechanism utilizing intron definition, its high sequence conservation may be a reflection of constraints imposed by a unique mechanism that coordinates splicing of this last PKD1 intron with polyadenylation.

Conserved intergenic sequences revealed by CTAG-profiling in Salmonella: thermodynamic modeling for function prediction

NASA Astrophysics Data System (ADS)

Tang, Le; Zhu, Songling; Mastriani, Emilio; Fang, Xin; Zhou, Yu-Jie; Li, Yong-Guo; Johnston, Randal N.; Guo, Zheng; Liu, Gui-Rong; Liu, Shu-Lin

2017-03-01

Highly conserved short sequences help identify functional genomic regions and facilitate genomic annotation. We used Salmonella as the model to search the genome for evolutionarily conserved regions and focused on the tetranucleotide sequence CTAG for its potentially important functions. In Salmonella, CTAG is highly conserved across the lineages and large numbers of CTAG-containing short sequences fall in intergenic regions, strongly indicating their biological importance. Computer modeling demonstrated stable stem-loop structures in some of the CTAG-containing intergenic regions, and substitution of a nucleotide of the CTAG sequence would radically rearrange the free energy and disrupt the structure. The postulated degeneration of CTAG takes distinct patterns among Salmonella lineages and provides novel information about genomic divergence and evolution of these bacterial pathogens. Comparison of the vertically and horizontally transmitted genomic segments showed different CTAG distribution landscapes, with the genome amelioration process to remove CTAG taking place inward from both terminals of the horizontally acquired segment.

CTCF and cohesin regulate chromatin loop stability with distinct dynamics

PubMed Central

Hansen, Anders S; Pustova, Iryna; Cattoglio, Claudia; Tjian, Robert; Darzacq, Xavier

2017-01-01

Folding of mammalian genomes into spatial domains is critical for gene regulation. The insulator protein CTCF and cohesin control domain location by folding domains into loop structures, which are widely thought to be stable. Combining genomic and biochemical approaches we show that CTCF and cohesin co-occupy the same sites and physically interact as a biochemically stable complex. However, using single-molecule imaging we find that CTCF binds chromatin much more dynamically than cohesin (~1–2 min vs. ~22 min residence time). Moreover, after unbinding, CTCF quickly rebinds another cognate site unlike cohesin for which the search process is long (~1 min vs. ~33 min). Thus, CTCF and cohesin form a rapidly exchanging 'dynamic complex' rather than a typical stable complex. Since CTCF and cohesin are required for loop domain formation, our results suggest that chromatin loops are dynamic and frequently break and reform throughout the cell cycle. DOI: http://dx.doi.org/10.7554/eLife.25776.001 PMID:28467304

The complete mitochondrial genome and its remarkable secondary structure for a stonefly Acroneuria hainana Wu (Insecta: Plecoptera, Perlidae).

PubMed

Huang, Mingchao; Wang, Yuyu; Liu, Xingyue; Li, Weihai; Kang, Zehui; Wang, Kai; Li, Xuankun; Yang, Ding

2015-02-15

The Plecoptera (stoneflies) is a hemimetabolous order of insects, whose larvae are usually used as indicators for fresh water biomonitoring. Herein, we describe the complete mitochondrial (mt) genome of a stonefly species, namely Acroneuria hainana Wu belonging to the family Perlidae. This mt genome contains 13 PCGs, 22 tRNA-coding genes and 2 rRNA-coding genes that are conserved in most insect mt genomes, and it also has the identical gene order with the insect ancestral gene order. However, there are three special initiation codons of ND1, ND5 and COI in PCGs: TTG, GTG and CGA, coding for L, V and R, respectively. Additionally, the 899-bp control region, with 73.30% A+T content, has two long repeated sequences which are found at the 3'-end closing to the tRNA(Ile) gene. Both of them can be folded into a stem-loop structure, whose adjacent upstream and downstream sequences can be also folded into stem-loop structures. It is presumed that the four special structures in series could be associated with the D-loop replication. It might be able to adjust the replication speed of two replicate directions. Copyright © 2014 Elsevier B.V. All rights reserved.

Fluorescence Competition Assay Measurements of Free Energy Changes for RNA Pseudoknots†

PubMed Central

2009-01-01

RNA pseudoknots have important functions, and thermodynamic stability is a key to predicting pseudoknots in RNA sequences and to understanding their functions. Traditional methods, such as UV melting and differential scanning calorimetry, for measuring RNA thermodynamics are restricted to temperature ranges around the melting temperature for a pseudoknot. Here, we report RNA pseudoknot free energy changes at 37 °C measured by fluorescence competition assays. Sequence-dependent studies for the loop 1−stem 2 region reveal (1) the individual nearest-neighbor hydrogen bonding (INN-HB) model provides a reasonable estimate for the free energy change when a Watson−Crick base pair in stem 2 is changed, (2) the loop entropy can be estimated by a statistical polymer model, although some penalty for certain loop sequences is necessary, and (3) tertiary interactions can significantly stabilize pseudoknots and extending the length of stem 2 may alter tertiary interactions such that the INN-HB model does not predict the net effect of adding a base pair. The results can inform writing of algorithms for predicting and/or designing RNA secondary structures. PMID:19921809

Coronal loops and active region structure

NASA Technical Reports Server (NTRS)

Webb, D. F.; Zirin, H.

1981-01-01

Synoptic H-alpha Ca K, magnetograph and Skylab soft X-ray and EUV data were compared for the purpose of identifying the basic coronal magnetic structure of loops in a 'typical' active region and studying its evolution. A complex of activity in July 1973, especially McMath 12417, was emphasized. The principal results are: (1) most of the brightest loops connected the bright f plage to either the sunspot penumbra or to p satellite spots; no non-flaring X-ray loops end in umbrae; (2) short, bright loops had one or both ends in regions of emergent flux, strong field or high field gradients; (3) stable, strongly sheared loop arcades formed over filaments; (4) EFRs were always associated with compact X-ray arcades; and (5) loops connecting to other active regions had their bases in outlying plage of weak field strength in McM 417 where H-alpha fibrils marked the direction of the loops

Engineering the thermostability of β-glucuronidase from Penicillium purpurogenum Li-3 by loop transplant.

PubMed

Feng, Xudong; Tang, Heng; Han, Beijia; Zhang, Liang; Lv, Bo; Li, Chun

2016-12-01

In this study, we proposed a loop transplant strategy to improve the thermostability of Penicillium purpurogenum Li-3 β-glucuronidase expressed in Escherichia coli (abbreviated to PGUS-E). Firstly, three unstable surface loops of PGUS-E to be replaced were identified with regards to B-factor values and in-depth structure analysis: loops 205-211, 258-263, and 25-31. Then, based on B-factor analysis, eight stable loops for substitution were selected from two typical thermophilic glycosidases which had low homology with PGUS-E (less than 25 %). By analyzing the common features of these stable loops, it was found that they shared a common residue skeleton DXXTX(X)R, based on this, three chimera loops were also manually designed: RSQTSND, RSSTQRD, and DDQTSR. All these loops were introduced to replace the unstable loops of PGUS-E by homology structure modeling, and only mutants with increased hydrogen bonds number and good compatibility with the local mutated region were further subjected to experimental verification. By using this strategy, 10 mutants were experimentally generated, among which three mutants, M1, M3, and M8, were obtained which showed 11.8, 3.3, and 9.4 times higher half-life at 70 °C than that of wild-type (8.5 min). Finally, the MD simulation indicated that the increased hydrogen bonds, decreased flexibility of N-terminal, and increased π-π stacking interaction were responsible for the improved thermostability.

Sjögren Syndrome Antigen B (SSB)/La Promotes Global MicroRNA Expression by Binding MicroRNA Precursors through Stem-Loop Recognition*

PubMed Central

Liang, Chunyang; Xiong, Ke; Szulwach, Keith E.; Zhang, Yi; Wang, Zhaohui; Peng, Junmin; Fu, Mingui; Jin, Peng; Suzuki, Hiroshi I.; Liu, Qinghua

2013-01-01

MicroRNAs (miRNA) control numerous physiological and pathological processes. Typically, the primary miRNA (pri-miRNA) transcripts are processed by nuclear Drosha complex into ∼70-nucleotide stem-loop precursor miRNAs (pre-miRNA), which are further cleaved by cytoplasmic Dicer complex into ∼21-nucleotide mature miRNAs. However, it is unclear how nascent pre-miRNAs are protected from ribonucleases, such as MCPIP1, that degrade pre-miRNAs to abort miRNA production. Here, we identify Sjögren syndrome antigen B (SSB)/La as a pre-miRNA-binding protein that regulates miRNA processing in vitro. All three RNA-binding motifs (LAM, RRM1, and RRM2) of La/SSB are required for efficient pre-miRNA binding. Intriguingly, La/SSB recognizes the characteristic stem-loop structure of pre-miRNAs, of which the majority lack a 3′ UUU terminus. Moreover, La/SSB associates with endogenous pri-/pre-miRNAs and promotes miRNA biogenesis by stabilizing pre-miRNAs from nuclease (e.g. MCPIP1)-mediated decay in mammalian cells. Accordingly, we observed positive correlations between the expression status of La/SSB and Dicer in human cancer transcriptome and prognosis. These studies identify an important function of La/SSB as a global regulator of miRNA expression, and implicate stem-loop recognition as a major mechanism that mediates association between La/SSB and diverse RNA molecules. PMID:23129761

Sjogren syndrome antigen B (SSB)/La promotes global microRNA expression by binding microRNA precursors through stem-loop recognition.

PubMed

Liang, Chunyang; Xiong, Ke; Szulwach, Keith E; Zhang, Yi; Wang, Zhaohui; Peng, Junmin; Fu, Mingui; Jin, Peng; Suzuki, Hiroshi I; Liu, Qinghua

2013-01-04

MicroRNAs (miRNA) control numerous physiological and pathological processes. Typically, the primary miRNA (pri-miRNA) transcripts are processed by nuclear Drosha complex into ~70-nucleotide stem-loop precursor miRNAs (pre-miRNA), which are further cleaved by cytoplasmic Dicer complex into ~21-nucleotide mature miRNAs. However, it is unclear how nascent pre-miRNAs are protected from ribonucleases, such as MCPIP1, that degrade pre-miRNAs to abort miRNA production. Here, we identify Sjögren syndrome antigen B (SSB)/La as a pre-miRNA-binding protein that regulates miRNA processing in vitro. All three RNA-binding motifs (LAM, RRM1, and RRM2) of La/SSB are required for efficient pre-miRNA binding. Intriguingly, La/SSB recognizes the characteristic stem-loop structure of pre-miRNAs, of which the majority lack a 3' UUU terminus. Moreover, La/SSB associates with endogenous pri-/pre-miRNAs and promotes miRNA biogenesis by stabilizing pre-miRNAs from nuclease (e.g. MCPIP1)-mediated decay in mammalian cells. Accordingly, we observed positive correlations between the expression status of La/SSB and Dicer in human cancer transcriptome and prognosis. These studies identify an important function of La/SSB as a global regulator of miRNA expression, and implicate stem-loop recognition as a major mechanism that mediates association between La/SSB and diverse RNA molecules.

Structural specificity of Rn nuclease I as probed on yeast tRNA(Phe) and tRNA(Asp).

PubMed Central

Przykorska, A; el Adlouni, C; Keith, G; Szarkowski, J W; Dirheimer, G

1992-01-01

A single-strand-specific nuclease from rye germ (Rn nuclease I) was characterized as a tool for secondary and tertiary structure investigation of RNAs. To test the procedure, yeast tRNA(Phe) and tRNA(Asp) for which the tertiary structures are known, as well as the 3'-half of tRNA(Asp) were used as substrates. In tRNA(Phe) the nuclease introduced main primary cuts at positions U33 and A35 of the anticodon loop and G18 and G19 of the D loop. No primary cuts were observed within the double stranded stems. In tRNA(Asp) the main cuts occurred at positions U33, G34, U35, C36 of the anticodon loop and G18 and C20:1 positions in the D loop. No cuts were observed in the T loop in intact tRNA(Asp) but strong primary cleavages occurred at positions psi 55, C56, A57 within that loop in the absence of the tertiary interactions between T and D loops (use of 3'-half tRNA(Asp)). These results show that Rn nuclease I is specific for exposed single-stranded regions. Images PMID:1542562

Hairpin stabilized fluorescent silver nanoclusters for quantitative detection of NAD+ and monitoring NAD+/NADH based enzymatic reactions.

PubMed

Jain, Priyamvada; Chakma, Babina; Patra, Sanjukta; Goswami, Pranab

2017-03-01

A set of 90 mer long ssDNA candidates, with different degrees of cytosine (C-levels) (% and clusters) was analyzed for their function as suitable Ag-nanocluster (AgNC) nucleation scaffolds. The sequence (P4) with highest C-level (42.2%) emerged as the only candidate supporting the nucleation process as evident from its intense fluorescence peak at λ 660 nm . Shorter DNA subsets derived from P4 with only stable hairpin structures could support the AgNC formation. The secondary hairpin structures were confirmed by PAGE, and CD studies. The number of base pairs in the stem region also contributes to the stability of the hairpins. A shorter 29 mer sequence (Sub 3) (ΔG = -1.3 kcal/mol) with 3-bp in the stem of a 7-mer loop conferred highly stable AgNC. NAD + strongly quenched the fluorescence of Sub 3-AgNC in a concentration dependent manner. Time resolved photoluminescence studies revealed the quenching involves a combined static and dynamic interaction where the binding constant and number of binding sites for NAD + were 0.201 L mol -1 and 3.6, respectively. A dynamic NAD + detection range of 50-500 μM with a limit of detection of 22.3 μM was discerned. The NAD + mediated quenching of AgNC was not interfered by NADH, NADP + , monovalent and divalent ions, or serum samples. The method was also used to follow alcohol dehydrogenase and lactate dehydrogenase catalyzed physiological reactions in a turn-on and turn-off assay, respectively. The proposed method with ssDNA-AgNC could therefore be extended to monitor other NAD + /NADH based enzyme catalyzed reactions in a turn-on/turn-off approach. Copyright © 2016 Elsevier B.V. All rights reserved.

Prokaryotic and eukaryotic features observed on the secondary structures of Giardia SSU rRNAs and its phylogenetic implications.

PubMed

Hwang, Ui Wook

2007-04-01

Phylogenetic position of a diplomonad protist Giardia, a principle cause of diarrhea, among eukaryotes has been vigorously debated so far. Through the comparisons of primary and secondary structures of SSU rRNAs of G. intestinalis, G. microti, G. ardeae, and G. muris, I found two major indel regions (a 6-nt indel and a 22-26-nt indel), which correspond to the helix 10 of the V2 region and helices E23-8 to E23-9 of the V4 region, respectively. As generally shown in eukaryotes, G. intestinalis and G. microti have commonly a relatively longer helix 10 (a 7-bp stem and a 4-nt loop), and also the eukaryote-specific helices E23-6 to E23-9. On the other hand, G. muris and G. ardeae have a shorter helix 10: a 2-bp stem and a 6-nt loop in G. ardeae and a 3-bp stem and a 6-nt loop in G. muris. In the V4, they have a single long helix (like the P23-1 helix in prokaryotes) instead of the helices E23-6 to E23-9. Among the four Giardia species, co-appearance of prokaryote- and eukaryote-typical features might be significant evidence to suggest that Giardia (Archezoa) is a living fossil showing an "intermediate stage" during the evolution from prokaryotes to eukaryotes.

Hinge-Like Motions in RNA Kink-Turns: The Role of the Second A-Minor Motif and Nominally Unpaired Bases

PubMed Central

Rázga, Filip; Koča, Jaroslav; Šponer, Jiří; Leontis, Neocles B.

2005-01-01

Kink-turn (K-turn) motifs are asymmetric internal loops found at conserved positions in diverse RNAs, with sharp bends in phosphodiester backbones producing V-shaped structures. Explicit-solvent molecular dynamics simulations were carried out for three K-turns from 23S rRNA, i.e., Kt-38 located at the base of the A-site finger, Kt-42 located at the base of the L7/L12 stalk, and Kt-58 located in domain III, and for the K-turn of human U4 snRNA. The simulations reveal hinge-like K-turn motions on the nanosecond timescale. The first conserved A-minor interaction between the K-turn stems is entirely stable in all simulations. The angle between the helical arms of Kt-38 and Kt-42 is regulated by local variations of the second A-minor (type I) interaction between the stems. Its variability ranges from closed geometries to open ones stabilized by insertion of long-residency waters between adenine and cytosine. The simulated A-minor geometries fully agree with x-ray data. Kt-58 and Kt-U4 exhibit similar elbow-like motions caused by conformational change of the adenosine from the nominally unpaired region. Despite the observed substantial dynamics of K-turns, key tertiary interactions are stable and no sign of unfolding is seen. We suggest that some K-turns are flexible elements mediating large-scale ribosomal motions during the protein synthesis cycle. PMID:15722438

Solution structure of an ATP-binding RNA aptamer reveals a novel fold.

PubMed Central

Dieckmann, T; Suzuki, E; Nakamura, G K; Feigon, J

1996-01-01

In vitro selection has been used to isolate several RNA aptamers that bind specifically to biological cofactors. A well-characterized example in the ATP-binding RNA aptamer family, which contains a conserved 11-base loop opposite a bulged G and flanked by regions of double-stranded RNA. The nucleotides in the consensus sequence provide a binding pocket for ATP (or AMP), which binds with a Kd in the micromolar range. Here we present the three-dimensional solution structure of a 36-nucleotide ATP-binding RNA aptamer complexed with AMP, determined from NMR-derived distance and dihedral angle restraints. The conserved loop and bulged G form a novel compact, folded structure around the AMP. The backbone tracing of the loop nucleotides can be described by a Greek zeta (zeta). Consecutive loop nucleotides G, A, A form a U-turn at the bottom of the zeta, and interact with the AMP to form a structure similar to a GNRA tetraloop, with AMP standing in for the final A. Two asymmetric G. G base pairs close the stems flanking the internal loop. Mutated aptamers support the existence of the tertiary interactions within the consensus nucleotides and with the AMP found in the calculated structures. PMID:8756406

Active Site Sharing and Subterminal Hairpin Recognition in a New Class of DNA Transposases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ronning, Donald R.; Guynet, Catherine; Ton-Hoang, Bao

2010-07-20

Many bacteria harbor simple transposable elements termed insertion sequences (IS). In Helicobacter pylori, the chimeric IS605 family elements are particularly interesting due to their proximity to genes encoding gastric epithelial invasion factors. Protein sequences of IS605 transposases do not bear the hallmarks of other well-characterized transposases. We have solved the crystal structure of full-length transposase (TnpA) of a representative member, ISHp608. Structurally, TnpA does not resemble any characterized transposase; rather, it is related to rolling circle replication (RCR) proteins. Consistent with RCR, Mg{sup 2+} and a conserved tyrosine, Tyr127, are essential for DNA nicking and the formation of a covalentmore » intermediate between TnpA and DNA. TnpA is dimeric, contains two shared active sites, and binds two DNA stem loops representing the conserved inverted repeats near each end of ISHp608. The cocrystal structure with stem-loop DNA illustrates how this family of transposases specifically recognizes and pairs ends, necessary steps during transposition.« less

Gene silencing efficiency and INF-β induction effects of splicing miRNA 155-based artificial miRNA with pre-miRNA stem-loop structures.

PubMed

Sin, Onsam; Mabiala, Prudence; Liu, Ye; Sun, Ying; Hu, Tao; Liu, Qingzhen; Guo, Deyin

2012-02-01

Artificial microRNA (miRNA) expression vectors have been developed and used for RNA interference. The secondary structure of artificial miRNA is important for RNA interference efficacy. We designed two groups of six artificial splicing miRNA 155-based miRNAs (SM155-based miRNAs) with the same target in the coding region or 3' UTR of a target gene and studied their RNA silencing efficiency and interferon β (IFN-β) induction effects. SM155-based miRNA with a mismatch at the +1 position and a bulge at the +11, +12 positions in a miRNA precursor stem-loop structure showed the highest gene silencing efficiency and lowest IFN-β induction effect (increased IFN-β mRNA level by 10% in both target cases), regardless of the specificity of the target sequence, suggesting that pSM155-based miRNA with this design could be a valuable miRNA expression vector.

The interaction between the iron-responsive element binding protein and its cognate RNA is highly dependent upon both RNA sequence and structure.

PubMed

Jaffrey, S R; Haile, D J; Klausner, R D; Harford, J B

1993-09-25

To assess the influence of RNA sequence/structure on the interaction RNAs with the iron-responsive element binding protein (IRE-BP), twenty eight altered RNAs were tested as competitors for an RNA corresponding to the ferritin H chain IRE. All changes in the loop of the predicted IRE hairpin and in the unpaired cytosine residue characteristically found in IRE stems significantly decreased the apparent affinity of the RNA for the IRE-BP. Similarly, alteration in the spacing and/or orientation of the loop and the unpaired cytosine of the stem by either increasing or decreasing the number of base pairs separating them significantly reduced efficacy as a competitor. It is inferred that the IRE-BP forms multiple contacts with its cognate RNA, and that these contacts, acting in concert, provide the basis for the high affinity of this interaction.

R-chie: a web server and R package for visualizing RNA secondary structures

PubMed Central

Lai, Daniel; Proctor, Jeff R.; Zhu, Jing Yun A.; Meyer, Irmtraud M.

2012-01-01

Visually examining RNA structures can greatly aid in understanding their potential functional roles and in evaluating the performance of structure prediction algorithms. As many functional roles of RNA structures can already be studied given the secondary structure of the RNA, various methods have been devised for visualizing RNA secondary structures. Most of these methods depict a given RNA secondary structure as a planar graph consisting of base-paired stems interconnected by roundish loops. In this article, we present an alternative method of depicting RNA secondary structure as arc diagrams. This is well suited for structures that are difficult or impossible to represent as planar stem-loop diagrams. Arc diagrams can intuitively display pseudo-knotted structures, as well as transient and alternative structural features. In addition, they facilitate the comparison of known and predicted RNA secondary structures. An added benefit is that structure information can be displayed in conjunction with a corresponding multiple sequence alignments, thereby highlighting structure and primary sequence conservation and variation. We have implemented the visualization algorithm as a web server R-chie as well as a corresponding R package called R4RNA, which allows users to run the software locally and across a range of common operating systems. PMID:22434875

Closed-Loop Control of Complex Networks: A Trade-Off between Time and Energy

NASA Astrophysics Data System (ADS)

Sun, Yong-Zheng; Leng, Si-Yang; Lai, Ying-Cheng; Grebogi, Celso; Lin, Wei

2017-11-01

Controlling complex nonlinear networks is largely an unsolved problem at the present. Existing works focus either on open-loop control strategies and their energy consumptions or on closed-loop control schemes with an infinite-time duration. We articulate a finite-time, closed-loop controller with an eye toward the physical and mathematical underpinnings of the trade-off between the control time and energy as well as their dependence on the network parameters and structure. The closed-loop controller is tested on a large number of real systems including stem cell differentiation, food webs, random ecosystems, and spiking neuronal networks. Our results represent a step forward in developing a rigorous and general framework to control nonlinear dynamical networks with a complex topology.

Highly sensitive self-complementary DNA nanoswitches triggered by polyelectrolytes

NASA Astrophysics Data System (ADS)

Wu, Jincai; Yu, Feng; Zhang, Zheng; Chen, Yong; Du, Jie; Maruyama, Atsushi

2015-12-01

Dimerization of two homologous strands of genomic DNA/RNA is an essential feature of retroviral replication. Herein we show that a cationic comb-type copolymer (CCC), poly(l-lysine)-graft-dextran, accelerates the dimerization of self-complementary stem-loop DNA, frequently found in functional DNA/RNA molecules, such as aptamers. Furthermore, an anionic polymer poly(sodium vinylsulfonate) (PVS) dissociates CCC from the duplex shortly within a few seconds. Then single stem-loop DNA spontaneously transforms from its dimer. Thus we can easily control the dimer and stem-loop DNA by switching on/off CCC activity. Both polyelectrolytes and DNA concentrations are in the nanomole per liter range. The polyelectrolyte-assisted transconformation and sequences design strategy ensures the reversible state control with rapid response and effective switching under physiologically relevant conditions. A further application of this sensitive assembly is to construct an aptamer-type drug delivery system, bind or release functional molecules responding to its transconformation.Dimerization of two homologous strands of genomic DNA/RNA is an essential feature of retroviral replication. Herein we show that a cationic comb-type copolymer (CCC), poly(l-lysine)-graft-dextran, accelerates the dimerization of self-complementary stem-loop DNA, frequently found in functional DNA/RNA molecules, such as aptamers. Furthermore, an anionic polymer poly(sodium vinylsulfonate) (PVS) dissociates CCC from the duplex shortly within a few seconds. Then single stem-loop DNA spontaneously transforms from its dimer. Thus we can easily control the dimer and stem-loop DNA by switching on/off CCC activity. Both polyelectrolytes and DNA concentrations are in the nanomole per liter range. The polyelectrolyte-assisted transconformation and sequences design strategy ensures the reversible state control with rapid response and effective switching under physiologically relevant conditions. A further application of this sensitive assembly is to construct an aptamer-type drug delivery system, bind or release functional molecules responding to its transconformation. Electronic supplementary information (ESI) available: I. Sequences of DIS25, DIS25-2a and DIS25-3a. II. Structural formula of poly(l-lysine)-graft-dextran (PLL-g-Dex). 1H-NMR spectra of PLL-g-Dex in D2O. III. Gel electrophoretic analysis of dimerization of DIS25 with various N/P ratios. IV. The effect of polyelectrolyte on the fluorescence polarity of TAMRA-labeled duplex. V. UV absorption/Tm profiles of DIS25. VI. Arrhenius plots for spontaneous dissociation of the DIS25 dimer and PLL-g-Dex-assisted dimerization of DIS25.VII. Switching between double stem-loop DIS42 and extended multiplex drived by PLL-g-Dex and PVS. See DOI: 10.1039/c5nr05193b

Terminal structures of West Nile virus genomic RNA and their interactions with viral NS5 protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dong Hongping; Zhang Bo; Shi Peiyong

2008-11-10

Genome cyclization is essential for flavivirus replication. We used RNases to probe the structures formed by the 5'-terminal 190 nucleotides and the 3'-terminal 111 nucleotides of the West Nile virus (WNV) genomic RNA. When analyzed individually, the two RNAs adopt stem-loop structures as predicted by the thermodynamic-folding program. However, when mixed together, the two RNAs form a duplex that is mediated through base-pairings of two sets of RNA elements (5'CS/3'CSI and 5'UAR/3'UAR). Formation of the RNA duplex facilitates a conformational change that leaves the 3'-terminal nucleotides of the genome (position - 8 to - 16) to be single-stranded. Viral NS5more » binds specifically to the 5'-terminal stem-loop (SL1) of the genomic RNA. The 5'SL1 RNA structure is essential for WNV replication. The study has provided further evidence to suggest that flavivirus genome cyclization and NS5/5'SL1 RNA interaction facilitate NS5 binding to the 3' end of the genome for the initiation of viral minus-strand RNA synthesis.« less

Deciphering the structural framework of glycine receptor anchoring by gephyrin

PubMed Central

Kim, Eun Young; Schrader, Nils; Smolinsky, Birthe; Bedet, Cécile; Vannier, Christian; Schwarz, Günter; Schindelin, Hermann

2006-01-01

Glycine is the major inhibitory neurotransmitter in the spinal cord and brain stem. Gephyrin is required to achieve a high concentration of glycine receptors (GlyRs) in the postsynaptic membrane, which is crucial for efficient glycinergic signal transduction. The interaction between gephyrin and the GlyR involves the E-domain of gephyrin and a cytoplasmic loop located between transmembrane segments three and four of the GlyR β subunit. Here, we present crystal structures of the gephyrin E-domain with and without the GlyR β-loop at 2.4 and 2.7 Å resolutions, respectively. The GlyR β-loop is bound in a symmetric ‘key and lock' fashion to each E-domain monomer in a pocket adjacent to the dimer interface. Structure-guided mutagenesis followed by in vitro binding and in vivo colocalization assays demonstrate that a hydrophobic interaction formed by Phe 330 of gephyrin and Phe 398 and Ile 400 of the GlyR β-loop is crucial for binding. PMID:16511563

All-atomic Molecular Dynamic Studies of Human CDK8: Insight into the A-loop, Point Mutations and Binding with Its Partner CycC

PubMed Central

Xu, Wu; Amire-Brahimi, Benjamin; Xie, Xiao-Jun; Huang, Liying; Ji, Jun-Yuan

2014-01-01

The Mediator, a conserved multisubunit protein complex in eukaryotic organisms, regulates gene expression by bridging sequence-specific DNA-binding transcription factors to the general RNA polymerase II machinery. In yeast, Mediator complex is organized in three core modules (head, middle and tail) and a separable ‘CDK8 submodule’ consisting of four subunits including Cyclin-dependent kinase CDK8 (CDK8), Cyclin C (CycC), MED12, and MED13. The 3-D structure of human CDK8-CycC complex has been recently experimentally determined. To take advantage of this structure and the improved theoretical calculation methods, we have performed molecular dynamic simulations to study dynamics of CDK8 and two CDK8 point mutations (D173A and D189N), which have been identified in human cancers, with and without full length of the A-loop as well as the binding between CDK8 and CycC. We found that CDK8 structure gradually loses two helical structures during the 50-ns molecular dynamic simulation, likely due to the presence of the full-length A-loop. In addition, our studies showed the hydrogen bond occupation of the CDK8 A-loop increases during the first 20-ns MD simulation and stays stable during the later 30-ns MD simulation. Four residues in the A-loop of CDK8 have high hydrogen bond occupation, while the rest residues have low or no hydrogen bond occupation. The hydrogen bond dynamic study of the A-loop residues exhibits three types of changes: increasing, decreasing, and stable. Furthermore, the 3-D structures of CDK8 point mutations D173A, D189N, T196A and T196D have been built by molecular modeling and further investigated by 50-ns molecular dynamic simulations. D173A has the highest average potential energy, while T196D has the lowest average potential energy, indicating that T196D is the most stable structure. Finally, we calculated theoretical binding energy of CDK8 and CycC by MM/PBSA and MM/GBSA methods, and the negative values obtained from both methods demonstrate stability of CDK8-CycC complex. Taken together, these analyses will improve our understanding of the exact functions of CDK8 and the interaction with its partner CycC. PMID:24754906

Conformation and Stability of Intramolecular Telomeric G-Quadruplexes: Sequence Effects in the Loops

PubMed Central

Sattin, Giovanna; Artese, Anna; Nadai, Matteo; Costa, Giosuè; Parrotta, Lucia; Alcaro, Stefano; Palumbo, Manlio; Richter, Sara N.

2013-01-01

Telomeres are guanine-rich sequences that protect the ends of chromosomes. These regions can fold into G-quadruplex structures and their stabilization by G-quadruplex ligands has been employed as an anticancer strategy. Genetic analysis in human telomeres revealed extensive allelic variation restricted to loop bases, indicating that the variant telomeric sequences maintain the ability to fold into G-quadruplex. To assess the effect of mutations in loop bases on G-quadruplex folding and stability, we performed a comprehensive analysis of mutant telomeric sequences by spectroscopic techniques, molecular dynamics simulations and gel electrophoresis. We found that when the first position in the loop was mutated from T to C or A the resulting structure adopted a less stable antiparallel topology; when the second position was mutated to C or A, lower thermal stability and no evident conformational change were observed; in contrast, substitution of the third position from A to C induced a more stable and original hybrid conformation, while mutation to T did not significantly affect G-quadruplex topology and stability. Our results indicate that allelic variations generate G-quadruplex telomeric structures with variable conformation and stability. This aspect needs to be taken into account when designing new potential anticancer molecules. PMID:24367632

Principles for computational design of binding antibodies

PubMed Central

Pszolla, M. Gabriele; Lapidoth, Gideon D.; Norn, Christoffer; Dym, Orly; Unger, Tamar; Albeck, Shira; Tyka, Michael D.; Fleishman, Sarel J.

2017-01-01

Natural proteins must both fold into a stable conformation and exert their molecular function. To date, computational design has successfully produced stable and atomically accurate proteins by using so-called “ideal” folds rich in regular secondary structures and almost devoid of loops and destabilizing elements, such as cavities. Molecular function, such as binding and catalysis, however, often demands nonideal features, including large and irregular loops and buried polar interaction networks, which have remained challenging for fold design. Through five design/experiment cycles, we learned principles for designing stable and functional antibody variable fragments (Fvs). Specifically, we (i) used sequence-design constraints derived from antibody multiple-sequence alignments, and (ii) during backbone design, maintained stabilizing interactions observed in natural antibodies between the framework and loops of complementarity-determining regions (CDRs) 1 and 2. Designed Fvs bound their ligands with midnanomolar affinities and were as stable as natural antibodies, despite having >30 mutations from mammalian antibody germlines. Furthermore, crystallographic analysis demonstrated atomic accuracy throughout the framework and in four of six CDRs in one design and atomic accuracy in the entire Fv in another. The principles we learned are general, and can be implemented to design other nonideal folds, generating stable, specific, and precise antibodies and enzymes. PMID:28973872

Nucleotide sequence of the gag gene and gag-pol junction of feline leukemia virus.

PubMed Central

Laprevotte, I; Hampe, A; Sherr, C J; Galibert, F

1984-01-01

The nucleotide sequence of the gag gene of feline leukemia virus and its flanking sequences were determined and compared with the corresponding sequences of two strains of feline sarcoma virus and with that of the Moloney strain of murine leukemia virus. A high degree of nucleotide sequence homology between the feline leukemia virus and murine leukemia virus gag genes was observed, suggesting that retroviruses of domestic cats and laboratory mice have a common, proximal evolutionary progenitor. The predicted structure of the complete feline leukemia virus gag gene precursor suggests that the translation of nonglycosylated and glycosylated gag gene polypeptides is initiated at two different AUG codons. These initiator codons fall in the same reading frame and are separated by a 222-base-pair segment which encodes an amino terminal signal peptide. The nucleotide sequence predicts the order of amino acids in each of the individual gag-coded proteins (p15, p12, p30, p10), all of which derive from the gag gene precursor. Stable stem-and-loop secondary structures are proposed for two regions of viral RNA. The first falls within sequences at the 5' end of the viral genome, together with adjacent palindromic sequences which may play a role in dimer linkage of RNA subunits. The second includes coding sequences at the gag-pol junction and is proposed to be involved in translation of the pol gene product. Sequence analysis of the latter region shows that the gag and pol genes are translated in different reading frames. Classical consensus splice donor and acceptor sequences could not be localized to regions which would permit synthesis of the expected gag-pol precursor protein. Alternatively, we suggest that the pol gene product (RNA-dependent DNA polymerase) could be translated by a frameshift suppressing mechanism which could involve cleavage modification of stems and loops in a manner similar to that observed in tRNA processing. PMID:6328019

N -Methylation as a Strategy for Enhancing the Affinity and Selectivity of RNA-binding Peptides: Application to the HIV-1 Frameshift-Stimulating RNA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hilimire, Thomas A.; Bennett, Ryan P.; Stewart, Ryan A.

Human Immunodeficiency Virus (HIV) type 1 uses a -1 programmed ribosomal frameshift (-1 PRF) event to translate its enzymes from the same transcript used to encode the virus’ structural proteins. The frequency of this event is highly regulated, and significant deviation from the normal 5-10% frequency has been demonstrated to decrease viral infectivity. Frameshifting is primarily regulated by the Frameshift Stimulatory Signal RNA (FSS-RNA), a thermodynamically stable, highly conserved stem loop that has been proposed as a therapeutic target. We describe the design, synthesis, and testing of a series of N-methyl peptides able to bind the HIV-1 FSS RNA stemmore » loop with low nanomolar afinity and high selectivity. Surface plasmon resonance (SPR) data indicates increased affinity is a reflection of a substantially enhanced on rate. Compounds readily penetrate cell membranes and inhibit HIV infectivity in a pseudotyped virus assay. Viral infectivity inhibition correlates with compound-dependent changes in the ratios of Gag and Gag-Pol in virus particles. As the first compounds with both single digit nanomolar affinities for the FSS RNA and an ability to inhibit HIV in cells, these studies support the use of N-methylation for enhancing the affinity, selectivity, and bioactivity of RNA-binding peptides.« less

N -Methylation as a Strategy for Enhancing the Affinity and Selectivity of RNA-binding Peptides: Application to the HIV-1 Frameshift-Stimulating RNA

DOE PAGES

Hilimire, Thomas A.; Bennett, Ryan P.; Stewart, Ryan A.; ...

2015-10-23

Human Immunodeficiency Virus (HIV) type 1 uses a -1 programmed ribosomal frameshift (-1 PRF) event to translate its enzymes from the same transcript used to encode the virus’ structural proteins. The frequency of this event is highly regulated, and significant deviation from the normal 5-10% frequency has been demonstrated to decrease viral infectivity. Frameshifting is primarily regulated by the Frameshift Stimulatory Signal RNA (FSS-RNA), a thermodynamically stable, highly conserved stem loop that has been proposed as a therapeutic target. We describe the design, synthesis, and testing of a series of N-methyl peptides able to bind the HIV-1 FSS RNA stemmore » loop with low nanomolar afinity and high selectivity. Surface plasmon resonance (SPR) data indicates increased affinity is a reflection of a substantially enhanced on rate. Compounds readily penetrate cell membranes and inhibit HIV infectivity in a pseudotyped virus assay. Viral infectivity inhibition correlates with compound-dependent changes in the ratios of Gag and Gag-Pol in virus particles. As the first compounds with both single digit nanomolar affinities for the FSS RNA and an ability to inhibit HIV in cells, these studies support the use of N-methylation for enhancing the affinity, selectivity, and bioactivity of RNA-binding peptides.« less

Terminator Detection by Support Vector Machine Utilizing aStochastic Context-Free Grammar

DOE Office of Scientific and Technical Information (OSTI.GOV)

Francis-Lyon, Patricia; Cristianini, Nello; Holbrook, Stephen

2006-12-30

A 2-stage detector was designed to find rho-independent transcription terminators in the Escherichia coli genome. The detector includes a Stochastic Context Free Grammar (SCFG) component and a Support Vector Machine (SVM) component. To find terminators, the SCFG searches the intergenic regions of nucleotide sequence for local matches to a terminator grammar that was designed and trained utilizing examples of known terminators. The grammar selects sequences that are the best candidates for terminators and assigns them a prefix, stem-loop, suffix structure using the Cocke-Younger-Kasaami (CYK) algorithm, modified to incorporate energy affects of base pairing. The parameters from this inferred structure aremore » passed to the SVM classifier, which distinguishes terminators from non-terminators that score high according to the terminator grammar. The SVM was trained with negative examples drawn from intergenic sequences that include both featureless and RNA gene regions (which were assigned prefix, stem-loop, suffix structure by the SCFG), so that it successfully distinguishes terminators from either of these. The classifier was found to be 96.4% successful during testing.« less

Molecular dynamics simulations of electrostatics and hydration distributions around RNA and DNA motifs

NASA Astrophysics Data System (ADS)

Marlowe, Ashley E.; Singh, Abhishek; Semichaevsky, Andrey V.; Yingling, Yaroslava G.

2009-03-01

Nucleic acid nanoparticles can self-assembly through the formation of complementary loop-loop interactions or stem-stem interactions. Presence and concentration of ions can significantly affect the self-assembly process and the stability of the nanostructure. In this presentation we use explicit molecular dynamics simulations to examine the variations in cationic distributions and hydration environment around DNA and RNA helices and loop-loop interactions. Our simulations show that the potassium and sodium ionic distributions are different around RNA and DNA motifs which could be indicative of ion mediated relative stability of loop-loop complexes. Moreover in RNA loop-loop motifs ions are consistently present and exchanged through a distinct electronegative channel. We will also show how we used the specific RNA loop-loop motif to design a RNA hexagonal nanoparticle.

The complete mitogenome sequence of the Japanese oak silkmoth, Antheraea yamamai (Lepidoptera: Saturniidae).

PubMed

Kim, Seong Ryeol; Kim, Man Il; Hong, Mee Yeon; Kim, Kee Young; Kang, Pil Don; Hwang, Jae Sam; Han, Yeon Soo; Jin, Byung Rae; Kim, Iksoo

2009-09-01

The 15,338-bp long complete mitochondrial genome (mitogenome) of the Japanese oak silkmoth, Antheraea yamamai (Lepidoptera: Saturniidae) was determined. This genome has a gene arrangement identical to those of all other sequenced lepidopteran insects, but differs from the most common type, as the result of the movement of tRNA(Met) to a position 5'-upstream of tRNA(Ile). No typical start codon of the A. yamamai COI gene is available. Instead, a tetranucleotide, TTAG, which is found at the beginning context of all sequenced lepidopteran insects was tentatively designated as the start codon for A. yamamai COI gene. Three of the 13 protein-coding genes (PCGs) harbor the incomplete termination codon, T or TA. All tRNAs formed stable stem-and-loop structures, with the exception of tRNA(Ser)(AGN), the DHU arm of which formed a simple loop as has been observed in many other metazoan mt tRNA(Ser)(AGN). The 334-bp long A + T-rich region is noteworthy in that it harbors tRNA-like structures, as has also been seen in the A + T-rich regions of other insect mitogenomes. Phylogenetic analyses of the available species of Bombycoidea, Pyraloidea, and Tortricidea bolstered the current morphology-based hypothesis that Bombycoidea and Pyraloidea are monophyletic (Obtectomera). As has been previously suggested, Bombycidae (Bombyx mori and B. mandarina) and Saturniidae (A. yamamai and Caligula boisduvalii) formed a reciprocal monophyletic group.

Invariant U2 snRNA nucleotides form a stem loop to recognize the intron early in splicing

PubMed Central

Perriman, Rhonda; Ares, Manuel

2010-01-01

U2 snRNA-intron branchpoint pairing is a critical step in pre-mRNA recognition by the splicing apparatus, but the mechanism by which these two RNAs engage each other is unknown. Here we identify a new U2 snRNA structure, the branchpoint interaction stem-loop (BSL), that presents the U2 nucleotides that will contact the intron. We provide evidence that the BSL forms prior to interaction with the intron, and is disrupted by the DExD/H protein Prp5p during engagement of the snRNA with the intron. In vitro splicing complex assembly in a BSL-destabilized mutant extract suggests that the BSL is required at a previously unrecognized step between commitment complex and prespliceosome formation. The extreme evolutionary conservation of the BSL suggests it represents an ancient structural solution to the problem of intron branchpoint recognition by dynamic RNA elements that must serve multiple functions at other times during splicing. PMID:20471947

Analysis of Physicochemical and Structural Properties Determining HIV-1 Coreceptor Usage

PubMed Central

Bozek, Katarzyna; Lengauer, Thomas; Sierra, Saleta; Kaiser, Rolf; Domingues, Francisco S.

2013-01-01

The relationship of HIV tropism with disease progression and the recent development of CCR5-blocking drugs underscore the importance of monitoring virus coreceptor usage. As an alternative to costly phenotypic assays, computational methods aim at predicting virus tropism based on the sequence and structure of the V3 loop of the virus gp120 protein. Here we present a numerical descriptor of the V3 loop encoding its physicochemical and structural properties. The descriptor allows for structure-based prediction of HIV tropism and identification of properties of the V3 loop that are crucial for coreceptor usage. Use of the proposed descriptor for prediction results in a statistically significant improvement over the prediction based solely on V3 sequence with 3 percentage points improvement in AUC and 7 percentage points in sensitivity at the specificity of the 11/25 rule (95%). We additionally assessed the predictive power of the new method on clinically derived ‘bulk’ sequence data and obtained a statistically significant improvement in AUC of 3 percentage points over sequence-based prediction. Furthermore, we demonstrated the capacity of our method to predict therapy outcome by applying it to 53 samples from patients undergoing Maraviroc therapy. The analysis of structural features of the loop informative of tropism indicates the importance of two loop regions and their physicochemical properties. The regions are located on opposite strands of the loop stem and the respective features are predominantly charge-, hydrophobicity- and structure-related. These regions are in close proximity in the bound conformation of the loop potentially forming a site determinant for the coreceptor binding. The method is available via server under http://structure.bioinf.mpi-inf.mpg.de/. PMID:23555214

Radiation Enhanced Absorption of Frank Loops by Nanovoids in Cu

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Youxing; Zhang, Xinghang; Wang, Jian

Neutron and heavy ion irradiation generally induces voids in metallic materials, and continuous radiations typically result in void swelling and mechanical failure of the irradiated materials. Recent experiments showed that nanovoids in nanotwinned copper could act as sinks for radiation-induced Frank loops, significantly mitigating radiation damage [Y. Chen et al., Nat. Commun. 6:7036 (2015)]. In this paper, we report on structural evolution of Frank loops under cascades and address the role of nanovoids in absorbing Frank loops in detail by using molecular dynamics simulations. Results show that a stand-alone Frank loop is stable under cascades. When Frank loops are adjacentmore » to nanovoids, the diffusion of a group of atoms from the loop into nanovoids is accomplished via the formation and propagation of dislocation loops. The loop-nanovoid interactions result in the shrinkage of the nanovoids and the Frank loops.« less

Radiation Enhanced Absorption of Frank Loops by Nanovoids in Cu

DOE PAGES

Chen, Youxing; Zhang, Xinghang; Wang, Jian

2016-11-01

Neutron and heavy ion irradiation generally induces voids in metallic materials, and continuous radiations typically result in void swelling and mechanical failure of the irradiated materials. Recent experiments showed that nanovoids in nanotwinned copper could act as sinks for radiation-induced Frank loops, significantly mitigating radiation damage [Y. Chen et al., Nat. Commun. 6:7036 (2015)]. In this paper, we report on structural evolution of Frank loops under cascades and address the role of nanovoids in absorbing Frank loops in detail by using molecular dynamics simulations. Results show that a stand-alone Frank loop is stable under cascades. When Frank loops are adjacentmore » to nanovoids, the diffusion of a group of atoms from the loop into nanovoids is accomplished via the formation and propagation of dislocation loops. The loop-nanovoid interactions result in the shrinkage of the nanovoids and the Frank loops.« less

Molecular dynamics correctly models the unusual major conformation of the GAGU RNA internal loop and with NMR reveals an unusual minor conformation.

PubMed

Spasic, Aleksandar; Kennedy, Scott D; Needham, Laura; Manoharan, Muthiah; Kierzek, Ryszard; Turner, Douglas H; Mathews, David H

2018-05-01

The RNA "GAGU" duplex, (5'GAC GAGU GUCA) 2 , contains the internal loop (5'-GAGU-3') 2 , which has two conformations in solution as determined by NMR spectroscopy. The major conformation has a loop structure consisting of trans -Watson-Crick/Hoogsteen GG pairs, A residues stacked on each other, U residues bulged outside the helix, and all sugars with a C2'- endo conformation. This differs markedly from the internal loops, (5'-G AG C-3') 2 , (5'-A AG U-3') 2 , and (5'-UAGG-3') 2 , which all have cis -Watson-Crick/Watson-Crick AG "imino" pairs flanked by cis -Watson-Crick/Watson-Crick canonical pairs resulting in maximal hydrogen bonding. Here, molecular dynamics was used to test whether the Amber force field (ff99 + bsc0 + OL3) approximates molecular interactions well enough to keep stable the unexpected conformation of the GAGU major duplex structure and the NMR structures of the duplexes containing (5'-G AG C-3') 2 , (5'-A AG U-3') 2 , and (5'-U AG G-3') 2 internal loops. One-microsecond simulations were repeated four times for each of the duplexes starting in their NMR conformations. With the exception of (5'-UAGG-3') 2 , equivalent simulations were also run starting with alternative conformations. Results indicate that the Amber force field keeps the NMR conformations of the duplexes stable for at least 1 µsec. They also demonstrate an unexpected minor conformation for the (5'-GAGU-3') 2 loop that is consistent with newly measured NMR spectra of duplexes with natural and modified nucleotides. Thus, unrestrained simulations led to the determination of the previously unknown minor conformation. The stability of the native (5'-GAGU-3') 2 internal loop as compared to other loops can be explained by changes in hydrogen bonding and stacking as the flanking bases are changed. © 2018 Spasic et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Electronic polarization stabilizes tertiary structure prediction of HP-36.

PubMed

Duan, Li L; Zhu, Tong; Zhang, Qing G; Tang, Bo; Zhang, John Z H

2014-04-01

Molecular dynamic (MD) simulations with both implicit and explicit solvent models have been carried out to study the folding dynamics of HP-36 protein. Starting from the extended conformation, the secondary structure of all three helices in HP-36 was formed in about 50 ns and remained stable in the remaining simulation. However, the formation of the tertiary structure was difficult. Although some intermediates were close to the native structure, the overall conformation was not stable. Further analysis revealed that the large structure fluctuation of loop and hydrophobic core regions was devoted mostly to the instability of the structure during MD simulation. The backbone root-mean-square deviation (RMSD) of the loop and hydrophobic core regions showed strong correlation with the backbone RMSD of the whole protein. The free energy landscape indicated that the distribution of main chain torsions in loop and turn regions was far away from the native state. Starting from an intermediate structure extracted from the initial AMBER simulation, HP-36 was found to generally fold to the native state under the dynamically adjusted polarized protein-specific charge (DPPC) simulation, while the peptide did not fold into the native structure when AMBER force filed was used. The two best folded structures were extracted and taken into further simulations in water employing AMBER03 charge and DPPC for 25 ns. Result showed that introducing polarization effect into interacting potential could stabilize the near-native protein structure.

Structure modulation of helix 69 from Escherichia coli 23S ribosomal RNA by pseudouridylations.

PubMed

Jiang, Jun; Aduri, Raviprasad; Chow, Christine S; SantaLucia, John

2014-04-01

Helix 69 (H69) is a 19-nt stem-loop region from the large subunit ribosomal RNA. Three pseudouridine (Ψ) modifications clustered in H69 are conserved across phylogeny and known to affect ribosome function. To explore the effects of Ψ on the conformations of Escherichia coli H69 in solution, nuclear magnetic resonance spectroscopy was used to reveal the structural differences between H69 with (ΨΨΨ) and without (UUU) Ψ modifications. Comparison of the two structures shows that H69 ΨΨΨ has the following unique features: (i) the loop region is closed by a Watson-Crick base pair between Ψ1911 and A1919, which is potentially reinforced by interactions involving Ψ1911N1H and (ii) Ψ modifications at loop residues 1915 and 1917 promote base stacking from Ψ1915 to A1918. In contrast, the H69 UUU loop region, which lacks Ψ modifications, is less organized. Structure modulation by Ψ leads to alteration in conformational behavior of the 5' half of the H69 loop region, observed as broadening of C1914 non-exchangeable base proton resonances in the H69 ΨΨΨ nuclear magnetic resonance spectra, and plays an important biological role in establishing the ribosomal intersubunit bridge B2a and mediating translational fidelity.

Combined hairpin-antisense compositions and methods for modulating expression

DOEpatents

Shanklin, John; Nguyen, Tam

2014-08-05

A nucleotide construct comprising a nucleotide sequence that forms a stem and a loop, wherein the loop comprises a nucleotide sequence that modulates expression of a target, wherein the stem comprises a nucleotide sequence that modulates expression of a target, and wherein the target modulated by the nucleotide sequence in the loop and the target modulated by the nucleotide sequence in the stem may be the same or different. Vectors, methods of regulating target expression, methods of providing a cell, and methods of treating conditions comprising the nucleotide sequence are also disclosed.

Combined hairpin-antisense compositions and methods for modulating expression

DOEpatents

Shanklin, John; Nguyen, Tam Huu

2015-11-24

A nucleotide construct comprising a nucleotide sequence that forms a stem and a loop, wherein the loop comprises a nucleotide sequence that modulates expression of a target, wherein the stem comprises a nucleotide sequence that modulates expression of a target, and wherein the target modulated by the nucleotide sequence in the loop and the target modulated by the nucleotide sequence in the stem may be the same or different. Vectors, methods of regulating target expression, methods of providing a cell, and methods of treating conditions comprising the nucleotide sequence are also disclosed.

Folding thermodynamics of pseudoknotted chain conformations

PubMed Central

Kopeikin, Zoia; Chen, Shi-Jie

2008-01-01

We develop a statistical mechanical framework for the folding thermodynamics of pseudoknotted structures. As applications of the theory, we investigate the folding stability and the free energy landscapes for both the thermal and the mechanical unfolding of pseudoknotted chains. For the mechanical unfolding process, we predict the force-extension curves, from which we can obtain the information about structural transitions in the unfolding process. In general, a pseudoknotted structure unfolds through multiple structural transitions. The interplay between the helix stems and the loops plays an important role in the folding stability of pseudoknots. For instance, variations in loop sizes can lead to the destabilization of some intermediate states and change the (equilibrium) folding pathways (e.g., two helix stems unfold either cooperatively or sequentially). In both thermal and mechanical unfolding, depending on the nucleotide sequence, misfolded intermediate states can emerge in the folding process. In addition, thermal and mechanical unfoldings often have different (equilibrium) pathways. For example, for certain sequences, the misfolded intermediates, which generally have longer tails, can fold, unfold, and refold again in the pulling process, which means that these intermediates can switch between two different average end-end extensions. PMID:16674261

S-ovalbumin, an ovalbumin conformer with properties analogous to those of loop-inserted serpins.

PubMed Central

Huntington, J. A.; Patston, P. A.; Gettins, P. G.

1995-01-01

Most serpins are inhibitors of serine proteinases and are thought to undergo a conformational change upon complex formation with proteinase that involves partial insertion of the reactive center loop into a beta-sheet of the inhibitor. Ovalbumin, although a serpin, is not an inhibitor of serine proteinases. It has been proposed that this deficiency arises from the presence of a charged residue, arginine, at a critical point (P14) in the reactive center region, which prevents loop insertion into the beta-sheet and thereby precludes inhibitory properties. To test whether loop insertion is prevented in ovalbumin we have examined the properties of two forms of ovalbumin: the native protein and S-ovalbumin, a form that forms spontaneously from native ovalbumin and has increased stability. Calorimetric measurements showed that S-ovalbumin was more stable than ovalbumin by about 3 kcal mol-1. CD spectra, which indicated that S-ovalbumin had less alpha-helix than native ovalbumin, and 1H NMR spectra, which indicated very similar overall structures, suggest limited conformational differences between the two forms. From comparison of the susceptibility of the reactive center region of each protein to proteolysis by porcine pancreatic elastase and by subtilisin Carlsberg, we concluded that the limited native-to-S conformational change specifically affected the reactive center region. These data are consistent with a structure for S-ovalbumin in which part of the reactive center loop has inserted into beta-sheet A to give a more stable structure, analogously to other serpins. However, the rate of loop insertion appears to be very much lower than for inhibitory serpins.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7613461

Mitochondrial Genome of the Stonefly Kamimuria wangi (Plecoptera: Perlidae) and Phylogenetic Position of Plecoptera Based on Mitogenomes

PubMed Central

Yu-Han, Qian; Hai-Yan, Wu; Xiao-Yu, Ji; Wei-Wei, Yu; Yu-Zhou, Du

2014-01-01

This study determined the mitochondrial genome sequence of the stonefly, Kamimuria wangi. In order to investigate the relatedness of stonefly to other members of Neoptera, a phylogenetic analysis was undertaken based on 13 protein-coding genes of mitochondrial genomes in 13 representative insects. The mitochondrial genome of the stonefly is a circular molecule consisting of 16,179 nucleotides and contains the 37 genes typically found in other insects. A 10-bp poly-T stretch was observed in the A+T-rich region of the K. wangi mitochondrial genome. Downstream of the poly-T stretch, two regions were located with potential ability to form stem-loop structures; these were designated stem-loop 1 (positions 15848–15651) and stem-loop 2 (15965–15998). The arrangement of genes and nucleotide composition of the K. wangi mitogenome are similar to those in Pteronarcys princeps, suggesting a conserved genome evolution within the Plecoptera. Phylogenetic analysis using maximum likelihood and Bayesian inference of 13 protein-coding genes supported a novel relationship between the Plecoptera and Ephemeroptera. The results contradict the existence of a monophyletic Plectoptera and Plecoptera as sister taxa to Embiidina, and thus requires further analyses with additional mitogenome sampling at the base of the Neoptera. PMID:24466028

Mitochondrial genome of the stonefly Kamimuria wangi (Plecoptera: Perlidae) and phylogenetic position of plecoptera based on mitogenomes.

PubMed

Yu-Han, Qian; Hai-Yan, Wu; Xiao-Yu, Ji; Wei-Wei, Yu; Yu-Zhou, Du

2014-01-01

This study determined the mitochondrial genome sequence of the stonefly, Kamimuria wangi. In order to investigate the relatedness of stonefly to other members of Neoptera, a phylogenetic analysis was undertaken based on 13 protein-coding genes of mitochondrial genomes in 13 representative insects. The mitochondrial genome of the stonefly is a circular molecule consisting of 16,179 nucleotides and contains the 37 genes typically found in other insects. A 10-bp poly-T stretch was observed in the A+T-rich region of the K. wangi mitochondrial genome. Downstream of the poly-T stretch, two regions were located with potential ability to form stem-loop structures; these were designated stem-loop 1 (positions 15848-15651) and stem-loop 2 (15965-15998). The arrangement of genes and nucleotide composition of the K. wangi mitogenome are similar to those in Pteronarcys princeps, suggesting a conserved genome evolution within the Plecoptera. Phylogenetic analysis using maximum likelihood and Bayesian inference of 13 protein-coding genes supported a novel relationship between the Plecoptera and Ephemeroptera. The results contradict the existence of a monophyletic Plectoptera and Plecoptera as sister taxa to Embiidina, and thus requires further analyses with additional mitogenome sampling at the base of the Neoptera.

Inhibition of Influenza H7 Hemagglutinin-Mediated Entry

PubMed Central

Antanasijevic, Aleksandar; Cheng, Han; Wardrop, Duncan J.; Rong, Lijun; Caffrey, Michael

2013-01-01

The recent outbreak of H7N9 influenza in China is of high concern to public health. H7 hemagglutinin (HA) plays a critical role in influenza entry and thus HA presents an attractive target for antivirals. Previous studies have suggested that the small molecule tert-butyl hydroquinone (TBHQ) inhibits the entry of influenza H3 HA by binding to the stem loop of HA and stabilizing the neutral pH conformation of HA, thereby disrupting the membrane fusion step. Based on amino acid sequence, structure and immunogenicity, H7 is a related Group 2 HA. In this work we show, using a pseudovirus entry assay, that TBHQ inhibits H7 HA-mediated entry, as well as H3 HA-mediated entry, with an IC50∼6 µM. Using NMR, we show that TBHQ binds to the H7 stem loop region. STD NMR experiments indicate that the aromatic ring of TBHQ makes extensive contact with the H7 HA surface. Limited proteolysis experiments indicate that TBHQ inhibits influenza entry by stabilizing the H7 HA neutral pH conformation. Together, this work suggests that the stem loop region of H7 HA is an attractive target for therapeutic intervention and that TBHQ, which is a widely used food preservative, is a promising lead compound. PMID:24194835

Detection of negative and positive RNA strand of poliovirus Sabin 1 and echovirus E19 by a stem-loop reverse transcription PCR.

PubMed

Fikatas, A; Dimitriou, T G; Kyriakopoulou, Z; Moschonas, G D; Amoutzias, G D; Mossialos, D; Gartzonika, C; Levidiotou-Stefanou, S; Markoulatos, P

2017-09-01

In this report a strand specific RT-PCR was established for the detection of the replicative negative RNA strand of poliovirus sabin 1 (Sabin1) and Echovirus 19 (E19) strains. The key for the successful conduction of the assay was the use of a specific reverse transcription primer targeting the 5'-UTR of enteroviruses that consisted of a stem-loop structure at the 5'-end and an enteroviral-specific sequence at the 3'-end. The stem loop RT-PCR was found to be an accurate and sensitive method, detecting even 10 -2 CCID 50 of poliovirus sabin 1 (Sabin1) and E19 strains 6 h postinfection (p.i.), while CPE appeared 3 days later. This assay was also validated in SiHa and Caski cell lines that are not used for the detection of enteroviruses. The negative RNA strand was detected 6 h and 12 h p.i. in SiHa and Caski cells, when these cell lines were inoculated with 10 5 and 1 CCID 50 respectively, whereas CPE was observed 5 days p.i for SiHa cells and 8 days p.i for Caski cells and that only at 10 5 CCID 50 . The results show that this approach may be used for replacing the time-consuming cell cultures in order to detect the active replication of enteroviruses. Enteroviruses are positive stranded RNA viruses that may cause severe diseases. The conventional method for detection of active viral replication involves virus isolation in sensitive cell cultures followed by titration and seroneutralization. In this report, we describe the use of a stem-loop secondary structured oligonucleotide in RT-PCR assay for the detection of the replicative negative strand of the positive-stranded RNA of poliovirus sabin 1 and E19 strains. This approach proved to be a useful tool that may be used for replacing the time-consuming cell culture assays in order to detect the active replication of enteroviruses. © 2017 The Society for Applied Microbiology.

Stable loop in the crystal structure of the intercalated four-stranded cytosine-rich metazoan telomere

NASA Technical Reports Server (NTRS)

Kang, C.; Berger, I.; Lockshin, C.; Ratliff, R.; Moyzis, R.; Rich, A.

1995-01-01

In most metazoans, the telomeric cytosine-rich strand repeating sequence is d(TAACCC). The crystal structure of this sequence was solved to 1.9-A resolution. Four strands associate via the cytosine-containing parts to form a four-stranded intercalated structure held together by C.C+ hydrogen bonds. The base-paired strands are parallel to each other, and the two duplexes are intercalated into each other in opposite orientations. One TAA end forms a highly stabilized loop with the 5' thymine Hoogsteen-base-paired to the third adenine. The 5' end of this loop is in close proximity to the 3' end of one of the other intercalated cytosine strands. Instead of being entirely in a DNA duplex, this structure suggests the possibility of an alternative conformation for the cytosine-rich telomere strands.

URS DataBase: universe of RNA structures and their motifs.

PubMed

Baulin, Eugene; Yacovlev, Victor; Khachko, Denis; Spirin, Sergei; Roytberg, Mikhail

2016-01-01

The Universe of RNA Structures DataBase (URSDB) stores information obtained from all RNA-containing PDB entries (2935 entries in October 2015). The content of the database is updated regularly. The database consists of 51 tables containing indexed data on various elements of the RNA structures. The database provides a web interface allowing user to select a subset of structures with desired features and to obtain various statistical data for a selected subset of structures or for all structures. In particular, one can easily obtain statistics on geometric parameters of base pairs, on structural motifs (stems, loops, etc.) or on different types of pseudoknots. The user can also view and get information on an individual structure or its selected parts, e.g. RNA-protein hydrogen bonds. URSDB employs a new original definition of loops in RNA structures. That definition fits both pseudoknot-free and pseudoknotted secondary structures and coincides with the classical definition in case of pseudoknot-free structures. To our knowledge, URSDB is the first database supporting searches based on topological classification of pseudoknots and on extended loop classification.Database URL: http://server3.lpm.org.ru/urs/. © The Author(s) 2016. Published by Oxford University Press.

URS DataBase: universe of RNA structures and their motifs

PubMed Central

Baulin, Eugene; Yacovlev, Victor; Khachko, Denis; Spirin, Sergei; Roytberg, Mikhail

2016-01-01

The Universe of RNA Structures DataBase (URSDB) stores information obtained from all RNA-containing PDB entries (2935 entries in October 2015). The content of the database is updated regularly. The database consists of 51 tables containing indexed data on various elements of the RNA structures. The database provides a web interface allowing user to select a subset of structures with desired features and to obtain various statistical data for a selected subset of structures or for all structures. In particular, one can easily obtain statistics on geometric parameters of base pairs, on structural motifs (stems, loops, etc.) or on different types of pseudoknots. The user can also view and get information on an individual structure or its selected parts, e.g. RNA–protein hydrogen bonds. URSDB employs a new original definition of loops in RNA structures. That definition fits both pseudoknot-free and pseudoknotted secondary structures and coincides with the classical definition in case of pseudoknot-free structures. To our knowledge, URSDB is the first database supporting searches based on topological classification of pseudoknots and on extended loop classification. Database URL: http://server3.lpm.org.ru/urs/ PMID:27242032

High-resolution reversible folding of hyperstable RNA tetraloops using molecular dynamics simulations

PubMed Central

Chen, Alan A.; García, Angel E.

2013-01-01

We report the de novo folding of three hyperstable RNA tetraloops to 1–3 Å rmsd from their experimentally determined structures using molecular dynamics simulations initialized in the unfolded state. RNA tetraloops with loop sequences UUCG, GCAA, or CUUG are hyperstable because of the formation of noncanonical loop-stabilizing interactions, and they are all faithfully reproduced to angstrom-level accuracy in replica exchange molecular dynamics simulations, including explicit solvent and ion molecules. This accuracy is accomplished using unique RNA parameters, in which biases that favor rigid, highly stacked conformations are corrected to accurately capture the inherent flexibility of ssRNA loops, accurate base stacking energetics, and purine syn-anti interconversions. In a departure from traditional quantum chemistrycentric approaches to force field optimization, our parameters are calibrated directly from thermodynamic and kinetic measurements of intra- and internucleotide structural transitions. The ability to recapitulate the signature noncanonical interactions of the three most abundant hyperstable stem loop motifs represents a significant milestone to the accurate prediction of RNA tertiary structure using unbiased all-atom molecular dynamics simulations. PMID:24043821

Noncanoncial signal recognition particle RNAs in a major eukaryotic phylum revealed by purification of SRP from the human pathogen Cryptococcus neoformans

PubMed Central

Dumesic, Phillip A.; Rosenblad, Magnus A.; Samuelsson, Tore; Nguyen, Tiffany; Moresco, James J.; Yates, John R.; Madhani, Hiten D.

2015-01-01

Despite conservation of the signal recognition particle (SRP) from bacteria to man, computational approaches have failed to identify SRP components from genomes of many lower eukaryotes, raising the possibility that they have been lost or altered in those lineages. We report purification and analysis of SRP in the human pathogen Cryptococcus neoformans, providing the first description of SRP in basidiomycetous yeast. The C. neoformans SRP RNA displays a predicted structure in which the universally conserved helix 8 contains an unprecedented stem-loop insertion. Guided by this sequence, we computationally identified 152 SRP RNAs throughout the phylum Basidiomycota. This analysis revealed additional helix 8 alterations including single and double stem-loop insertions as well as loop diminutions affecting RNA structural elements that are otherwise conserved from bacteria to man. Strikingly, these SRP RNA features in Basidiomycota are accompanied by phylum-specific alterations in the RNA-binding domain of Srp54, the SRP protein subunit that directly interacts with helix 8. Our findings reveal unexpected fungal SRP diversity and suggest coevolution of the two most conserved SRP features—SRP RNA helix 8 and Srp54—in basidiomycetes. Because members of this phylum include important human and plant pathogens, these noncanonical features provide new targets for antifungal compound development. PMID:26275773

Genetic instability associated with loop or stem–loop structures within transcription units can be independent of nucleotide excision repair

PubMed Central

Burns, John A; Chowdhury, Moinuddin A; Cartularo, Laura; Berens, Christian; Scicchitano, David A

2018-01-01

Abstract Simple sequence repeats (SSRs) are found throughout the genome, and under some conditions can change in length over time. Germline and somatic expansions of trinucleotide repeats are associated with a series of severely disabling illnesses, including Huntington's disease. The underlying mechanisms that effect SSR expansions and contractions have been experimentally elusive, but models suggesting a role for DNA repair have been proposed, in particular the involvement of transcription-coupled nucleotide excision repair (TCNER) that removes transcription-blocking DNA damage from the transcribed strand of actively expressed genes. If the formation of secondary DNA structures that are associated with SSRs were to block RNA polymerase progression, TCNER could be activated, resulting in the removal of the aberrant structure and a concomitant change in the region's length. To test this, TCNER activity in primary human fibroblasts was assessed on defined DNA substrates containing extrahelical DNA loops that lack discernible internal base pairs or DNA stem–loops that contain base pairs within the stem. The results show that both structures impede transcription elongation, but there is no corresponding evidence that nucleotide excision repair (NER) or TCNER operates to remove them. PMID:29474673

Construction of stable capillary networks using a microfluidic device.

PubMed

Sudo, Ryo

2015-01-01

Construction of stable capillary networks is required to provide sufficient oxygen and nutrients to the deep region of thick tissues, which is important in the context of 3D tissue engineering. Although conventional in vitro culture models have been used to investigate the mechanism of capillary formation, recent advances in microfluidics technologies allowed us to control biophysical and biochemical culture environments more precisely, which led to the construction of functional and stable capillary networks. In this study, endothelial cells and mesenchymal stem cells were co-cultured in microfluidic devices to construct stable capillary networks, which resulted in the construction of luminal structures covered by pericytes. Interactions between endothelial cells and mesenchymal stem cells are also discussed in the context of capillary formation.

The rolling-circle melting-pot model for porcine circovirus DNA replication

USDA-ARS?s Scientific Manuscript database

A stem-loop structure, formed by a pair of inverted repeats during DNA replication, is a conserved feature at the origin of DNA replication (Ori) among plant and animal viruses, bacteriophages and plasmids that replicate their genomes via the rolling-circle replication (RCR) mechanism. Porcine circo...

Modified nucleoside dependent Watson-Crick and wobble codon binding by tRNALysUUU species.

PubMed

Yarian, C; Marszalek, M; Sochacka, E; Malkiewicz, A; Guenther, R; Miskiewicz, A; Agris, P F

2000-11-07

Nucleoside modifications are important to the structure of all tRNAs and are critical to the function of some tRNA species. The transcript of human tRNA(Lys3)(UUU) with a UUU anticodon, and the corresponding anticodon stem and loop domain (ASL(Lys3)(UUU)), are unable to bind to poly-A programmed ribosomes. To determine if specific anticodon domain modified nucleosides of tRNA(Lys) species would restore ribosomal binding and also affect thermal stability, we chemically synthesized ASL(Lys) heptadecamers and site-specifically incorporated the anticodon domain modified nucleosides pseudouridine (Psi(39)), 5-methylaminomethyluridine (mnm(5)U(34)) and N6-threonylcarbamoyl-adenosine (t(6)A(37)). Incorporation of t(6)A(37) and mnm(5)U(34) contributed structure to the anticodon loop, apparent by increases in DeltaS, and significantly enhanced the ability of ASL(Lys3)(UUU) to bind poly-A programmed ribosomes. Neither ASL(Lys3)(UUU)-t(6)A(37) nor ASL(Lys3)(UUU)-mnm(5)U(34) bound AAG programmed ribosomes. Only the presence of both t(6)A(37) and mnm(5)U(34) enabled ASL(Lys3)(UUU) to bind AAG programmed ribosomes, as well as increased its affinity for poly-A programmed ribosomes to the level of native Escherichia coli tRNA(Lys). The completely unmodified anticodon stem and loop of human tRNA(Lys1,2)(CUU) with a wobble position-34 C bound AAG, but did not wobble to AAA, even when the ASL was modified with t(6)A(37). The data suggest that tRNA(Lys)(UUU) species require anticodon domain modifications in the loop to impart an ordered structure to the anticodon for ribosomal binding to AAA and require a combination of modified nucleosides to bind AAG.

Formation of prismatic loops from C15 Laves phase interstitial clusters in body-centered cubic iron

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Yongfeng; Bai, Xian-Ming; Tonks, Michael R.

2015-03-01

This Letter reports the transition of C15 phase self-interstitial clusters to loops in body-centered-cubic Iron. Molecular dynamics simulations are performed to evaluate the relative stabilities of difference interstitial cluster configurations including C15 phase structure and <100> and <111>/2 loops. Within a certain size range, C15 cluster are found more stable than loops, and the relative stabilities are reversed beyond that range. In accordance to the crossover in relative stabilities, C15 clusters may grow by absorbing individual interstitials at small sizes and transitions into loops eventually. The transition takes place by nucleation and reaction of <111>/2 loop segments. These observations explainmore » the absence of C15 phase interstitial clusters predicted by density-functional-theory calculations in previous experimental observations. More importantly, the current results provide a new formation mechanism of <100> loops which requires no interaction of loops.« less

A competent catalytic active site is necessary for substrate induced dimer assembly in triosephosphate isomerase.

PubMed

Jimenez-Sandoval, Pedro; Vique-Sanchez, Jose Luis; Hidalgo, Marisol López; Velazquez-Juarez, Gilberto; Diaz-Quezada, Corina; Arroyo-Navarro, Luis Fernando; Moran, Gabriela Montero; Fattori, Juliana; Jessica Diaz-Salazar, A; Rudiño-Pinera, Enrique; Sotelo-Mundo, Rogerio; Figueira, Ana Carolina Migliorini; Lara-Gonzalez, Samuel; Benítez-Cardoza, Claudia G; Brieba, Luis G

2017-11-01

The protozoan parasite Trichomonas vaginalis contains two nearly identical triosephosphate isomerases (TvTIMs) that dissociate into stable monomers and dimerize upon substrate binding. Herein, we compare the role of the "ball and socket" and loop 3 interactions in substrate assisted dimer assembly in both TvTIMs. We found that point mutants at the "ball" are only 39 and 29-fold less catalytically active than their corresponding wild-type counterparts, whereas Δloop 3 deletions are 1502 and 9400-fold less active. Point and deletion mutants dissociate into stable monomers. However, point mutants assemble as catalytic competent dimers upon binding of the transition state substrate analog PGH, whereas loop 3 deletions remain monomeric. A comparison between crystal structures of point and loop 3 deletion monomeric mutants illustrates that the catalytic residues in point mutants and wild-type TvTIMs are maintained in the same orientation, whereas the catalytic residues in deletion mutants show an increase in thermal mobility and present structural disorder that may hamper their catalytic role. The high enzymatic activity present in monomeric point mutants correlates with the formation of dimeric TvTIMs upon substrate binding. In contrast, the low activity and lack of dimer assembly in deletion mutants suggests a role of loop 3 in promoting the formation of the active site as well as dimer assembly. Our results suggest that in TvTIMs the active site is assembled during dimerization and that the integrity of loop 3 and ball and socket residues is crucial to stabilize the dimer. Copyright © 2017 Elsevier B.V. All rights reserved.

Structure modulation of helix 69 from Escherichia coli 23S ribosomal RNA by pseudouridylations

PubMed Central

Jiang, Jun; Aduri, Raviprasad; Chow, Christine S.; SantaLucia, John

2014-01-01

Helix 69 (H69) is a 19-nt stem-loop region from the large subunit ribosomal RNA. Three pseudouridine (Ψ) modifications clustered in H69 are conserved across phylogeny and known to affect ribosome function. To explore the effects of Ψ on the conformations of Escherichia coli H69 in solution, nuclear magnetic resonance spectroscopy was used to reveal the structural differences between H69 with (ΨΨΨ) and without (UUU) Ψ modifications. Comparison of the two structures shows that H69 ΨΨΨ has the following unique features: (i) the loop region is closed by a Watson–Crick base pair between Ψ1911 and A1919, which is potentially reinforced by interactions involving Ψ1911N1H and (ii) Ψ modifications at loop residues 1915 and 1917 promote base stacking from Ψ1915 to A1918. In contrast, the H69 UUU loop region, which lacks Ψ modifications, is less organized. Structure modulation by Ψ leads to alteration in conformational behavior of the 5' half of the H69 loop region, observed as broadening of C1914 non-exchangeable base proton resonances in the H69 ΨΨΨ nuclear magnetic resonance spectra, and plays an important biological role in establishing the ribosomal intersubunit bridge B2a and mediating translational fidelity. PMID:24371282

A dihydropyridine receptor alpha1s loop region critical for skeletal muscle contraction is intrinsically unstructured and binds to a SPRY domain of the type 1 ryanodine receptor.

PubMed

Cui, Yanfang; Tae, Han-Shen; Norris, Nicole C; Karunasekara, Yamuna; Pouliquin, Pierre; Board, Philip G; Dulhunty, Angela F; Casarotto, Marco G

2009-03-01

The II-III loop of the dihydropyridine receptor (DHPR) alpha(1s) subunit is a modulator of the ryanodine receptor (RyR1) Ca(2+) release channel in vitro and is essential for skeletal muscle contraction in vivo. Despite its importance, the structure of this loop has not been reported. We have investigated its structure using a suite of NMR techniques which revealed that the DHPR II-III loop is an intrinsically unstructured protein (IUP) and as such belongs to a burgeoning structural class of functionally important proteins. The loop does not possess a stable tertiary fold: it is highly flexible, with a strong N-terminal helix followed by nascent helical/turn elements and unstructured segments. Its residual structure is loosely globular with the N and C termini in close proximity. The unstructured nature of the II-III loop may allow it to easily modify its interaction with RyR1 following a surface action potential and thus initiate rapid Ca(2+) release and contraction. The in vitro binding partner for the II-III was investigated. The II-III loop interacts with the second of three structurally distinct SPRY domains in RyR1, whose function is unknown. This interaction occurs through two preformed N-terminal alpha-helical regions and a C-terminal hydrophobic element. The A peptide corresponding to the helical N-terminal region is a common probe of RyR function and binds to the same SPRY domain as the full II-III loop. Thus the second SPRY domain is an in vitro binding site for the II-III loop. The possible in vivo role of this region is discussed.

Short loop length and high thermal stability determine genomic instability induced by G-quadruplex-forming minisatellites

PubMed Central

Piazza, Aurèle; Adrian, Michael; Samazan, Frédéric; Heddi, Brahim; Hamon, Florian; Serero, Alexandre; Lopes, Judith; Teulade-Fichou, Marie-Paule; Phan, Anh Tuân; Nicolas, Alain

2015-01-01

G-quadruplexes (G4) are polymorphic four-stranded structures formed by certain G-rich nucleic acids, with various biological roles. However, structural features dictating their formation and/or functionin vivo are unknown. InS. cerevisiae, the pathological persistency of G4 within the CEB1 minisatellite induces its rearrangement during leading-strand replication. We now show that several other G4-forming sequences remain stable. Extensive mutagenesis of the CEB25 minisatellite motif reveals that only variants with very short (≤ 4 nt) G4 loops preferentially containing pyrimidine bases trigger genomic instability. Parallel biophysical analyses demonstrate that shortening loop length does not change the monomorphic G4 structure of CEB25 variants but drastically increases its thermal stability, in correlation with thein vivo instability. Finally, bioinformatics analyses reveal that the threat for genomic stability posed by G4 bearing short pyrimidine loops is conserved inC. elegans and humans. This work provides a framework explanation for the heterogeneous instability behavior of G4-forming sequencesin vivo, highlights the importance of structure thermal stability, and questions the prevailing assumption that G4 structures with short or longer loops are as likely to formin vivo. PMID:25956747

Trajectory tracking control for underactuated stratospheric airship

NASA Astrophysics Data System (ADS)

Zheng, Zewei; Huo, Wei; Wu, Zhe

2012-10-01

Stratospheric airship is a new kind of aerospace system which has attracted worldwide developing interests for its broad application prospects. Based on the trajectory linearization control (TLC) theory, a novel trajectory tracking control method for an underactuated stratospheric airship is presented in this paper. Firstly, the TLC theory is described sketchily, and the dynamic model of the stratospheric airship is introduced with kinematics and dynamics equations. Then, the trajectory tracking control strategy is deduced in detail. The designed control system possesses a cascaded structure which consists of desired attitude calculation, position control loop and attitude control loop. Two sub-loops are designed for the position and attitude control loops, respectively, including the kinematics control loop and dynamics control loop. Stability analysis shows that the controlled closed-loop system is exponentially stable. Finally, simulation results for the stratospheric airship to track typical trajectories are illustrated to verify effectiveness of the proposed approach.

Structural dynamics of the lac repressor-DNA complex revealed by a multiscale simulation.

PubMed

Villa, Elizabeth; Balaeff, Alexander; Schulten, Klaus

2005-05-10

A multiscale simulation of a complex between the lac repressor protein (LacI) and a 107-bp-long DNA segment is reported. The complex between the repressor and two operator DNA segments is described by all-atom molecular dynamics; the size of the simulated system comprises either 226,000 or 314,000 atoms. The DNA loop connecting the operators is modeled as a continuous elastic ribbon, described mathematically by the nonlinear Kirchhoff differential equations with boundary conditions obtained from the coordinates of the terminal base pairs of each operator. The forces stemming from the looped DNA are included in the molecular dynamics simulations; the loop structure and the forces are continuously recomputed because the protein motions during the simulations shift the operators and the presumed termini of the loop. The simulations reveal the structural dynamics of the LacI-DNA complex in unprecedented detail. The multiple domains of LacI exhibit remarkable structural stability during the simulation, moving much like rigid bodies. LacI is shown to absorb the strain from the looped DNA mainly through its mobile DNA-binding head groups. Even with large fluctuating forces applied, the head groups tilt strongly and keep their grip on the operator DNA, while the remainder of the protein retains its V-shaped structure. A simulated opening of the cleft of LacI by 500-pN forces revealed the interactions responsible for locking LacI in the V-conformation.

Determinants of RNA binding and translational repression by the Bicaudal-C regulatory protein.

PubMed

Zhang, Yan; Park, Sookhee; Blaser, Susanne; Sheets, Michael D

2014-03-14

Bicaudal-C (Bic-C) RNA binding proteins function as important translational repressors in multiple biological contexts within metazoans. However, their RNA binding sites are unknown. We recently demonstrated that Bic-C functions in spatially regulated translational repression of the xCR1 mRNA during Xenopus development. This repression contributes to normal development by confining the xCR1 protein, a regulator of key signaling pathways, to specific cells of the embryo. In this report, we combined biochemical approaches with in vivo mRNA reporter assays to define the minimal Bic-C target site within the xCR1 mRNA. This 32-nucleotide Bic-C target site is predicted to fold into a stem-loop secondary structure. Mutational analyses provided evidence that this stem-loop structure is important for Bic-C binding. The Bic-C target site was sufficient for Bic-C mediated repression in vivo. Thus, we describe the first RNA binding site for a Bic-C protein. This identification provides an important step toward understanding the mechanisms by which evolutionarily conserved Bic-C proteins control cellular function in metazoans.

Mutational analysis of three predicted 5'-proximal stem-loop structures in the genome of tick-borne encephalitis virus indicates different roles in RNA replication and translation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rouha, Harald; Hoenninger, Verena M.; Thurner, Caroline

2011-08-15

Flavivirus gene expression is modulated by RNA secondary structure elements at the terminal ends of the viral RNA molecule. For tick-borne encephalitis virus (TBEV), four stem-loop (SL) elements have been predicted in the first 180 nucleotides of the viral genome: 5'-SL1, 5'-SL2, 5'-SL3 and 5'-SL4. The last three of these appear to be unique to tick-borne flaviviruses. Here, we report their characterization by mutagenesis in a TBEV luciferase reporter system. By manipulating their thermodynamic properties, we found that an optimal stability of the 5'-SL2 is required for efficient RNA replication. 5'-SL3 formation is also important for viral RNA replication, butmore » although it contains the viral start codon, its formation is dispensable for RNA translation. 5'-SL4 appears to facilitate both RNA translation and replication. Our data suggest that maintenance of the balanced thermodynamic stability of these SL elements is important for temporal regulation of its different functions.« less

Dual Stem Loops within the Poliovirus Internal Ribosomal Entry Site Control Neurovirulence

PubMed Central

Gromeier, Matthias; Bossert, Birgit; Arita, Mineo; Nomoto, Akio; Wimmer, Eckard

1999-01-01

In the human central nervous system, susceptibility to poliovirus (PV) infection is largely confined to a specific subpopulation of neuronal cells. PV tropism is likely to be determined by cell-external components such as the PV receptor CD155, as well as cell-internal constraints such as the availability of a suitable microenvironment for virus propagation. We reported previously that the exchange of the cognate internal ribosomal entry site (IRES) within the 5′ nontranslated region of PV with its counterpart from human rhinovirus type 2 (HRV2) can eliminate the neuropathogenic phenotype in a transgenic mouse model for poliomyelitis without diminishing the growth properties in HeLa cells. We now show that attenuation of neurovirulence of PV/HRV2 chimeras is not confined to CD155 transgenic mice but is evident also after intraspinal inoculation into Cynomolgus monkeys. We have dissected the PV and HRV2 IRES elements to determine those structures responsible for neurovirulence (or attenuation) of these chimeric viruses. We report that two adjacent stem loop structures within the IRES cooperatively determine neuropathogenicity. PMID:9882296

Complete mitochondrial genome of the stonefly Cryptoperla stilifera Sivec (Plecoptera: Peltoperlidae) and the phylogeny of Polyneopteran insects.

PubMed

Wu, Hai-Yan; Ji, Xiao-Yu; Yu, Wei-Wei; Du, Yu-Zhou

2014-03-10

We present the complete mitogenome of a stonefly, Cryptoperla stilifera Sivec (Plecoptera; Peltoperlidae). The mitogenome was a circular molecule consisting of 15,633 nucleotides, 37 genes and a A+T-rich region. C. stilifera mitogenome was similar to Pteronarcys princeps mitogenome (Plecoptera; Pteronarcyidae). All transfer RNA genes (tRNAs) had typical cloverleaf secondary structures except for trnSer (AGN), where the stem-loop structure of the dihydrouridine (DHU) arm was missing. The A+T-rich region of C. stilifera had two stem-loops and each had two interlink. Three conserved sequence blocks (CSBs) were present in the A+T-rich regions of C. stilifera, Peltoperla tarteri and Peltoperla arcuata. Moreover, many polynucleotide stretches (Poly N, N=A, T and C) in the A+T-rich region of C. stilifera Phylogenetic relationships of Polyneopteran species were constructed based on the nucleotide sequences of 13 protein coding genes (PCGs). Both maximum likelihood (ML) and Bayesian inference (BI) analyses supported Grylloblattodea as the sister group to Plecoptera+Dermaptera and Embiidina and Phasmatodea as sister groups. Copyright © 2014 Elsevier B.V. All rights reserved.

SELEX and SHAPE reveal that sequence motifs and an extended hairpin in the 5' portion of Turnip crinkle virus satellite RNA C mediate fitness in plants.

PubMed

Bayne, Charlie F; Widawski, Max E; Gao, Feng; Masab, Mohammed H; Chattopadhyay, Maitreyi; Murawski, Allison M; Sansevere, Robert M; Lerner, Bryan D; Castillo, Rinaldys J; Griesman, Trevor; Fu, Jiantao; Hibben, Jennifer K; Garcia-Perez, Alma D; Simon, Anne E; Kushner, David B

2018-07-01

Noncoding RNAs use their sequence and/or structure to mediate function(s). The 5' portion (166 nt) of the 356-nt noncoding satellite RNA C (satC) of Turnip crinkle virus (TCV) was previously modeled to contain a central region with two stem-loops (H6 and H7) and a large connecting hairpin (H2). We now report that in vivo functional selection (SELEX) experiments assessing sequence/structure requirements in H2, H6, and H7 reveal that H6 loop sequence motifs were recovered at nonrandom rates and only some residues are proposed to base-pair with accessible complementary sequences within the 5' central region. In vitro SHAPE of SELEX winners indicates that the central region is heavily base-paired, such that along with the lower stem and H2 region, one extensive hairpin exists composing the entire 5' region. As these SELEX winners are highly fit, these characteristics facilitate satRNA amplification in association with TCV in plants. Copyright © 2018 Elsevier Inc. All rights reserved.

The highly conserved codon following the slippery sequence supports -1 frameshift efficiency at the HIV-1 frameshift site.

PubMed

Mathew, Suneeth F; Crowe-McAuliffe, Caillan; Graves, Ryan; Cardno, Tony S; McKinney, Cushla; Poole, Elizabeth S; Tate, Warren P

2015-01-01

HIV-1 utilises -1 programmed ribosomal frameshifting to translate structural and enzymatic domains in a defined proportion required for replication. A slippery sequence, U UUU UUA, and a stem-loop are well-defined RNA features modulating -1 frameshifting in HIV-1. The GGG glycine codon immediately following the slippery sequence (the 'intercodon') contributes structurally to the start of the stem-loop but has no defined role in current models of the frameshift mechanism, as slippage is inferred to occur before the intercodon has reached the ribosomal decoding site. This GGG codon is highly conserved in natural isolates of HIV. When the natural intercodon was replaced with a stop codon two different decoding molecules-eRF1 protein or a cognate suppressor tRNA-were able to access and decode the intercodon prior to -1 frameshifting. This implies significant slippage occurs when the intercodon is in the (perhaps distorted) ribosomal A site. We accommodate the influence of the intercodon in a model of frame maintenance versus frameshifting in HIV-1.

A numerical study of the thermal stability of low-lying coronal loops

NASA Technical Reports Server (NTRS)

Klimchuk, J. A.; Antiochos, S. K.; Mariska, J. T.

1986-01-01

The nonlinear evolution of loops that are subjected to a variety of small but finite perturbations was studied. Only the low-lying loops are considered. The analysis was performed numerically using a one-dimensional hydrodynamical model developed at the Naval Research Laboratory. The computer codes solve the time-dependent equations for mass, momentum, and energy transport. The primary interest is the active region filaments, hence a geometry appropriate to those structures was considered. The static solutions were subjected to a moderate sized perturbation and allowed to evolve. The results suggest that both hot and cool loops of the geometry considered are thermally stable against amplitude perturbations of all kinds.

Detection of Naja atra Cardiotoxin Using Adenosine-Based Molecular Beacon.

PubMed

Shi, Yi-Jun; Chen, Ying-Jung; Hu, Wan-Ping; Chang, Long-Sen

2017-01-07

This study presents an adenosine (A)-based molecular beacon (MB) for selective detection of Naja atra cardiotoxin (CTX) that functions by utilizing the competitive binding between CTX and the poly(A) stem of MB to coralyne. The 5'- and 3'-end of MB were labeled with a reporter fluorophore and a non-fluorescent quencher, respectively. Coralyne induced formation of the stem-loop MB structure through A₂-coralyne-A₂ coordination, causing fluorescence signal turn-off due to fluorescence resonance energy transfer between the fluorophore and quencher. CTX3 could bind to coralyne. Moreover, CTX3 alone induced the folding of MB structure and quenching of MB fluorescence. Unlike that of snake venom α-neurotoxins, the fluorescence signal of coralyne-MB complexes produced a bell-shaped concentration-dependent curve in the presence of CTX3 and CTX isotoxins; a turn-on fluorescence signal was noted when CTX concentration was ≤80 nM, while a turn-off fluorescence signal was noted with a further increase in toxin concentrations. The fluorescence signal of coralyne-MB complexes yielded a bell-shaped curve in response to varying concentrations of N. atra crude venom but not those of Bungarus multicinctus and Protobothrops mucrosquamatus venoms. Moreover, N. nigricollis venom also functioned as N. atra venom to yield a bell-shaped concentration-dependent curve of MB fluorescence signal, again supporting that the hairpin-shaped MB could detect crude venoms containing CTXs. Taken together, our data validate that a platform composed of coralyne-induced stem-loop MB structure selectively detects CTXs.

Challenging Cognitive Construals: A Dynamic Alternative to Stable Misconceptions

ERIC Educational Resources Information Center

Gouvea, Julia S.; Simon, Matt R.

2018-01-01

In biology education research, it has been common to model cognition in terms of relatively stable knowledge structures (e.g., mental models, alternative frameworks, deeply held misconceptions). For example, John D. Coley and Kimberley D. Tanner recently proposed that many student difficulties in biology stem from underlying cognitive frameworks…

Therapeutic mechanisms of high-frequency stimulation in Parkinson's disease and neural restoration via loop-based reinforcement.

PubMed

Santaniello, Sabato; McCarthy, Michelle M; Montgomery, Erwin B; Gale, John T; Kopell, Nancy; Sarma, Sridevi V

2015-02-10

High-frequency deep brain stimulation (HFS) is clinically recognized to treat parkinsonian movement disorders, but its mechanisms remain elusive. Current hypotheses suggest that the therapeutic merit of HFS stems from increasing the regularity of the firing patterns in the basal ganglia (BG). Although this is consistent with experiments in humans and animal models of Parkinsonism, it is unclear how the pattern regularization would originate from HFS. To address this question, we built a computational model of the cortico-BG-thalamo-cortical loop in normal and parkinsonian conditions. We simulated the effects of subthalamic deep brain stimulation both proximally to the stimulation site and distally through orthodromic and antidromic mechanisms for several stimulation frequencies (20-180 Hz) and, correspondingly, we studied the evolution of the firing patterns in the loop. The model closely reproduced experimental evidence for each structure in the loop and showed that neither the proximal effects nor the distal effects individually account for the observed pattern changes, whereas the combined impact of these effects increases with the stimulation frequency and becomes significant for HFS. Perturbations evoked proximally and distally propagate along the loop, rendezvous in the striatum, and, for HFS, positively overlap (reinforcement), thus causing larger poststimulus activation and more regular patterns in striatum. Reinforcement is maximal for the clinically relevant 130-Hz stimulation and restores a more normal activity in the nuclei downstream. These results suggest that reinforcement may be pivotal to achieve pattern regularization and restore the neural activity in the nuclei downstream and may stem from frequency-selective resonant properties of the loop.

Therapeutic mechanisms of high-frequency stimulation in Parkinson’s disease and neural restoration via loop-based reinforcement

PubMed Central

Santaniello, Sabato; McCarthy, Michelle M.; Montgomery, Erwin B.; Gale, John T.; Kopell, Nancy; Sarma, Sridevi V.

2015-01-01

High-frequency deep brain stimulation (HFS) is clinically recognized to treat parkinsonian movement disorders, but its mechanisms remain elusive. Current hypotheses suggest that the therapeutic merit of HFS stems from increasing the regularity of the firing patterns in the basal ganglia (BG). Although this is consistent with experiments in humans and animal models of Parkinsonism, it is unclear how the pattern regularization would originate from HFS. To address this question, we built a computational model of the cortico-BG-thalamo-cortical loop in normal and parkinsonian conditions. We simulated the effects of subthalamic deep brain stimulation both proximally to the stimulation site and distally through orthodromic and antidromic mechanisms for several stimulation frequencies (20–180 Hz) and, correspondingly, we studied the evolution of the firing patterns in the loop. The model closely reproduced experimental evidence for each structure in the loop and showed that neither the proximal effects nor the distal effects individually account for the observed pattern changes, whereas the combined impact of these effects increases with the stimulation frequency and becomes significant for HFS. Perturbations evoked proximally and distally propagate along the loop, rendezvous in the striatum, and, for HFS, positively overlap (reinforcement), thus causing larger poststimulus activation and more regular patterns in striatum. Reinforcement is maximal for the clinically relevant 130-Hz stimulation and restores a more normal activity in the nuclei downstream. These results suggest that reinforcement may be pivotal to achieve pattern regularization and restore the neural activity in the nuclei downstream and may stem from frequency-selective resonant properties of the loop. PMID:25624501

In situ structures of the genome and genome-delivery apparatus in a single-stranded RNA virus.

PubMed

Dai, Xinghong; Li, Zhihai; Lai, Mason; Shu, Sara; Du, Yushen; Zhou, Z Hong; Sun, Ren

2017-01-05

Packaging of the genome into a protein capsid and its subsequent delivery into a host cell are two fundamental processes in the life cycle of a virus. Unlike double-stranded DNA viruses, which pump their genome into a preformed capsid, single-stranded RNA (ssRNA) viruses, such as bacteriophage MS2, co-assemble their capsid with the genome; however, the structural basis of this co-assembly is poorly understood. MS2 infects Escherichia coli via the host 'sex pilus' (F-pilus); it was the first fully sequenced organism and is a model system for studies of translational gene regulation, RNA-protein interactions, and RNA virus assembly. Its positive-sense ssRNA genome of 3,569 bases is enclosed in a capsid with one maturation protein monomer and 89 coat protein dimers arranged in a T = 3 icosahedral lattice. The maturation protein is responsible for attaching the virus to an F-pilus and delivering the viral genome into the host during infection, but how the genome is organized and delivered is not known. Here we describe the MS2 structure at 3.6 Å resolution, determined by electron-counting cryo-electron microscopy (cryoEM) and asymmetric reconstruction. We traced approximately 80% of the backbone of the viral genome, built atomic models for 16 RNA stem-loops, and identified three conserved motifs of RNA-coat protein interactions among 15 of these stem-loops with diverse sequences. The stem-loop at the 3' end of the genome interacts extensively with the maturation protein, which, with just a six-helix bundle and a six-stranded β-sheet, forms a genome-delivery apparatus and joins 89 coat protein dimers to form a capsid. This atomic description of genome-capsid interactions in a spherical ssRNA virus provides insight into genome delivery via the host sex pilus and mechanisms underlying ssRNA-capsid co-assembly, and inspires speculation about the links between nucleoprotein complexes and the origins of viruses.

The structure of high-temperature solar flare plasma in non-thermal flare models

NASA Technical Reports Server (NTRS)

Emslie, A. G.

1985-01-01

Analytic differential emission measure distributions have been derived for coronal plasma in flare loops heated both by collisions of high-energy suprathermal electrons with background plasma, and by ohmic heating by the beam-normalizing return current. For low densities, reverse current heating predominates, while for higher densities collisional heating predominates. There is thus a minimum peak temperature in an electron-heated loop. In contrast to previous approximate analyses, it is found that a stable reverse current can dominate the heating rate in a flare loop, especially in the low corona. Two 'scaling laws' are found which relate the peak temperature in the loop to the suprathermal electron flux. These laws are testable observationally and constitute a new diagnostic procedure for examining modes of energy transport in flaring loops.

Automated classification of RNA 3D motifs and the RNA 3D Motif Atlas

PubMed Central

Petrov, Anton I.; Zirbel, Craig L.; Leontis, Neocles B.

2013-01-01

The analysis of atomic-resolution RNA three-dimensional (3D) structures reveals that many internal and hairpin loops are modular, recurrent, and structured by conserved non-Watson–Crick base pairs. Structurally similar loops define RNA 3D motifs that are conserved in homologous RNA molecules, but can also occur at nonhomologous sites in diverse RNAs, and which often vary in sequence. To further our understanding of RNA motif structure and sequence variability and to provide a useful resource for structure modeling and prediction, we present a new method for automated classification of internal and hairpin loop RNA 3D motifs and a new online database called the RNA 3D Motif Atlas. To classify the motif instances, a representative set of internal and hairpin loops is automatically extracted from a nonredundant list of RNA-containing PDB files. Their structures are compared geometrically, all-against-all, using the FR3D program suite. The loops are clustered into motif groups, taking into account geometric similarity and structural annotations and making allowance for a variable number of bulged bases. The automated procedure that we have implemented identifies all hairpin and internal loop motifs previously described in the literature. All motif instances and motif groups are assigned unique and stable identifiers and are made available in the RNA 3D Motif Atlas (http://rna.bgsu.edu/motifs), which is automatically updated every four weeks. The RNA 3D Motif Atlas provides an interactive user interface for exploring motif diversity and tools for programmatic data access. PMID:23970545

Structural variations of single and tandem mismatches in RNA duplexes: a joint MD simulation and crystal structure database analysis.

PubMed

Halder, Sukanya; Bhattacharyya, Dhananjay

2012-10-04

Internal loops within RNA duplex regions are formed by single or tandem basepairing mismatches with flanking canonical Watson-Crick basepairs on both sides. They are the most common motif observed in RNA secondary structures and play integral functional and structural roles. In this report, we have studied the structural features of 1 × 1, 2 × 2, and 3 × 3 internal loops using all-atom molecular dynamics (MD) simulation technique with explicit solvent model. As MD simulation is intricately dependent on the choice of force-field and these are often rather approximate, we have used both the most popular force-fields for nucleic acids-CHARMM27 and AMBER94-for a comparative analysis. We find that tandem noncanonical basepairs forming 2 × 2 and 3 × 3 internal loops are considerably more stable than the single mismatches forming 1 × 1 internal loops, irrespective of the force field. We have also analyzed crystal structure database to study the conservation of these helical fragments in the corresponding sets of RNA structures. We observe that the nature of stability in MD simulations mimic their fluctuating natures in crystal data sets also, probably indicating reliable natures of both the force fields to reproduce experimental results. We also notice significant structural changes in the wobble G:U basepairs present in these double helical stretches, leading to a biphasic stability for these wobble pairs to release the deformational strains introduced by internal loops within duplex regions.

Importance of the perforating arteries in the proximal part of the PICA for surgical approaches to the brain stem and fourth ventricle--an anatomical study.

PubMed

Kayaci, Selim; Caglar, Yusuf Sukru; Bas, Orhan; Ozveren, Mehmet Faik

2013-10-01

The purpose of this study is to examine the perforating arteries (PAs) in the proximal part of the posterior inferior cerebellar artery (PICA) for surgical approaches to the brain stem and fourth ventricle, and to stress their importance in microsurgical procedures. Twenty-six adult cadaver obtained from routine autopsies were used. During the examination, the PAs and the segmental structure of the proximal part of the PICAs and their relation to the neighbouring anatomical structures were demonstrated. We classified the PICAs into 4 types on the basis of the distance of the middle point of the width of the caudal loop to the midline, and their presence or absence as Group A (symmetrical, anterior medullary type: 26.9%), Group B (lateral medullary type: 15.4%), Group C (asymmetrical type: 38.5%), and Group D (unilateral type: 19.2%). The number of the PAs in the tonsillomedullary segment and the caudal loop was higher than those originating from the other segments. Approaches to the medial or lateral of the PICA should be made in a way that protects the PAs (avoiding retraction of the PICA). Otherwise the PAs will be damaged and as a result brain stem ischaemia may occur, which can have serious clinical outcomes. Copyright © 2013 Elsevier B.V. All rights reserved.

Free energy profile of RNA hairpins: a molecular dynamics simulation study.

PubMed

Deng, Nan-Jie; Cieplak, Piotr

2010-02-17

RNA hairpin loops are one of the most abundant secondary structure elements and participate in RNA folding and protein-RNA recognition. To characterize the free energy surface of RNA hairpin folding at an atomic level, we calculated the potential of mean force (PMF) as a function of the end-to-end distance, by using umbrella sampling simulations in explicit solvent. Two RNA hairpins containing tetraloop cUUCGg and cUUUUg are studied with AMBER ff99 and CHARMM27 force fields. Experimentally, the UUCG hairpin is known to be significantly more stable than UUUU. In this study, the calculations using AMBER force field give a qualitatively correct description for the folding of two RNA hairpins, as the calculated PMF confirms the global stability of the folded structures and the resulting relative folding free energy is in quantitative agreement with the experimental result. The hairpin stabilities are also correctly differentiated by the more rapid molecular mechanics-Poisson Boltzmann-surface area approach, but the relative free energy estimated from this method is overestimated. The free energy profile shows that the native state basin and the unfolded state plateau are separated by a wide shoulder region, which samples a variety of native-like structures with frayed terminal basepair. The calculated PMF lacks major barriers that are expected near the transition regions, and this is attributed to the limitation of the 1-D reaction coordinate. The PMF results are compared with other studies of small RNA hairpins using kinetics method and coarse grained models. The two RNA hairpins described by CHARMM27 are significantly more deformable than those represented by AMBER. Compared with the AMBER results, the CHARMM27 calculated DeltaG(fold) for the UUUU tetraloop is in better agreement with the experimental results. However, the CHARMM27 calculation does not confirm the global stability of the experimental UUCG structure; instead, the extended conformations are predicted to be thermodynamically stable in solution. This finding is further supported by separate unrestrained CHARMM27 simulations, in which the UUCG hairpin unfolds spontaneously within 10 ns. The instability of the UUCG hairpin originates from the loop region, and propagates to the stem. The results of this study provide a molecular picture of RNA hairpin unfolding and reveal problems in the force field descriptions for the conformational energy of certain RNA hairpin. Copyright 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Molecular mechanisms for the regulation of histone mRNA stem-loop–binding protein by phosphorylation

PubMed Central

Zhang, Jun; Tan, Dazhi; DeRose, Eugene F.; Perera, Lalith; Dominski, Zbigniew; Marzluff, William F.; Tong, Liang; Hall, Traci M. Tanaka

2014-01-01

Replication-dependent histone mRNAs end with a conserved stem loop that is recognized by stem-loop–binding protein (SLBP). The minimal RNA-processing domain of SLBP is phosphorylated at an internal threonine, and Drosophila SLBP (dSLBP) also is phosphorylated at four serines in its 18-aa C-terminal tail. We show that phosphorylation of dSLBP increases RNA-binding affinity dramatically, and we use structural and biophysical analyses of dSLBP and a crystal structure of human SLBP phosphorylated on the internal threonine to understand the striking improvement in RNA binding. Together these results suggest that, although the C-terminal tail of dSLBP does not contact the RNA, phosphorylation of the tail promotes SLBP conformations competent for RNA binding and thereby appears to reduce the entropic penalty for the association. Increased negative charge in this C-terminal tail balances positively charged residues, allowing a more compact ensemble of structures in the absence of RNA. PMID:25002523

Plate with decentralised velocity feedback loops: Power absorption and kinetic energy considerations

NASA Astrophysics Data System (ADS)

Gardonio, P.; Miani, S.; Blanchini, F.; Casagrande, D.; Elliott, S. J.

2012-04-01

This paper is focused on the vibration effects produced by an array of decentralised velocity feedback loops that are evenly distributed over a rectangular thin plate to minimise its flexural response. The velocity feedback loops are formed by collocated ideal velocity sensor and point force actuator pairs, which are unconditionally stable and produce 'sky-hook' damping on the plate. The study compares how the overall flexural vibration of the plate and the local absorption of vibration power by the feedback loops vary with the control gains. The analysis is carried out both considering a typical frequency-domain formulation based on kinetic energy and structural power physical quantities, which is normally used to study vibration and noise problems, and a time-domain formulation also based on kinetic energy and structural power, which is usually implemented to investigate control problems. The time-domain formulation shows to be much more computationally efficient and robust with reference to truncation errors. Thus it has been used to perform a parametric study to assess if, and under which conditions, the minimum of the kinetic energy and the maximum of the absorbed power cost functions match with reference to: (a) the number of feedback control loops, (b) the structural damping in the plate, (c) the mutual distance of a pair of control loops and (d) the mutual gains implemented in a pair of feedback loops.

Conditions for Stabilizability of Linear Switched Systems

NASA Astrophysics Data System (ADS)

Minh, Vu Trieu

2011-06-01

This paper investigates some conditions that can provide stabilizability for linear switched systems with polytopic uncertainties via their closed loop linear quadratic state feedback regulator. The closed loop switched systems can stabilize unstable open loop systems or stable open loop systems but in which there is no solution for a common Lyapunov matrix. For continuous time switched linear systems, we show that if there exists solution in an associated Riccati equation for the closed loop systems sharing one common Lyapunov matrix, the switched linear systems are stable. For the discrete time switched systems, we derive a Linear Matrix Inequality (LMI) to calculate a common Lyapunov matrix and solution for the stable closed loop feedback systems. These closed loop linear quadratic state feedback regulators guarantee the global asymptotical stability for any switched linear systems with any switching signal sequence.

Cambium

NASA Technical Reports Server (NTRS)

Savidge, Rodney

2009-01-01

The Cambium investigation is one in a pair of investigations which utilizes the Advanced Biological Research System (ABRS). Cambium seeks definitive evidence that gravity has a direct effect on cambial cells (cells located under the inner bark where secondary growth occurs) in willow, Salix babylonica. The Cambium investigation uses willow plants flown on the International Space Station to better understand the fundamental processes by which plants produce cellulose and lignin, the two main structural materials found in plant matter. On Earth, the nature of wood within tree stems varies depending on position, and that within-tree variation includes differences in cell types and chemistry including lignin and cellulose, two major components of wood influencing wood strength, usefulness and carbon content. Reaction wood is an extreme example of such variation, and it is believed that reaction wood develops as a reaction to the influence of gravity. For the Cambium experiment, young willow plants will be launched to the ISS where their stems will be looped in an attempt to induce reaction wood formation. After on-orbit growth, the plants will be preserved and returned to Earth for analysis. Understanding the role of gravity in wood formation is expected to enable wiser management of forests for carbon sequestration as well as better utilization of trees for wood products. Detailed Research Description: The Cambium experiment will provide an understanding of physiological processes such as gene expression, metabolism and general plant development that are affected in plant systems exposed to space flight. Cambium seeks definitive evidence that gravity has a direct effect on the cambial cells (cells located under the inner bark where secondary growth occurs) that contribute to xylogenesis (reaction wood formation) in willow plants, Salix babylonica. Tension wood fibers differentiate on the upper sides of stems when the stem is altered from its normal (vertical) growth position by looping. This reaction wood response does not occur if the orientation of the plant stem with respect to gravity is not altered. If a localized zone of tension wood should be formed in looped stems under microgravity conditions, this would be the first conclusive evidence that gravity is not required. On the other hand, if a zone of tension wood is not produced in looped stems (subjected to tension on one side, compression on the other) in microgravity, this would be the first definitive evidence that gravity has a direct effect on the cambial cells which contribute to reaction wood formation. Following return to Earth the plants will be analyzed by microscopy and chemical methods.

Structural insights into translational recoding by frameshift suppressor tRNASufJ

PubMed Central

Fagan, Crystal E.; Maehigashi, Tatsuya; Dunkle, Jack A.; Miles, Stacey J.

2014-01-01

The three-nucleotide mRNA reading frame is tightly regulated during translation to ensure accurate protein expression. Translation errors that lead to aberrant protein production can result from the uncoupled movement of the tRNA in either the 5′ or 3′ direction on mRNA. Here, we report the biochemical and structural characterization of +1 frameshift suppressor tRNASufJ, a tRNA known to decode four, instead of three, nucleotides. Frameshift suppressor tRNASufJ contains an insertion 5′ to its anticodon, expanding the anticodon loop from seven to eight nucleotides. Our results indicate that the expansion of the anticodon loop of either ASLSufJ or tRNASufJ does not affect its affinity for the A site of the ribosome. Structural analyses of both ASLSufJ and ASLThr bound to the Thermus thermophilus 70S ribosome demonstrate both ASLs decode in the zero frame. Although the anticodon loop residues 34–37 are superimposable with canonical seven-nucleotide ASLs, the single C31.5 insertion between nucleotides 31 and 32 in ASLSufJ imposes a conformational change of the anticodon stem, that repositions and tilts the ASL toward the back of the A site. Further modeling analyses reveal that this tilting would cause a distortion in full-length A-site tRNASufJ during tRNA selection and possibly impede gripping of the anticodon stem by 16S rRNA nucleotides in the P site. Together, these data implicate tRNA distortion as a major driver of noncanonical translation events such as frameshifting. PMID:25352689

Structator: fast index-based search for RNA sequence-structure patterns

PubMed Central

2011-01-01

Background The secondary structure of RNA molecules is intimately related to their function and often more conserved than the sequence. Hence, the important task of searching databases for RNAs requires to match sequence-structure patterns. Unfortunately, current tools for this task have, in the best case, a running time that is only linear in the size of sequence databases. Furthermore, established index data structures for fast sequence matching, like suffix trees or arrays, cannot benefit from the complementarity constraints introduced by the secondary structure of RNAs. Results We present a novel method and readily applicable software for time efficient matching of RNA sequence-structure patterns in sequence databases. Our approach is based on affix arrays, a recently introduced index data structure, preprocessed from the target database. Affix arrays support bidirectional pattern search, which is required for efficiently handling the structural constraints of the pattern. Structural patterns like stem-loops can be matched inside out, such that the loop region is matched first and then the pairing bases on the boundaries are matched consecutively. This allows to exploit base pairing information for search space reduction and leads to an expected running time that is sublinear in the size of the sequence database. The incorporation of a new chaining approach in the search of RNA sequence-structure patterns enables the description of molecules folding into complex secondary structures with multiple ordered patterns. The chaining approach removes spurious matches from the set of intermediate results, in particular of patterns with little specificity. In benchmark experiments on the Rfam database, our method runs up to two orders of magnitude faster than previous methods. Conclusions The presented method's sublinear expected running time makes it well suited for RNA sequence-structure pattern matching in large sequence databases. RNA molecules containing several stem-loop substructures can be described by multiple sequence-structure patterns and their matches are efficiently handled by a novel chaining method. Beyond our algorithmic contributions, we provide with Structator a complete and robust open-source software solution for index-based search of RNA sequence-structure patterns. The Structator software is available at http://www.zbh.uni-hamburg.de/Structator. PMID:21619640

Small, synthetic, GC-rich mRNA stem-loop modules 5' proximal to the AUG start-codon predictably tune gene expression in yeast.

PubMed

Lamping, Erwin; Niimi, Masakazu; Cannon, Richard D

2013-07-29

A large range of genetic tools has been developed for the optimal design and regulation of complex metabolic pathways in bacteria. However, fewer tools exist in yeast that can precisely tune the expression of individual enzymes in novel metabolic pathways suitable for industrial-scale production of non-natural compounds. Tuning expression levels is critical for reducing the metabolic burden of over-expressed proteins, the accumulation of toxic intermediates, and for redirecting metabolic flux from native pathways involving essential enzymes without negatively affecting the viability of the host. We have developed a yeast membrane protein hyper-expression system with critical advantages over conventional, plasmid-based, expression systems. However, expression levels are sometimes so high that they adversely affect protein targeting/folding or the growth and/or phenotype of the host. Here we describe the use of small synthetic mRNA control modules that allowed us to predictably tune protein expression levels to any desired level. Down-regulation of expression was achieved by engineering small GC-rich mRNA stem-loops into the 5' UTR that inhibited translation initiation of the yeast ribosomal 43S preinitiation complex (PIC). Exploiting the fact that the yeast 43S PIC has great difficulty scanning through GC-rich mRNA stem-loops, we created yeast strains containing 17 different RNA stem-loop modules in the 5' UTR that expressed varying amounts of the fungal multidrug efflux pump reporter Cdr1p from Candida albicans. Increasing the length of mRNA stem-loops (that contained only GC-pairs) near the AUG start-codon led to a surprisingly large decrease in Cdr1p expression; ~2.7-fold for every additional GC-pair added to the stem, while the mRNA levels remained largely unaffected. An mRNA stem-loop of seven GC-pairs (∆G = -15.8 kcal/mol) reduced Cdr1p expression levels by >99%, and even the smallest possible stem-loop of only three GC-pairs (∆G = -4.4 kcal/mol) inhibited Cdr1p expression by ~50%. We have developed a simple cloning strategy to fine-tune protein expression levels in yeast that has many potential applications in metabolic engineering and the optimization of protein expression in yeast. This study also highlights the importance of considering the use of multiple cloning-sites carefully to preclude unwanted effects on gene expression.

Small, synthetic, GC-rich mRNA stem-loop modules 5′ proximal to the AUG start-codon predictably tune gene expression in yeast

PubMed Central

2013-01-01

Background A large range of genetic tools has been developed for the optimal design and regulation of complex metabolic pathways in bacteria. However, fewer tools exist in yeast that can precisely tune the expression of individual enzymes in novel metabolic pathways suitable for industrial-scale production of non-natural compounds. Tuning expression levels is critical for reducing the metabolic burden of over-expressed proteins, the accumulation of toxic intermediates, and for redirecting metabolic flux from native pathways involving essential enzymes without negatively affecting the viability of the host. We have developed a yeast membrane protein hyper-expression system with critical advantages over conventional, plasmid-based, expression systems. However, expression levels are sometimes so high that they adversely affect protein targeting/folding or the growth and/or phenotype of the host. Here we describe the use of small synthetic mRNA control modules that allowed us to predictably tune protein expression levels to any desired level. Down-regulation of expression was achieved by engineering small GC-rich mRNA stem-loops into the 5′ UTR that inhibited translation initiation of the yeast ribosomal 43S preinitiation complex (PIC). Results Exploiting the fact that the yeast 43S PIC has great difficulty scanning through GC-rich mRNA stem-loops, we created yeast strains containing 17 different RNA stem-loop modules in the 5′ UTR that expressed varying amounts of the fungal multidrug efflux pump reporter Cdr1p from Candida albicans. Increasing the length of mRNA stem-loops (that contained only GC-pairs) near the AUG start-codon led to a surprisingly large decrease in Cdr1p expression; ~2.7-fold for every additional GC-pair added to the stem, while the mRNA levels remained largely unaffected. An mRNA stem-loop of seven GC-pairs (∆G = −15.8 kcal/mol) reduced Cdr1p expression levels by >99%, and even the smallest possible stem-loop of only three GC-pairs (∆G = −4.4 kcal/mol) inhibited Cdr1p expression by ~50%. Conclusion We have developed a simple cloning strategy to fine-tune protein expression levels in yeast that has many potential applications in metabolic engineering and the optimization of protein expression in yeast. This study also highlights the importance of considering the use of multiple cloning-sites carefully to preclude unwanted effects on gene expression. PMID:23895661

DNA–DNA kissing complexes as a new tool for the assembly of DNA nanostructures

PubMed Central

Barth, Anna; Kobbe, Daniela; Focke, Manfred

2016-01-01

Kissing-loop annealing of nucleic acids occurs in nature in several viruses and in prokaryotic replication, among other circumstances. Nucleobases of two nucleic acid strands (loops) interact with each other, although the two strands cannot wrap around each other completely because of the adjacent double-stranded regions (stems). In this study, we exploited DNA kissing-loop interaction for nanotechnological application. We functionalized the vertices of DNA tetrahedrons with DNA stem-loop sequences. The complementary loop sequence design allowed the hybridization of different tetrahedrons via kissing-loop interaction, which might be further exploited for nanotechnology applications like cargo transport and logical elements. Importantly, we were able to manipulate the stability of those kissing-loop complexes based on the choice and concentration of cations, the temperature and the number of complementary loops per tetrahedron either at the same or at different vertices. Moreover, variations in loop sequences allowed the characterization of necessary sequences within the loop as well as additional stability control of the kissing complexes. Therefore, the properties of the presented nanostructures make them an important tool for DNA nanotechnology. PMID:26773051

Homology modeling study toward identifying structural properties in the HA2 B-loop that would influence the HA1 receptor-binding site.

PubMed

Cueno, Marni E; Imai, Kenichi; Shimizu, Kazufumi; Ochiai, Kuniyasu

2013-07-01

Influenza hemagglutinin (HA) consists of a fibrous globular stem (HA2) inserted into the viral membrane supporting a globular head (HA1). HA1 receptor-binding has been hypothesized to be structurally correlated to the HA2 B-loop, however, this was never fully understood. Here, we elucidated the structural relationship between the HA2 B-loop and the HA1 receptor-binding site (RBS). Throughout this study, we analyzed 2486 H1N1 HA homology models obtained from human, swine and avian strains during 1976-2012. Quality of all homology models were verified before further analyses. We established that amino acid residue 882 is putatively strain-conserved and differs in the human (K882), swine (H882) and avian (N882) strains. Moreover, we observed that the amino acid at residue 882 and, similarly, its orientation has the potential to influence the HA1 RBS diameter measurements which we hypothesize may consequentially affect influenza H1N1 viral infectivity, immune escape, transmissibility, and evolution. Copyright © 2013 Elsevier Inc. All rights reserved.

Additional hydrogen bonds and base-pair kinetics in the symmetrical AMP-DNA aptamer complex.

PubMed Central

Nonin-Lecomte, S; Lin, C H; Patel, D J

2001-01-01

The solution structure of an adenosine monophosphate (AMP)-DNA aptamer complex has been determined previously [Lin, C. H., and Patel, D. J. (1997) Chem. Biol. 4:817-832]. On a symmetrical aptamer complex containing the same binding loop, but with better resolved spectra, we have identified two additional hydrogen bond-mediated associations in the binding loop. One of these involves a rapidly exchanging G imino proton. The phosphate group of the AMP ligand was identified as the acceptor by comparison with other aptamer complexes. Imino proton exchange measurements also yielded the dissociation constants of the stem and binding loop base pairs. This study shows that nuclear magnetic resonance-based imino proton exchange is a good probe for detection of weak hydrogen-bond associations. PMID:11721004

TEM study of {beta} Prime precipitate interaction mechanisms with dislocations and {beta} Prime interfaces with the aluminium matrix in Al-Mg-Si alloys

DOE Office of Scientific and Technical Information (OSTI.GOV)

Teichmann, Katharina; Marioara, Calin D.; Andersen, Sigmund J.

The interaction mechanisms between dislocations and semi-coherent, needle-shaped {beta} Prime precipitates in Al-Mg-Si alloys have been studied by High Resolution Transmission Electron Microscopy (HRTEM). Dislocation loops appearing as broad contrast rings around the precipitate cross-sections were identified in the Al matrix. A size dependency of the interaction mechanism was observed; the precipitates were sheared when the longest dimension of their cross-section was shorter than approximately 15 nm, and looped otherwise. A more narrow ring located between the Al matrix and bulk {beta} Prime indicates the presence of a transition interface layer. Together with the bulk {beta} Prime structure, this wasmore » further investigated by High Angle Annular Dark Field Scanning TEM (HAADF-STEM). In the bulk {beta} Prime a higher intensity could be correlated with a third of the Si-columns, as predicted from the published structure. The transition layer incorporates Si columns in the same arrangement as in bulk {beta} Prime , although it is structurally distinct from it. The Z-contrast information and arrangement of these Si-columns demonstrate that they are an extension of the Si-network known to structurally connect all the precipitate phases in the Al-Mg-Si(-Cu) system. The width of the interface layer was estimated to about 1 nm. - Highlights: Black-Right-Pointing-Pointer {beta} Prime is found to be looped at sizes larger than 15 nm (cross section diameter). Black-Right-Pointing-Pointer {beta} Prime is found to be sheared at sizes smaller than 15 nm (cross section diameter). Black-Right-Pointing-Pointer The recently determined crystal structure of {beta} Prime is confirmed by HAADF-STEM. Black-Right-Pointing-Pointer Between {beta} Prime and the Al-matrix a transition layer of about 1 nm is existent. Black-Right-Pointing-Pointer The {beta} Prime /matrix layer is structurally distinct from bulk {beta} Prime and the aluminium matrix.« less

Interaction between the phage HK022 Nun protein and the nut RNA of phage lambda.

PubMed

Chattopadhyay, S; Hung, S C; Stuart, A C; Palmer, A G; Garcia-Mena, J; Das, A; Gottesman, M E

1995-12-19

The nun gene product of prophage HK022 excludes phage lambda infection by blocking the expression of genes downstream from the lambda nut sequence. The Nun protein functions both by competing with lambda N transcription-antitermination protein and by actively inducing transcription termination on the lambda chromosome. We demonstrate that Nun binds directly to a stem-loop structure within nut RNA, boxB, which is also the target for the N antiterminator. The two proteins show comparable affinities for boxB and they compete with each other. Their interactions with boxB are similar, as shown by RNase protection experiments, NMR spectroscopy, and analysis of boxB mutants. Each protein binds the 5' strand of the boxB stem and the adjacent loop. The stem does not melt upon the binding of Nun or N, as the 3' strand remains sensitive to a double-strand-specific RNase. The binding of RNA partially protects Nun from proteolysis and changes its NMR spectra. Evidently, although Nun and N bind to the same surface of boxB RNA, their respective complexes interact differently with RNA polymerase, inducing transcription termination or antitermination, respectively.

An RNA internal loop acts as a hinge to facilitate ribozyme folding and catalysis.

PubMed Central

Szewczak, A A; Cech, T R

1997-01-01

RNA molecules commonly consist of helical regions separated by internal loops, and in many cases these internal loops have been found to assume stable structures. We have examined the function and dynamics of an internal loop, J5/5a, that joins the two halves of the P4-P6 domain of the Tetrahymena self-splicing group I intron. P4-P6 RNAs with mutations in the J5/5a region showed nondenaturing gel electrophoretic mobilities and levels of Fe(II)-EDTA cleavage protection intermediate between those of wild-type RNA and a mutant incapable of folding into the native P4-P6 tertiary structure. Mutants with the least structured J5/5a loops behaved the most like wild-type P4-P6, and required smaller amounts of Mg2+ to rescue folding. The activity of reconstituted introns containing mutant P4-P6 RNAs correlated similarly with the nature of the J5/5a mutation. Our results suggest that, in solution, the P4-P6 RNA is in a two-state equilibrium between folded and unfolded states. We conclude that this internal loop mainly acts as a flexible hinge, allowing the coaxially stacked helical regions on either side of it to interact via specific tertiary contacts. To a lesser extent, the specific bases within the loop contribute to folding. Furthermore, it is crucial that the junction remain unstructured in the unfolded state. These conclusions cannot be derived from a simple examination of the P4-P6 crystal structure (Cate JH et al., 1996, Science 273:1678-1685), showing once again that structure determination must be supplemented with mutational and thermodynamic analysis to provide a complete picture of a folded macromolecule. PMID:9257643

In silico Analysis of HIV-1 Env-gp120 Reveals Structural Bases for Viral Adaptation in Growth-Restrictive Cells

PubMed Central

Yokoyama, Masaru; Nomaguchi, Masako; Doi, Naoya; Kanda, Tadahito; Adachi, Akio; Sato, Hironori

2016-01-01

Variable V1/V2 and V3 loops on human immunodeficiency virus type 1 (HIV-1) envelope-gp120 core play key roles in modulating viral competence to recognize two infection receptors, CD4 and chemokine-receptors. However, molecular bases for the modulation largely remain unclear. To address these issues, we constructed structural models for a full-length gp120 in CD4-free and -bound states. The models showed topologies of gp120 surface loop that agree with those in reported structural data. Molecular dynamics simulation showed that in the unliganded state, V1/V2 loop settled into a thermodynamically stable arrangement near V3 loop for conformational masking of V3 tip, a potent neutralization epitope. In the CD4-bound state, however, V1/V2 loop was rearranged near the bound CD4 to support CD4 binding. In parallel, cell-based adaptation in the absence of anti-viral antibody pressures led to the identification of amino acid substitutions that individually enhance viral entry and growth efficiencies in association with reduced sensitivity to CCR5 antagonist TAK-779. Notably, all these substitutions were positioned on the receptors binding surfaces in V1/V2 or V3 loop. In silico structural studies predicted some physical changes of gp120 by substitutions with alterations in viral replication phenotypes. These data suggest that V1/V2 loop is critical for creating a gp120 structure that masks co-receptor binding site compatible with maintenance of viral infectivity, and for tuning a functional balance of gp120 between immune escape ability and infectivity to optimize HIV-1 replication fitness. PMID:26903989

Toxin MqsR Cleaves Single-Stranded mRNA with Various 5 Ends

DTIC Science & Technology

2016-08-24

either protein ORIGINAL RESEARCH Toxin MqsR cleaves single- stranded mRNA with various 5’ ends Nityananda Chowdhury1,*, Brian W. Kwan1,*, Louise C...in which a single 5′- GCU site was predicted to be single- stranded (ssRNA), double- stranded (dsRNA), in the loop of a stem - loop (slRNA), or in a...single- stranded 5′- GCU sites since cleavage was approximately 20- fold higher than cleavage seen with the 5′- GCU site in the stem - loop and

Investigation of RNA Hairpin Loop Folding with Time-Resolved Infrared Spectroscopy

NASA Astrophysics Data System (ADS)

Stancik, Aaron Lee

Ribonucleic acids (RNAs) are a group of functional biopolymers central to the molecular underpinnings of life. To complete the many processes they mediate, RNAs must fold into precise three-dimensional structures. Hairpin loops are the most ubiquitous and basic structural elements present in all folded RNAs, and are the foundation upon which all complex tertiary structures are built. A hairpin loop forms when a single stranded RNA molecule folds back on itself creating a helical stem of paired bases capped by a loop. This work investigates the formation of UNCG hairpin loops with the sequence 5'-GC(UNCG)GC-3' (N = A, U, G, or C) using both equilibrium infrared (IR) and time-resolved IR spectroscopy. Equilibrium IR melting data were used to determine thermodynamic parameters. Melting temperatures ranged from 50 to 60°C, and enthalpies of unfolding were on the order of 100 kJ/mol. In the time-resolved work, temperature jumps of up to 20°C at 2.5°C increments were obtained with transient relaxation kinetics spanning nanoseconds to hundreds of microseconds. The relaxation kinetics for all of the oligomers studied were fit to first or second order exponentials. Multiple vibrational transitions were probed on each oligomer for fully folded and partially denatured structures. In the time-resolved limit, in contrast to equilibrium melting, RNA does not fold according to two-state behavior. These results are some of the first to show that RNA hairpins fold according to a rugged energy landscape, which contradicts their relatively simple nature. In addition, this work has proven that time-resolved IR spectroscopy is a powerful and novel tool for investigating the earliest events of RNA folding, the formation of the hairpin loop.

Adaptive Sliding Mode Control of Dynamic Systems Using Double Loop Recurrent Neural Network Structure.

PubMed

Fei, Juntao; Lu, Cheng

2018-04-01

In this paper, an adaptive sliding mode control system using a double loop recurrent neural network (DLRNN) structure is proposed for a class of nonlinear dynamic systems. A new three-layer RNN is proposed to approximate unknown dynamics with two different kinds of feedback loops where the firing weights and output signal calculated in the last step are stored and used as the feedback signals in each feedback loop. Since the new structure has combined the advantages of internal feedback NN and external feedback NN, it can acquire the internal state information while the output signal is also captured, thus the new designed DLRNN can achieve better approximation performance compared with the regular NNs without feedback loops or the regular RNNs with a single feedback loop. The new proposed DLRNN structure is employed in an equivalent controller to approximate the unknown nonlinear system dynamics, and the parameters of the DLRNN are updated online by adaptive laws to get favorable approximation performance. To investigate the effectiveness of the proposed controller, the designed adaptive sliding mode controller with the DLRNN is applied to a -axis microelectromechanical system gyroscope to control the vibrating dynamics of the proof mass. Simulation results demonstrate that the proposed methodology can achieve good tracking property, and the comparisons of the approximation performance between radial basis function NN, RNN, and DLRNN show that the DLRNN can accurately estimate the unknown dynamics with a fast speed while the internal states of DLRNN are more stable.

Robust Translation of the Nucleoid Protein Fis Requires a Remote Upstream AU Element and Is Enhanced by RNA Secondary Structure

PubMed Central

Nafissi, Maryam; Chau, Jeannette; Xu, Jimin

2012-01-01

Synthesis of the Fis nucleoid protein rapidly increases in response to nutrient upshifts, and Fis is one of the most abundant DNA binding proteins in Escherichia coli under nutrient-rich growth conditions. Previous work has shown that control of Fis synthesis occurs at transcription initiation of the dusB-fis operon. We show here that while translation of the dihydrouridine synthase gene dusB is low, unusual mechanisms operate to enable robust translation of fis. At least two RNA sequence elements located within the dusB coding region are responsible for high fis translation. The most important is an AU element centered 35 nucleotides (nt) upstream of the fis AUG, which may function as a binding site for ribosomal protein S1. In addition, a 44-nt segment located upstream of the AU element and predicted to form a stem-loop secondary structure plays a prominent role in enhancing fis translation. On the other hand, mutations close to the AUG, including over a potential Shine-Dalgarno sequence, have little effect on Fis protein levels. The AU element and stem-loop regions are phylogenetically conserved within dusB-fis operons of representative enteric bacteria. PMID:22389479

Rewriting nature's assembly manual for a ssRNA virus.

PubMed

Patel, Nikesh; Wroblewski, Emma; Leonov, German; Phillips, Simon E V; Tuma, Roman; Twarock, Reidun; Stockley, Peter G

2017-11-14

Satellite tobacco necrosis virus (STNV) is one of the smallest viruses known. Its genome encodes only its coat protein (CP) subunit, relying on the polymerase of its helper virus TNV for replication. The genome has been shown to contain a cryptic set of dispersed assembly signals in the form of stem-loops that each present a minimal CP-binding motif AXXA in the loops. The genomic fragment encompassing nucleotides 1-127 is predicted to contain five such packaging signals (PSs). We have used mutagenesis to determine the critical assembly features in this region. These include the CP-binding motif, the relative placement of PS stem-loops, their number, and their folding propensity. CP binding has an electrostatic contribution, but assembly nucleation is dominated by the recognition of the folded PSs in the RNA fragment. Mutation to remove all AXXA motifs in PSs throughout the genome yields an RNA that is unable to assemble efficiently. In contrast, when a synthetic 127-nt fragment encompassing improved PSs is swapped onto the RNA otherwise lacking CP recognition motifs, assembly is partially restored, although the virus-like particles created are incomplete, implying that PSs outside this region are required for correct assembly. Swapping this improved region into the wild-type STNV1 sequence results in a better assembly substrate than the viral RNA, producing complete capsids and outcompeting the wild-type genome in head-to-head competition. These data confirm details of the PS-mediated assembly mechanism for STNV and identify an efficient approach for production of stable virus-like particles encapsidating nonnative RNAs or other cargoes. Copyright © 2017 the Author(s). Published by PNAS.

RNA binding specificity of Ebola virus transcription factor VP30.

PubMed

Schlereth, Julia; Grünweller, Arnold; Biedenkopf, Nadine; Becker, Stephan; Hartmann, Roland K

2016-09-01

The transcription factor VP30 of the non-segmented RNA negative strand Ebola virus balances viral transcription and replication. Here, we comprehensively studied RNA binding by VP30. Using a novel VP30:RNA electrophoretic mobility shift assay, we tested truncated variants of 2 potential natural RNA substrates of VP30 - the genomic Ebola viral 3'-leader region and its complementary antigenomic counterpart (each ∼155 nt in length) - and a series of other non-viral RNAs. Based on oligonucleotide interference, the major VP30 binding region on the genomic 3'-leader substrate was assigned to the internal expanded single-stranded region (∼ nt 125-80). Best binding to VP30 was obtained with ssRNAs of optimally ∼ 40 nt and mixed base composition; underrepresentation of purines or pyrimidines was tolerated, but homopolymeric sequences impaired binding. A stem-loop structure, particularly at the 3'-end or positioned internally, supports stable binding to VP30. In contrast, dsRNA or RNAs exposing large internal loops flanked by entirely helical arms on both sides are not bound. Introduction of a 5´-Cap(0) structure impaired VP30 binding. Also, ssDNAs bind substantially weaker than isosequential ssRNAs and heparin competes with RNA for binding to VP30, indicating that ribose 2'-hydroxyls and electrostatic contacts of the phosphate groups contribute to the formation of VP30:RNA complexes. Our results indicate a rather relaxed RNA binding specificity of filoviral VP30, which largely differs from that of the functionally related transcription factor of the Paramyxoviridae which binds to ssRNAs as short as 13 nt with a preference for oligo(A) sequences.

A controller design approach for large flexible space structures

NASA Technical Reports Server (NTRS)

Joshi, S. M.

1981-01-01

A controller design approach for large space structures is presented, which consists of a primary attitude controller and a secondary or damping enhancement controller. The secondary controller, which uses several Annular Momentum Control Device (AMCD's), is shown to make the closed loop system asymptotically stable under relatively simple conditions. The primary controller using torque actuators (or AMCD's) and colocated attitude and rate sensors is shown to be stable. It is shown that the same AMCD's can be used for simultaneous actuation of primary and secondary controllers. Numerical results are obtained for a large, thin, completely free plate model.

Folding of a transcriptionally acting PreQ1 riboswitch

PubMed Central

Rieder, Ulrike; Kreutz, Christoph; Micura, Ronald

2010-01-01

7-Aminomethyl-7-deazaguanine (preQ1) sensitive mRNA domains belong to the smallest riboswitches known to date. Although recent efforts have revealed the three-dimensional architecture of the ligand–aptamer complex less is known about the molecular details of the ligand-induced response mechanism that modulates gene expression. We present an in vitro investigation on the ligand-induced folding process of the preQ1 responsive RNA element from Fusobacterium nucleatum using biophysical methods, including fluorescence and NMR spectroscopy of site-specifically labeled riboswitch variants. We provide evidence that the full-length riboswitch domain adopts two different coexisting stem-loop structures in the expression platform. Upon addition of preQ1, the equilibrium of the competing hairpins is significantly shifted. This system therefore, represents a finely tunable antiterminator/terminator interplay that impacts the in vivo cellular response mechanism. A model is presented how a riboswitch that provides no obvious overlap between aptamer and terminator stem-loop solves this communication problem by involving bistable sequence determinants. PMID:20534493

Method and apparatus for controlling fluid flow

DOEpatents

Miller, J.R.

1980-06-27

A method and apparatus for precisely controlling the rate (and hence amount) of fluid flow are given. The controlled flow rate is finely adjustable, can be extremely small (on the order of microliter-atmospheres per second), can be adjusted to zero (flow stopped), and is stable to better than 1% with time. The dead volume of the valve can be made arbitrarily small, in fact essentially zero. The valve employs no wearing mechanical parts (including springs, stems, or seals). The valve is finely adjustable, has a flow rate dynamic range of many decades, can be made compatible with any fluid, and is suitable for incorporation into an open or closed loop servo-control system.

Structural insights into translational recoding by frameshift suppressor tRNA SufJ

DOE PAGES

Fagan, Crystal E.; Maehigashi, Tatsuya; Dunkle, Jack A.; ...

2014-10-28

The three-nucleotide mRNA reading frame is tightly regulated during translation to ensure accurate protein expression. Translation errors that lead to aberrant protein production can result from the uncoupled movement of the tRNA in either the 5' or 3' direction on mRNA. Here, we report the biochemical and structural characterization of +1 frameshift suppressor tRNA SufJ, a tRNA known to decode four, instead of three, nucleotides. Frameshift suppressor tRNA SufJ contains an insertion 5' to its anticodon, expanding the anticodon loop from seven to eight nucleotides. Our results indicate that the expansion of the anticodon loop of either ASL SufJ ormore » tRNA SufJ does not affect its affinity for the A site of the ribosome. Structural analyses of both ASL SufJ and ASL Thr bound to the Thermus thermophilus 70S ribosome demonstrate both ASLs decode in the zero frame. Although the anticodon loop residues 34–37 are superimposable with canonical seven-nucleotide ASLs, the single C31.5 insertion between nucleotides 31 and 32 in ASL SufJ imposes a conformational change of the anticodon stem, that repositions and tilts the ASL toward the back of the A site. Further modeling analyses reveal that this tilting would cause a distortion in full-length A-site tRNA SufJ during tRNA selection and possibly impede gripping of the anticodon stem by 16S rRNA nucleotides in the P site. Together, these data implicate tRNA distortion as a major driver of noncanonical translation events such as frameshifting.« less

A structural model for apolipoprotein C-II amyloid fibrils: experimental characterization and molecular dynamics simulations.

PubMed

Teoh, Chai Lean; Pham, Chi L L; Todorova, Nevena; Hung, Andrew; Lincoln, Craig N; Lees, Emma; Lam, Yuen Han; Binger, Katrina J; Thomson, Neil H; Radford, Sheena E; Smith, Trevor A; Müller, Shirley A; Engel, Andreas; Griffin, Michael D W; Yarovsky, Irene; Gooley, Paul R; Howlett, Geoffrey J

2011-02-04

The self-assembly of specific proteins to form insoluble amyloid fibrils is a characteristic feature of a number of age-related and debilitating diseases. Lipid-free human apolipoprotein C-II (apoC-II) forms characteristic amyloid fibrils and is one of several apolipoproteins that accumulate in amyloid deposits located within atherosclerotic plaques. X-ray diffraction analysis of aligned apoC-II fibrils indicated a simple cross-β-structure composed of two parallel β-sheets. Examination of apoC-II fibrils using transmission electron microscopy, scanning transmission electron microscopy, and atomic force microscopy indicated that the fibrils are flat ribbons composed of one apoC-II molecule per 4.7-Å rise of the cross-β-structure. Cross-linking results using single-cysteine substitution mutants are consistent with a parallel in-register structural model for apoC-II fibrils. Fluorescence resonance energy transfer analysis of apoC-II fibrils labeled with specific fluorophores provided distance constraints for selected donor-acceptor pairs located within the fibrils. These findings were used to develop a simple 'letter-G-like' β-strand-loop-β-strand model for apoC-II fibrils. Fully solvated all-atom molecular dynamics (MD) simulations showed that the model contained a stable cross-β-core with a flexible connecting loop devoid of persistent secondary structure. The time course of the MD simulations revealed that charge clusters in the fibril rearrange to minimize the effects of same-charge interactions inherent in parallel in-register models. Our structural model for apoC-II fibrils suggests that apoC-II monomers fold and self-assemble to form a stable cross-β-scaffold containing relatively unstructured connecting loops. Copyright © 2010 Elsevier Ltd. All rights reserved.

A molecular dynamics study of loop fluctuation in human papillomavirus type 16 virus-like particles: a possible indicator of immunogenicity.

PubMed

Joshi, Harshad; Cheluvaraja, Srinath; Somogyi, Endre; Brown, Darron R; Ortoleva, Peter

2011-11-28

Immunogenicity varies between the human papillomavirus (HPV) L1 monomer assemblies of various sizes (e.g., monomers, pentamers or whole capsids). The hypothesis that this can be attributed to the intensity of fluctuations of important loops containing neutralizing epitopes for the various assemblies is proposed for HPV L1 assemblies. Molecular dynamics simulations were utilized to begin testing this hypothesis. Fluctuations of loops that contain critical neutralizing epitopes (especially FG loop) were quantified via root-mean-square fluctuation and features in the frequency spectrum of dynamic changes in loop conformation. If this fluctuation-immunogenicity hypothesis is a universal aspect of immunogenicity (i.e., immune system recognition of an epitope within a loop is more reliable when it is presented via a more stable delivery structure), then fluctuation measures can serve as one predictor of immunogenicity as part of a computer-aided vaccine design strategy. Copyright © 2011 Elsevier Ltd. All rights reserved.

Topological and trivial magnetic oscillations in nodal loop semimetals

NASA Astrophysics Data System (ADS)

Oroszlány, László; Dóra, Balázs; Cserti, József; Cortijo, Alberto

2018-05-01

Nodal loop semimetals are close descendants of Weyl semimetals and possess a topologically dressed band structure. We argue by combining the conventional theory of magnetic oscillation with topological arguments that nodal loop semimetals host coexisting topological and trivial magnetic oscillations. These originate from mapping the topological properties of the extremal Fermi surface cross sections onto the physics of two dimensional semi-Dirac systems, stemming from merging two massless Dirac cones. By tuning the chemical potential and the direction of magnetic field, a sharp transition is identified from purely trivial oscillations, arising from the Landau levels of a normal two dimensional (2D) electron gas, to a phase where oscillations of topological and trivial origin coexist, originating from 2D massless Dirac and semi-Dirac points, respectively. These could in principle be directly identified in current experiments.

The Nematode Eukaryotic Translation Initiation Factor 4E/G Complex Works with a trans-Spliced Leader Stem-Loop To Enable Efficient Translation of Trimethylguanosine-Capped RNAs ▿ †

PubMed Central

Wallace, Adam; Filbin, Megan E.; Veo, Bethany; McFarland, Craig; Stepinski, Janusz; Jankowska-Anyszka, Marzena; Darzynkiewicz, Edward; Davis, Richard E.

2010-01-01

Eukaryotic mRNA translation begins with recruitment of the 40S ribosome complex to the mRNA 5′ end through the eIF4F initiation complex binding to the 5′ m7G-mRNA cap. Spliced leader (SL) RNA trans splicing adds a trimethylguanosine (TMG) cap and a sequence, the SL, to the 5′ end of mRNAs. Efficient translation of TMG-capped mRNAs in nematodes requires the SL sequence. Here we define a core set of nucleotides and a stem-loop within the 22-nucleotide nematode SL that stimulate translation of mRNAs with a TMG cap. The structure and core nucleotides are conserved in other nematode SLs and correspond to regions of SL1 required for early Caenorhabditis elegans development. These SL elements do not facilitate translation of m7G-capped RNAs in nematodes or TMG-capped mRNAs in mammalian or plant translation systems. Similar stem-loop structures in phylogenetically diverse SLs are predicted. We show that the nematode eukaryotic translation initiation factor 4E/G (eIF4E/G) complex enables efficient translation of the TMG-SL RNAs in diverse in vitro translation systems. TMG-capped mRNA translation is determined by eIF4E/G interaction with the cap and the SL RNA, although the SL does not increase the affinity of eIF4E/G for capped RNA. These results suggest that the mRNA 5′ untranslated region (UTR) can play a positive and novel role in translation initiation through interaction with the eIF4E/G complex in nematodes and raise the issue of whether eIF4E/G-RNA interactions play a role in the translation of other eukaryotic mRNAs. PMID:20154140

The nematode eukaryotic translation initiation factor 4E/G complex works with a trans-spliced leader stem-loop to enable efficient translation of trimethylguanosine-capped RNAs.

PubMed

Wallace, Adam; Filbin, Megan E; Veo, Bethany; McFarland, Craig; Stepinski, Janusz; Jankowska-Anyszka, Marzena; Darzynkiewicz, Edward; Davis, Richard E

2010-04-01

Eukaryotic mRNA translation begins with recruitment of the 40S ribosome complex to the mRNA 5' end through the eIF4F initiation complex binding to the 5' m(7)G-mRNA cap. Spliced leader (SL) RNA trans splicing adds a trimethylguanosine (TMG) cap and a sequence, the SL, to the 5' end of mRNAs. Efficient translation of TMG-capped mRNAs in nematodes requires the SL sequence. Here we define a core set of nucleotides and a stem-loop within the 22-nucleotide nematode SL that stimulate translation of mRNAs with a TMG cap. The structure and core nucleotides are conserved in other nematode SLs and correspond to regions of SL1 required for early Caenorhabditis elegans development. These SL elements do not facilitate translation of m(7)G-capped RNAs in nematodes or TMG-capped mRNAs in mammalian or plant translation systems. Similar stem-loop structures in phylogenetically diverse SLs are predicted. We show that the nematode eukaryotic translation initiation factor 4E/G (eIF4E/G) complex enables efficient translation of the TMG-SL RNAs in diverse in vitro translation systems. TMG-capped mRNA translation is determined by eIF4E/G interaction with the cap and the SL RNA, although the SL does not increase the affinity of eIF4E/G for capped RNA. These results suggest that the mRNA 5' untranslated region (UTR) can play a positive and novel role in translation initiation through interaction with the eIF4E/G complex in nematodes and raise the issue of whether eIF4E/G-RNA interactions play a role in the translation of other eukaryotic mRNAs.

Learning from ISS-modular adaptive NN control of nonlinear strict-feedback systems.

PubMed

Wang, Cong; Wang, Min; Liu, Tengfei; Hill, David J

2012-10-01

This paper studies learning from adaptive neural control (ANC) for a class of nonlinear strict-feedback systems with unknown affine terms. To achieve the purpose of learning, a simple input-to-state stability (ISS) modular ANC method is first presented to ensure the boundedness of all the signals in the closed-loop system and the convergence of tracking errors in finite time. Subsequently, it is proven that learning with the proposed stable ISS-modular ANC can be achieved. The cascade structure and unknown affine terms of the considered systems make it very difficult to achieve learning using existing methods. To overcome these difficulties, the stable closed-loop system in the control process is decomposed into a series of linear time-varying (LTV) perturbed subsystems with the appropriate state transformation. Using a recursive design, the partial persistent excitation condition for the radial basis function neural network (NN) is established, which guarantees exponential stability of LTV perturbed subsystems. Consequently, accurate approximation of the closed-loop system dynamics is achieved in a local region along recurrent orbits of closed-loop signals, and learning is implemented during a closed-loop feedback control process. The learned knowledge is reused to achieve stability and an improved performance, thereby avoiding the tremendous repeated training process of NNs. Simulation studies are given to demonstrate the effectiveness of the proposed method.

A diverse and intricate signalling network regulates stem cell fate in the shoot apical meristem.

PubMed

Dodsworth, Steven

2009-12-01

At the shoot apex of plants is a small region known as the shoot apical meristem (SAM) that maintains a population of undifferentiated (stem) cells whilst providing cells for developing lateral organs and the stem. All aerial structures of the plant develop from the SAM post-embryogenesis, enabling plants to grow in a characteristic modular fashion with great phenotypic and developmental plasticity throughout their lifetime. The maintenance of the stem cell population is intimately balanced with cell recruitment into differentiating tissues through intercellular communication involving a complex signalling network. Recent studies have shown that diverse regulators function in SAM maintenance, many of which converge on the WUSCHEL (WUS) gene. In this review the diverse regulatory modules that function in SAM maintenance are discussed: transcriptional and epigenetic control, hormonal regulation, and the balance with organogenesis. The central role of WUS as an integrator of multiple signals is highlighted; in addition, accessory feedback loops emerge as a feature enabling dynamic regulation of the stem cell niche.

Mobile Element Evolution Playing Jigsaw—SINEs in Gastropod and Bivalve Mollusks

PubMed Central

Matetovici, Irina; Sajgo, Szilard; Ianc, Bianca; Ochis, Cornelia; Bulzu, Paul; Popescu, Octavian; Damert, Annette

2016-01-01

SINEs (Short INterspersed Elements) are widely distributed among eukaryotes. Some SINE families are organized in superfamilies characterized by a shared central domain. These central domains are conserved across species, classes, and even phyla. Here we report the identification of two novel such superfamilies in the genomes of gastropod and bivalve mollusks. The central conserved domain of the first superfamily is present in SINEs in Caenogastropoda and Vetigastropoda as well as in all four subclasses of Bivalvia. We designated the domain MESC (Romanian for MElc—snail and SCoica—mussel) because it appears to be restricted to snails and mussels. The second superfamily is restricted to Caenogastropoda. Its central conserved domain—Snail—is related to the Nin-DC domain. Furthermore, we provide evidence that a 40-bp subdomain of the SINE V-domain is conserved in SINEs in mollusks and arthropods. It is predicted to form a stable stem-loop structure that is preserved in the context of the overall SINE RNA secondary structure in invertebrates. Our analysis also recovered short retrotransposons with a Long INterspersed Element (LINE)-derived 5′ end. These share the body and/or the tail with transfer RNA (tRNA)-derived SINEs within and across species. Finally, we identified CORE SINEs in gastropods and bivalves—extending the distribution range of this superfamily. PMID:26739168

Predicting loop–helix tertiary structural contacts in RNA pseudoknots

PubMed Central

Cao, Song; Giedroc, David P.; Chen, Shi-Jie

2010-01-01

Tertiary interactions between loops and helical stems play critical roles in the biological function of many RNA pseudoknots. However, quantitative predictions for RNA tertiary interactions remain elusive. Here we report a statistical mechanical model for the prediction of noncanonical loop–stem base-pairing interactions in RNA pseudoknots. Central to the model is the evaluation of the conformational entropy for the pseudoknotted folds with defined loop–stem tertiary structural contacts. We develop an RNA virtual bond-based conformational model (Vfold model), which permits a rigorous computation of the conformational entropy for a given fold that contains loop–stem tertiary contacts. With the entropy parameters predicted from the Vfold model and the energy parameters for the tertiary contacts as inserted parameters, we can then predict the RNA folding thermodynamics, from which we can extract the tertiary contact thermodynamic parameters from theory–experimental comparisons. These comparisons reveal a contact enthalpy (ΔH) of −14 kcal/mol and a contact entropy (ΔS) of −38 cal/mol/K for a protonated C+•(G–C) base triple at pH 7.0, and (ΔH = −7 kcal/mol, ΔS = −19 cal/mol/K) for an unprotonated base triple. Tests of the model for a series of pseudoknots show good theory–experiment agreement. Based on the extracted energy parameters for the tertiary structural contacts, the model enables predictions for the structure, stability, and folding pathways for RNA pseudoknots with known or postulated loop–stem tertiary contacts from the nucleotide sequence alone. PMID:20100813

Molecular dynamics studies of the 3D structure and planar ligand binding of a quadruplex dimer.

PubMed

Li, Ming-Hui; Luo, Quan; Xue, Xiang-Gui; Li, Ze-Sheng

2011-03-01

G-rich sequences can fold into a four-stranded structure called a G-quadruplex, and sequences with short loops are able to aggregate to form stable quadruplex multimers. Few studies have characterized the properties of this variety of quadruplex multimers. Using molecular modeling and molecular dynamics simulations, the present study investigated a dimeric G-quadruplex structure formed from a simple sequence of d(GGGTGGGTGGGTGGGT) (G1), and its interactions with a planar ligand of a perylene derivative (Tel03). A series of analytical methods, including free energy calculations and principal components analysis (PCA), was used. The results show that a dimer structure with stacked parallel monomer structures is maintained well during the entire simulation. Tel03 can bind to the dimer efficiently through end stacking, and the binding mode of the ligand stacked with the 3'-terminal thymine base is most favorable. PCA showed that the dominant motions in the free dimer occur on the loop regions, and the presence of the ligand reduces the flexibility of the loops. Our investigation will assist in understanding the geometric structure of stacked G-quadruplex multimers and may be helpful as a platform for rational drug design.

Detection of HbsAg and hATIII genetically modified goats (Caprahircus) by loop-mediated isothermal amplification.

PubMed

Tao, Chenyu; Zhang, Qingde; Zhai, Shanli; Liu, Bang

2013-11-01

In this study, sensitive and rapid detection systems were designed using a loop-mediated isothermal amplification (LAMP) method to detect the genetically modified goats. A set of 4 primers were designed for each exogenous nucleic acids HBsAg and hATIII. The DNA samples were first amplified with the outer and inner primers and released a single-stranded DNA,of which both ends were stem-loop structure. Then one inner primer hybridized with the loop, and initiated displacement synthesis in less than 1 h. The result could be visualized by both agarose gel electrophoresis and unaided eyes directly after adding SYBR GREEN 1. The detection limit of LAMP was ten copies of target molecules, indicating that LAMP was tenfold more sensitive than the classical PCR. Furthermore, all the samples of genetically modified goats were tested positively by LAMP, and the results demonstrated that the LAMP was a rapid and sensitive method for detecting the genetically modified organism.

Shoot development in grapevine (Vitis vinifera) is affected by the modular branching pattern of the stem and intra- and inter-shoot trophic competition.

PubMed

Lebon, Eric; Pellegrino, Anne; Tardieu, Francois; Lecoeur, Jeremie

2004-03-01

Shoot architecture variability in grapevine (Vitis vinifera) was analysed using a generic modelling approach based on thermal time developed for annual herbaceous species. The analysis of shoot architecture was based on various levels of shoot organization, including pre-existing and newly formed parts of the stem, and on the modular structure of the stem, which consists of a repeated succession of three phytomers (P0-P1-P2). Four experiments were carried out using the cultivar 'Grenache N': two on potted vines (one of which was carried out in a glasshouse) and two on mature vines in a vineyard. These experiments resulted in a broad diversity of environmental conditions, but none of the plants experienced soil water deficit. Development of the main axis was highly dependent on air temperature, being linearly related to thermal time for all stages of leaf development from budbreak to veraison. The stable progression of developmental stages along the main stem resulted in a thermal-time based programme of leaf development. Leaf expansion rate varied with trophic competition (shoot and cluster loads) and environmental conditions (solar radiation, VPD), accounting for differences in final leaf area. Branching pattern was highly variable. Classification of the branches according to ternary modular structure increased the accuracy of the quantitative analysis of branch development. The rate and duration of leaf production were higher for branches derived from P0 phytomers than for branches derived from P1 or P2 phytomers. Rates of leaf production, expressed as a -function of thermal time, were not stable and depended on trophic competition and environmental conditions such as solar radiation or VPD. The application to grapevine of a generic model developed in annual plants made it possible to identify constants in main stem development and to determine the hierarchical structure of branches with respect to the modular structure of the stem in response to intra- and inter-shoot trophic competition.

A molecular mechanism of P-loop pliability of Rho-kinase investigated by molecular dynamic simulation

NASA Astrophysics Data System (ADS)

Gohda, Keigo; Hakoshima, Toshio

2008-11-01

Rho-kinase is a leading player in the regulation of cytoskeletal events involving smooth muscle contraction and neurite growth-cone collapse and retraction, and is a promising drug target in the treatment of both vascular and neurological disorders. Recent crystal structure of Rho-kinase complexed with a small-molecule inhibitor fasudil has revealed structural details of the ATP-binding site, which represents the target site for the inhibitor, and showed that the conserved phenylalanine on the P-loop occupies the pocket, resulting in an increase of protein-ligand contacts. Thus, the P-loop pliability is considered to play an important role in inhibitor binding affinity and specificity. In this study, we carried out a molecular dynamic simulation for Rho-kinase-fasudil complexes with two different P-loop conformations, i.e., the extended and folded conformations, in order to understand the P-loop pliability and dynamics at atomic level. A PKA-fasudil complex was also used for comparison. In the MD simulation, the flip-flop movement of the P-loop conformation starting either from the extended or folded conformation was not able to be observed. However, a significant conformational change in a long loop region covering over the P-loop, and also alteration of ionic interaction-manner of fasudil with acidic residues in the ATP binding site were shown only in the Rho-kinase-fasudil complex with the extended P-loop conformation, while Rho-kinase with the folded P-loop conformation and PKA complexes did not show large fluctuations, suggesting that the Rho-kinase-fasudil complex with the extended P-loop conformation represents a meta-stable state. The information of the P-loop pliability at atomic level obtained in this study could provide valuable clues to designing potent and/or selective inhibitors for Rho-kinase.

A cyclometalated iridium(III) complex used as a conductor for the electrochemical sensing of IFN-γ

NASA Astrophysics Data System (ADS)

Miao, Xiangmin; Ko, Chung-Nga; Vellaisamy, Kasipandi; Li, Zongbing; Yang, Guanjun; Leung, Chung-Hang; Ma, Dik-Lung

2017-02-01

A novel iridium(III) complex was prepared and used as a conductor for sensitive and enzyme-free electrochemical detection of interferon gamma (IFN-γ). This assay is based on a dual signal amplification mechanism involving positively charged gold nanoparticles ((+)AuNPs) and hybridization chain reaction (HCR). To construct the sensor, nafion (Nf) and (+)AuNPs composite membrane was first immobilized onto the electrode surface. Subsequently, a loop-stem structured capture probe (CP) containing a special IFN-γ interact strand was modified onto the (+)AuNP surface via the formation of Au-S bonds. Upon addition of IFN-γ, the loop-stem structure of CP was opened, and the newly exposed “sticky” region of CP then hybridized with DNA hairpin-1 (H1), which in turn opened its hairpin structure for hybridizing with DNA hairpin-2 (H2). Happen of HCR between H1 and H2 thus generated a polymeric duplex DNA (dsDNA) chain. Meanwhile, the iridium(III) complex could interact with the grooves of the dsDNA polymer, producing a strong current signal that was proportional to IFN-γ concentration. Thus, sensitive detection of IFN-γ could be realized with a detection limit down to 16.3 fM. Moreover, satisfied results were achieved by using this method for the detection of IFN-γ in human serum samples.

Coherent random lasing controlled by Brownian motion of the active scatterer

NASA Astrophysics Data System (ADS)

Liang, Shuofeng; Yin, Leicheng; Zhang, ZhenZhen; Xia, Jiangying; Xie, Kang; Zou, Gang; Hu, Zhijia; Zhang, Qijin

2018-05-01

The stability of the scattering loop is fundamental for coherent random lasing in a dynamic scattering system. In this work, fluorescence of DPP (N, N-di [3-(isobutyl polyhedral oligomeric silsesquioxanes) propyl] perylene diimide) is scattered to produce RL and we realize the transition from incoherent RL to coherent RL by controlling the Brownian motion of the scatterers (dimer aggregates of DPP) and the stability of scattering loop. To produce coherent random lasers, the loop needs to maintain a stable state within the loop-stable time, which can be determined through controlled Brownian motion of scatterers in the scattering system. The result shows that the loop-stable time is within 5.83 × 10‑5 s to 1.61 × 10‑4 s based on the transition from coherent to incoherent random lasing. The time range could be tuned by finely controlling the viscosity of the solution. This work not only develops a method to predict the loop-stable time, but also develops the study between Brownian motion and random lasers, which opens the road to a variety of novel interdisciplinary investigations involving modern statistical mechanics and disordered photonics.

The Size of the Internal Loop in DNA Hairpins Influences Their Targeting with Partially Complementary Strands

PubMed Central

2015-01-01

Targeting of noncanonical DNA structures, such as hairpin loops, may have significant diagnostic and therapeutic potential. Oligonucleotides can be used for binding to mRNA, forming a DNA/RNA hybrid duplex that inhibits translation. This kind of modulation of gene expression is called the antisense approach. In order to determine the best strategy to target a common structural motif in mRNA, we have designed a set of stem-loop DNA molecules with sequence: d(GCGCTnGTAAT5GTTACTnGCGC), where n = 1, 3, or 5, “T5” is an end loop of five thymines. We used a combination of calorimetric and spectroscopy techniques to determine the thermodynamics for the reaction of a set of hairpins containing internal loops with their respective partially complementary strands. Our aim was to determine if internal- and end-loops are promising regions for targeting with their corresponding complementary strands. Indeed, all targeting reactions were accompanied by negative changes in free energy, indicating that reactions proceed spontaneously. Further investigation showed that these negative free energy terms result from a net balance of unfavorable entropy and favorable enthalpy contributions. In particular, unfolding of hairpins and duplexes is accompanied by positive changes in heat capacity, which may be a result of exposure of hydrophobic groups to the solvent. This study provides a new method for the targeting of mRNA in order to control gene expression. PMID:25486129

The Ndc80 internal loop is required for recruitment of the Ska complex to establish end-on microtubule attachment to kinetochores.

PubMed

Zhang, Gang; Kelstrup, Christian D; Hu, Xiao-Wen; Kaas Hansen, Mathilde J; Singleton, Martin R; Olsen, Jesper V; Nilsson, Jakob

2012-07-01

The Ndc80 complex establishes end-on attachment of kinetochores to microtubules, which is essential for chromosome segregation. The Ndc80 subunit is characterized by an N-terminal region that binds directly to microtubules, and a long coiled-coil region that interacts with Nuf2. A loop region in Ndc80 that generates a kink in the structure disrupts the long coiled-coil region but the exact function of this loop, has until now, not been clear. Here we show that this loop region is essential for end-on attachment of kinetochores to microtubules in human cells. Cells expressing loop mutants of Ndc80 are unable to align the chromosomes, and stable kinetochore fibers are absent. Through quantitative mass spectrometry and immunofluorescence we found that the binding of the spindle and kinetochore associated (Ska) complex depends on the loop region, explaining why end-on attachment is defective. This underscores the importance of the Ndc80 loop region in coordinating chromosome segregation through the recruitment of specific proteins to the kinetochore.

Meta-stable magnetic transitions and its field dependence in Co2.75Fe0.25O4 ferrite

NASA Astrophysics Data System (ADS)

Aswathi M., C.; Bhowmik, R. N.

2018-04-01

The Co2.75Fe0.25O4 ferrite has been prepared by chemical co-precipitation route. The as-prepared sample has been annealed at 500° C. X-ray diffraction pattern indicated cubic spinel structure in the sample. The sample showed ferrimagnetic nature with magnetic irreversibility and hysteresis loop. Magnetization data revealed high anisotropic nature and at least two prominent meta-stable magnetic transitions below the highest measurement temperature 350 K.

An sRNA and Cold Shock Protein Homolog-Based Feedforward Loop Post-transcriptionally Controls Cell Cycle Master Regulator CtrA

PubMed Central

Robledo, Marta; Schlüter, Jan-Philip; Loehr, Lars O.; Linne, Uwe; Albaum, Stefan P.; Jiménez-Zurdo, José I.; Becker, Anke

2018-01-01

Adjustment of cell cycle progression is crucial for bacterial survival and adaptation under adverse conditions. However, the understanding of modulation of cell cycle control in response to environmental changes is rather incomplete. In α-proteobacteria, the broadly conserved cell cycle master regulator CtrA underlies multiple levels of control, including coupling of cell cycle and cell differentiation. CtrA levels are known to be tightly controlled through diverse transcriptional and post-translational mechanisms. Here, small RNA (sRNA)-mediated post-transcriptional regulation is uncovered as an additional level of CtrA fine-tuning. Computational predictions as well as transcriptome and proteome studies consistently suggested targeting of ctrA and the putative cold shock chaperone cspA5 mRNAs by the trans-encoded sRNA (trans-sRNA) GspR (formerly SmelC775) in several Sinorhizobium species. GspR strongly accumulated in the stationary growth phase, especially in minimal medium (MM) cultures. Lack of the gspR locus confers a fitness disadvantage in competition with the wild type, while its overproduction hampers cell growth, suggesting that this riboregulator interferes with cell cycle progression. An eGFP-based reporter in vivo assay, involving wild-type and mutant sRNA and mRNA pairs, experimentally confirmed GspR-dependent post-transcriptional down-regulation of ctrA and cspA5 expression, which most likely occurs through base-pairing to the respective mRNA. The energetically favored secondary structure of GspR is predicted to comprise three stem-loop domains, with stem-loop 1 and stem-loop 3 targeting ctrA and cspA5 mRNA, respectively. Moreover, this work reports evidence for post-transcriptional control of ctrA by CspA5. Thus, this regulation and GspR-mediated post-transcriptional repression of ctrA and cspA5 expression constitute a coherent feed-forward loop, which may enhance the negative effect of GspR on CtrA levels. This novel regulatory circuit involving the riboregulator GspR, CtrA, and a cold shock chaperone may contribute to fine-tuning of ctrA expression. PMID:29740411

An sRNA and Cold Shock Protein Homolog-Based Feedforward Loop Post-transcriptionally Controls Cell Cycle Master Regulator CtrA.

PubMed

Robledo, Marta; Schlüter, Jan-Philip; Loehr, Lars O; Linne, Uwe; Albaum, Stefan P; Jiménez-Zurdo, José I; Becker, Anke

2018-01-01

Adjustment of cell cycle progression is crucial for bacterial survival and adaptation under adverse conditions. However, the understanding of modulation of cell cycle control in response to environmental changes is rather incomplete. In α-proteobacteria, the broadly conserved cell cycle master regulator CtrA underlies multiple levels of control, including coupling of cell cycle and cell differentiation. CtrA levels are known to be tightly controlled through diverse transcriptional and post-translational mechanisms. Here, small RNA (sRNA)-mediated post-transcriptional regulation is uncovered as an additional level of CtrA fine-tuning. Computational predictions as well as transcriptome and proteome studies consistently suggested targeting of ctrA and the putative cold shock chaperone cspA5 mRNAs by the trans- encoded sRNA ( trans- sRNA) GspR (formerly SmelC775) in several Sinorhizobium species. GspR strongly accumulated in the stationary growth phase, especially in minimal medium (MM) cultures. Lack of the gspR locus confers a fitness disadvantage in competition with the wild type, while its overproduction hampers cell growth, suggesting that this riboregulator interferes with cell cycle progression. An eGFP-based reporter in vivo assay, involving wild-type and mutant sRNA and mRNA pairs, experimentally confirmed GspR-dependent post-transcriptional down-regulation of ctrA and cspA5 expression, which most likely occurs through base-pairing to the respective mRNA. The energetically favored secondary structure of GspR is predicted to comprise three stem-loop domains, with stem-loop 1 and stem-loop 3 targeting ctrA and cspA5 mRNA, respectively. Moreover, this work reports evidence for post-transcriptional control of ctrA by CspA5. Thus, this regulation and GspR-mediated post-transcriptional repression of ctrA and cspA5 expression constitute a coherent feed-forward loop, which may enhance the negative effect of GspR on CtrA levels. This novel regulatory circuit involving the riboregulator GspR, CtrA, and a cold shock chaperone may contribute to fine-tuning of ctrA expression.

A feedback regulatory loop involving microRNA-9 and nuclear receptor TLX in neural stem cell fate determination.

PubMed

Zhao, Chunnian; Sun, GuoQiang; Li, Shengxiu; Shi, Yanhong

2009-04-01

MicroRNAs have been implicated as having important roles in stem cell biology. MicroRNA-9 (miR-9) is expressed specifically in neurogenic areas of the brain and may be involved in neural stem cell self-renewal and differentiation. We showed previously that the nuclear receptor TLX is an essential regulator of neural stem cell self-renewal. Here we show that miR-9 suppresses TLX expression to negatively regulate neural stem cell proliferation and accelerate neural differentiation. Introducing a TLX expression vector that is not prone to miR-9 regulation rescued miR-9-induced proliferation deficiency and inhibited precocious differentiation. In utero electroporation of miR-9 in embryonic brains led to premature differentiation and outward migration of the transfected neural stem cells. Moreover, TLX represses expression of the miR-9 pri-miRNA. By forming a negative regulatory loop with TLX, miR-9 provides a model for controlling the balance between neural stem cell proliferation and differentiation.

A feedback regulatory loop involving microRNA-9 and nuclear receptor TLX in neural stem cell fate determination

PubMed Central

Zhao, Chunnian; Sun, GuoQiang; Li, Shengxiu; Shi, Yanhong

2009-01-01

Summary MicroRNAs are important players in stem cell biology. Among them, microRNA-9 (miR-9) is expressed specifically in neurogenic areas of the brain. Whether miR-9 plays a role in neural stem cell self-renewal and differentiation is unknown. We showed previously that nuclear receptor TLX is an essential regulator of neural stem cell self-renewal. Here we show that miR-9 suppresses TLX expression to negatively regulate neural stem cell proliferation and accelerate neural differentiation. Introducing a TLX expression vector lacking the miR-9 recognition site rescued miR-9-induced proliferation deficiency and inhibited precocious differentiation. In utero electroporation of miR-9 in embryonic brains led to premature differentiation and outward migration of the transfected neural stem cells. Moreover, TLX represses miR-9 pri-miRNA expression. MiR-9, by forming a negative regulatory loop with TLX, establishes a model for controlling the balance between neural stem cell proliferation and differentiation. PMID:19330006

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pallan, Pradeep S.; Marshall, William S.; Harp, Joel

To understand the role of structural elements of RNA pseudoknots in controlling the extent of -1-type ribosomal frameshifting, we determined the crystal structure of a high-efficiency frameshifting mutant of the pseudoknot from potato leaf roll virus (PLRV). Correlations of the structure with available in vitro frameshifting data for PLRV pseudoknot mutants implicate sequence and length of a stem-loop linker as modulators of frameshifting efficiency. Although the sequences and overall structures of the RNA pseudoknots from PLRV and beet western yellow virus (BWYV) are similar, nucleotide deletions in the linker and adjacent minor groove loop abolish frameshifting only with the latter.more » Conversely, mutant PLRV pseudoknots with up to four nucleotides deleted in this region exhibit nearly wild-type frameshifting efficiencies. The crystal structure helps rationalize the different tolerances for deletions in the PLRV and BWYV RNAs, and we have used it to build a three-dimensional model of the PRLV pseudoknot with a four-nucleotide deletion. The resulting structure defines a minimal RNA pseudoknot motif composed of 22 nucleotides capable of stimulating -1-type ribosomal frameshifts.« less

Crystal structure of a polyhistidine-tagged recombinant catalytic subunit of cAMP-dependent protein kinase complexed with the peptide inhibitor PKI(5-24) and adenosine.

PubMed

Narayana, N; Cox, S; Shaltiel, S; Taylor, S S; Xuong, N

1997-04-15

The crystal structure of the hexahistidine-tagged mouse recombinant catalytic subunit (H6-rC) of cAMP-dependent protein kinase (cAPK), complexed with a 20-residue peptide inhibitor from the heat-stable protein kinase inhibitor PKI(5-24) and adenosine, was determined at 2.2 A resolution. Novel crystallization conditions were required to grow the ternary complex crystals. The structure was refined to a final crystallographic R-factor of 18.2% with good stereochemical parameters. The "active" enzyme adopts a "closed" conformation as found in rC:PKI(5-24) [Knighton et al. (1991a,b) Science 253, 407-414, 414-420] and packs in a similar manner with the peptide providing a major contact surface. This structure clearly defines the subsites of the unique nucleotide binding site found in the protein kinase family. The adenosine occupies a mostly hydrophobic pocket at the base of the cleft between the two lobes and is completely buried. The missing triphosphate moiety of ATP is filled with a water molecule (Wtr 415) which replaces the gamma-phosphate of ATP. The glycine-rich loop between beta1 and beta2 helps to anchor the phosphates while the ribose ring is buried beneath beta-strand 2. Another ordered water molecule (Wtr 375) is pentacoordinated with polar atoms from adenosine, Leu 49 in beta-strand 1, Glu 127 in the linker strand between the two lobes, Tyr 330, and a third water molecule, Wtr 359. The conserved nucleotide fold can be defined as a lid comprised of beta-strand 1, the glycine-rich loop, and beta-strand 2. The adenine ring is buried beneath beta-strand 1 and the linker strand (120-127) that joins the small and large lobes. The C-terminal tail containing Tyr 330, a segment that lies outside the conserved core, covers this fold and anchors it in a closed conformation. The main-chain atoms of the flexible glycine-rich loop (residues 50-55) in the ATP binding domain have a mean B-factor of 41.4 A2. This loop is quite mobile, in striking contrast to the other conserved loops that converge at the active site cleft. The catalytic loop (residues 166-171) and the Mg2+ positioning loop (residues 184-186) are a stable part of the large lobe and have low B-factors in all structures solved to date. The stability of the glycine-rich loop is highly dependent on the ligands that occupy the active site cleft with maximum stability achieved in the ternary complex containing Mg x ATP and the peptide inhibitor. In this ternary complex the gamma-phosphate is secured between both lobes by hydrogen bonds to the backbone amide of Ser 53 in the glycine-rich loop and the amino group of Lys 168 in the catalytic loop. In the adenosine ternary complex the water molecule replacing the gamma-phosphate hydrogen bonds between Lys 168 and Asp 166 and makes no contact with the small lobe. This glycine-rich loop is thus the most mobile component of the active site cleft, with the tip of the loop being highly sensitive to what occupies the gamma-subsite.

Contribution of the mu loop to the structure and function of rat glutathione transferase M1-1

PubMed Central

Hearne, Jennifer L.; Colman, Roberta F.

2006-01-01

The “mu loop,” an 11-residue loop spanning amino acid residues 33–43, is a characteristic structural feature of the mu class of glutathione transferases. To assess the contribution of the mu loop to the structure and function of rat GST M1-1, amino acid residues 35–44 (35GDAPDYDRSQ44) were excised by deletion mutagenesis, resulting in the “Deletion Enzyme.” Kinetic studies reveal that the Km values of the Deletion Enzyme are markedly increased compared with those of the wild-type enzyme: 32-fold for 1-chloro-2,4-dinitrobenzene, 99-fold for glutathione, and 880-fold for monobromobimane, while the Vmax value for each substrate is increased only modestly. Results from experiments probing the structure of the Deletion Enzyme, in comparison with that of the wild-type enzyme, suggest that the secondary and quaternary structures have not been appreciably perturbed. Thermostability studies indicate that the Deletion Enzyme is as stable as the wild-type enzyme at 4°C and 10°C, but it rapidly loses activity at 25°C, unlike the wild-type enzyme. In the temperature range of 4°C through 25°C, the loss of activity of the Deletion Enzyme is not the result of a change in its structure, as determined by circular dichroism spectroscopy and sedimentation equilibrium centrifugation. Collectively, these results indicate that the mu loop is not essential for GST M1-1 to maintain its structure nor is it required for the enzyme to retain some catalytic activity. However, it is an important determinant of the enzyme's affinity for its substrates. PMID:16672236

cry1Aa lacks stability elements at its 5'-UTR but integrity of its transcription terminator is critical to prevent decay of its transcript.

PubMed

Ramírez-Prado, Jorge Humberto; Martínez-Márquez, Eva Isabel; Olmedo-Alvarez, Gabriela

2006-07-01

We analyzed the influence of the 5' and 3' untranslated regions of the Bacillus thuringiensis cry1Aa on its mRNA stability. Although the cry1Aa gene has a stable transcript (8 min), its 5' UTR did not provide stability to the reporter gene uidA. Stability of cry1Aa could be increased to 40 min by addition of an SP82 stability element at the 5' UTR, suggesting that once the 5' and 3' ends were protected initiation of decay could be effectively blocked. We generated mutations in the transcription terminator and found that changes that reduced the stability of the stem, a larger loop, or elimination of the U-trail sharply decreased the half-life of the transcript. Therefore, unlike some stable bacterial transcripts, cry1Aa lacks special features at the end 5' to prevent decay, but its terminator is the main determinant of its stability.

Highly sensitive self-complementary DNA nanoswitches triggered by polyelectrolytes.

PubMed

Wu, Jincai; Yu, Feng; Zhang, Zheng; Chen, Yong; Du, Jie; Maruyama, Atsushi

2016-01-07

Dimerization of two homologous strands of genomic DNA/RNA is an essential feature of retroviral replication. Herein we show that a cationic comb-type copolymer (CCC), poly(L-lysine)-graft-dextran, accelerates the dimerization of self-complementary stem-loop DNA, frequently found in functional DNA/RNA molecules, such as aptamers. Furthermore, an anionic polymer poly(sodium vinylsulfonate) (PVS) dissociates CCC from the duplex shortly within a few seconds. Then single stem-loop DNA spontaneously transforms from its dimer. Thus we can easily control the dimer and stem-loop DNA by switching on/off CCC activity. Both polyelectrolytes and DNA concentrations are in the nanomole per liter range. The polyelectrolyte-assisted transconformation and sequences design strategy ensures the reversible state control with rapid response and effective switching under physiologically relevant conditions. A further application of this sensitive assembly is to construct an aptamer-type drug delivery system, bind or release functional molecules responding to its transconformation.

Stem-Loop RNA Hairpins in Giant Viruses: Invading rRNA-Like Repeats and a Template Free RNA

PubMed Central

Seligmann, Hervé; Raoult, Didier

2018-01-01

We examine the hypothesis that de novo template-free RNAs still form spontaneously, as they did at the origins of life, invade modern genomes, contribute new genetic material. Previously, analyses of RNA secondary structures suggested that some RNAs resembling ancestral (t)RNAs formed recently de novo, other parasitic sequences cluster with rRNAs. Here positive control analyses of additional RNA secondary structures confirm ancestral and de novo statuses of RNA grouped according to secondary structure. Viroids with branched stems resemble de novo RNAs, rod-shaped viroids resemble rRNA secondary structures, independently of GC contents. 5′ UTR leading regions of West Nile and Dengue flavivirid viruses resemble de novo and rRNA structures, respectively. An RNA homologous with Megavirus, Dengue and West Nile genomes, copperhead snake microsatellites and levant cotton repeats, not templated by Mimivirus' genome, persists throughout Mimivirus' infection. Its secondary structure clusters with candidate de novo RNAs. The saltatory phyletic distribution and secondary structure of Mimivirus' peculiar RNA suggest occasional template-free polymerization of this sequence, rather than noncanonical transcriptions (swinger polymerization, posttranscriptional editing). PMID:29449833

End-to-end crosstalk within the hepatitis C virus genome mediates the conformational switch of the 3′X-tail region

PubMed Central

Romero-López, Cristina; Barroso-delJesus, Alicia; García-Sacristán, Ana; Briones, Carlos; Berzal-Herranz, Alfredo

2014-01-01

The hepatitis C virus (HCV) RNA genome contains multiple structurally conserved domains that make long-distance RNA–RNA contacts important in the establishment of viral infection. Microarray antisense oligonucelotide assays, improved dimethyl sulfate probing methods and 2′ acylation chemistry (selective 2’-hydroxyl acylation and primer extension, SHAPE) showed the folding of the genomic RNA 3′ end to be regulated by the internal ribosome entry site (IRES) element via direct RNA–RNA interactions. The essential cis-acting replicating element (CRE) and the 3′X-tail region adopted different 3D conformations in the presence and absence of the genomic RNA 5′ terminus. Further, the structural transition in the 3′X-tail from the replication-competent conformer (consisting of three stem-loops) to the dimerizable form (with two stem-loops), was found to depend on the presence of both the IRES and the CRE elements. Complex interplay between the IRES, the CRE and the 3′X-tail region would therefore appear to occur. The preservation of this RNA–RNA interacting network, and the maintenance of the proper balance between different contacts, may play a crucial role in the switch between different steps of the HCV cycle. PMID:24049069

Functional Loop Dynamics of the Streptavidin-Biotin Complex

PubMed Central

Song, Jianing; Li, Yongle; Ji, Changge; Zhang, John Z. H.

2015-01-01

Accelerated molecular dynamics (aMD) simulation is employed to study the functional dynamics of the flexible loop3-4 in the strong-binding streptavidin-biotin complex system. Conventional molecular (cMD) simulation is also performed for comparison. The present study reveals the following important properties of the loop dynamics: (1) The transition of loop3-4 from open to closed state is observed in 200 ns aMD simulation. (2) In the absence of biotin binding, the open-state streptavidin is more stable, which is consistent with experimental evidences. The free energy (ΔG) difference is about 5 kcal/mol between two states. But with biotin binding, the closed state is more stable due to electrostatic and hydrophobic interactions between the loop3-4 and biotin. (3) The closure of loop3-4 is concerted to the stable binding of biotin to streptavidin. When the loop3-4 is in its open-state, biotin moves out of the binding pocket, indicating that the interactions between the loop3-4 and biotin are essential in trapping biotin in the binding pocket. (4) In the tetrameric streptavidin system, the conformational change of the loop3-4 in each monomer is independent of each other. That is, there is no cooperative binding for biotin bound to the four subunits of the tetramer. PMID:25601277

Molecular recognition of pyr mRNA by the Bacillus subtilis attenuation regulatory protein PyrR

PubMed Central

Bonner, Eric R.; D’Elia, John N.; Billips, Benjamin K.; Switzer, Robert L.

2001-01-01

The pyrimidine nucleotide biosynthesis (pyr) operon in Bacillus subtilis is regulated by transcriptional attenuation. The PyrR protein binds in a uridine nucleotide-dependent manner to three attenuation sites at the 5′-end of pyr mRNA. PyrR binds an RNA-binding loop, allowing a terminator hairpin to form and repressing the downstream genes. The binding of PyrR to defined RNA molecules was characterized by a gel mobility shift assay. Titration indicated that PyrR binds RNA in an equimolar ratio. PyrR bound more tightly to the binding loops from the second (BL2 RNA) and third (BL3 RNA) attenuation sites than to the binding loop from the first (BL1 RNA) attenuation site. PyrR bound BL2 RNA 4–5-fold tighter in the presence of saturating UMP or UDP and 150- fold tighter with saturating UTP, suggesting that UTP is the more important co-regulator. The minimal RNA that bound tightly to PyrR was 28 nt long. Thirty-one structural variants of BL2 RNA were tested for PyrR binding affinity. Two highly conserved regions of the RNA, the terminal loop and top of the upper stem and a purine-rich internal bulge and the base pairs below it, were crucial for tight binding. Conserved elements of RNA secondary structure were also required for tight binding. PyrR protected conserved areas of the binding loop in hydroxyl radical footprinting experiments. PyrR likely recognizes conserved RNA sequences, but only if they are properly positioned in the correct secondary structure. PMID:11726695

Long-range tertiary interactions in single hammerhead ribozymes bias motional sampling toward catalytically active conformations

PubMed Central

McDowell, S. Elizabeth; Jun, Jesse M.; Walter, Nils G.

2010-01-01

Enzymes generally are thought to derive their functional activity from conformational motions. The limited chemical variation in RNA suggests that such structural dynamics may play a particularly important role in RNA function. Minimal hammerhead ribozymes are known to cleave efficiently only in ∼10-fold higher than physiologic concentrations of Mg2+ ions. Extended versions containing native loop–loop interactions, however, show greatly enhanced catalytic activity at physiologically relevant Mg2+ concentrations, for reasons that are still ill-understood. Here, we use Mg2+ titrations, activity assays, ensemble, and single molecule fluorescence resonance energy transfer (FRET) approaches, combined with molecular dynamics (MD) simulations, to ask what influence the spatially distant tertiary loop–loop interactions of an extended hammerhead ribozyme have on its structural dynamics. By comparing hammerhead variants with wild-type, partially disrupted, and fully disrupted loop–loop interaction sequences we find that the tertiary interactions lead to a dynamic motional sampling that increasingly populates catalytically active conformations. At the global level the wild-type tertiary interactions lead to more frequent, if transient, encounters of the loop-carrying stems, whereas at the local level they lead to an enrichment in favorable in-line attack angles at the cleavage site. These results invoke a linkage between RNA structural dynamics and function and suggest that loop–loop interactions in extended hammerhead ribozymes—and Mg2+ ions that bind to minimal ribozymes—may generally allow more frequent access to a catalytically relevant conformation(s), rather than simply locking the ribozyme into a single active state. PMID:20921269

Sensitivity of bandpass filters using recirculating delay-line structures

NASA Astrophysics Data System (ADS)

Heyde, Eric C.

1996-12-01

Recirculating delay lines have value notably as sensors and optical signal processors. Most useful applications depend on a high-finesse response from a network. A proof that, with given response parameters, more complex systems can produce behavior that is more stable to the effects of nonidealities than a single recirculating loop is presented.

Complete sequence and analysis of the mitochondrial genome of Hemiselmis andersenii CCMP644 (Cryptophyceae).

PubMed

Kim, Eunsoo; Lane, Christopher E; Curtis, Bruce A; Kozera, Catherine; Bowman, Sharen; Archibald, John M

2008-05-12

Cryptophytes are an enigmatic group of unicellular eukaryotes with plastids derived by secondary (i.e., eukaryote-eukaryote) endosymbiosis. Cryptophytes are unusual in that they possess four genomes-a host cell-derived nuclear and mitochondrial genome and an endosymbiont-derived plastid and 'nucleomorph' genome. The evolutionary origins of the host and endosymbiont components of cryptophyte algae are at present poorly understood. Thus far, a single complete mitochondrial genome sequence has been determined for the cryptophyte Rhodomonas salina. Here, the second complete mitochondrial genome of the cryptophyte alga Hemiselmis andersenii CCMP644 is presented. The H. andersenii mtDNA is 60,553 bp in size and encodes 30 structural RNAs and 36 protein-coding genes, all located on the same strand. A prominent feature of the genome is the presence of a approximately 20 Kbp long intergenic region comprised of numerous tandem and dispersed repeat units of between 22-336 bp. Adjacent to these repeats are 27 copies of palindromic sequences predicted to form stable DNA stem-loop structures. One such stem-loop is located near a GC-rich and GC-poor region and may have a regulatory function in replication or transcription. The H. andersenii mtDNA shares a number of features in common with the genome of the cryptophyte Rhodomonas salina, including general architecture, gene content, and the presence of a large repeat region. However, the H. andersenii mtDNA is devoid of inverted repeats and introns, which are present in R. salina. Comparative analyses of the suite of tRNAs encoded in the two genomes reveal that the H. andersenii mtDNA has lost or converted its original trnK(uuu) gene and possesses a trnS-derived 'trnK(uuu)', which appears unable to produce a functional tRNA. Mitochondrial protein coding gene phylogenies strongly support a variety of previously established eukaryotic groups, but fail to resolve the relationships among higher-order eukaryotic lineages. Comparison of the H. andersenii and R. salina mitochondrial genomes reveals a number of cryptophyte-specific genomic features, most notably the presence of a large repeat-rich intergenic region. However, unlike R. salina, the H. andersenii mtDNA does not possess introns and lacks a Lys-tRNA, which is presumably imported from the cytosol.

Complete Sequence and Analysis of the Mitochondrial Genome of Hemiselmis andersenii CCMP644 (Cryptophyceae)

PubMed Central

Kim, Eunsoo; Lane, Christopher E; Curtis, Bruce A; Kozera, Catherine; Bowman, Sharen; Archibald, John M

2008-01-01

Background Cryptophytes are an enigmatic group of unicellular eukaryotes with plastids derived by secondary (i.e., eukaryote-eukaryote) endosymbiosis. Cryptophytes are unusual in that they possess four genomes–a host cell-derived nuclear and mitochondrial genome and an endosymbiont-derived plastid and 'nucleomorph' genome. The evolutionary origins of the host and endosymbiont components of cryptophyte algae are at present poorly understood. Thus far, a single complete mitochondrial genome sequence has been determined for the cryptophyte Rhodomonas salina. Here, the second complete mitochondrial genome of the cryptophyte alga Hemiselmis andersenii CCMP644 is presented. Results The H. andersenii mtDNA is 60,553 bp in size and encodes 30 structural RNAs and 36 protein-coding genes, all located on the same strand. A prominent feature of the genome is the presence of a ~20 Kbp long intergenic region comprised of numerous tandem and dispersed repeat units of between 22–336 bp. Adjacent to these repeats are 27 copies of palindromic sequences predicted to form stable DNA stem-loop structures. One such stem-loop is located near a GC-rich and GC-poor region and may have a regulatory function in replication or transcription. The H. andersenii mtDNA shares a number of features in common with the genome of the cryptophyte Rhodomonas salina, including general architecture, gene content, and the presence of a large repeat region. However, the H. andersenii mtDNA is devoid of inverted repeats and introns, which are present in R. salina. Comparative analyses of the suite of tRNAs encoded in the two genomes reveal that the H. andersenii mtDNA has lost or converted its original trnK(uuu) gene and possesses a trnS-derived 'trnK(uuu)', which appears unable to produce a functional tRNA. Mitochondrial protein coding gene phylogenies strongly support a variety of previously established eukaryotic groups, but fail to resolve the relationships among higher-order eukaryotic lineages. Conclusion Comparison of the H. andersenii and R. salina mitochondrial genomes reveals a number of cryptophyte-specific genomic features, most notably the presence of a large repeat-rich intergenic region. However, unlike R. salina, the H. andersenii mtDNA does not possess introns and lacks a Lys-tRNA, which is presumably imported from the cytosol. PMID:18474103

The molecular variability analysis of the RNA 3 of fifteen isolates of Prunus necrotic ringspot virus sheds light on the minimal requirements for the synthesis of its subgenomic RNA.

PubMed

Aparicio, Frederic; Pallás, Vicente

2002-01-01

The nucleotide sequences of the RNA 3 of fifteen isolates of Prunus necrotic ringspot virus (PNRSV) varying in the symptomatology they cause in six different Prunus spp. were determined. Analysis of the molecular variability has allowed, in addition to study the phylogenetic relationships among them, to evaluate the minimal requirements for the synthesis of the subgenomic RNA in Ilarvirus genus and their comparison to other members of the Bromoviridae family. Computer assisted comparisons led recently to Jaspars (Virus Genes 17, 233-242, 1998) to propose that a hairpin structure in viral minus strand RNA is required for subgenomic promoter activity of viruses from at least two, and possibly all five, genera in the family of Bromoviridae. For PNRSV and Apple mosaic virus two stable hairpins were proposed whereas for the rest of Ilarviruses and the other four genera of the Bromoviridae family only one stable hairpin was predicted. Comparative analysis of this region among the fifteen PNRSV isolates characterized in this study revealed that two of them showed a 12-nt deletion that led to the disappearance of the most proximal hairpin to the initiation site. Interestingly, the only hairpin found in these two isolates is very similar in primary and secondary structure to the one previously shown in Brome mosaic virus to be required for the synthesis of the subgenomic RNA. In this hairpin, the molecular diversity was concentrated mostly at the loop whereas compensatory mutations were observed at the base of the stem strongly suggesting its functional relevance. The evolutionary implications of these observations are discussed.

The Activation Domain of the Bovine Papillomavirus E2 Protein Mediates Association of DNA-Bound Dimers to form DNA Loops

NASA Astrophysics Data System (ADS)

Knight, Jonathan D.; Li, Rong; Botchan, Michael

1991-04-01

The E2 transactivator protein of bovine papillomavirus binds its specific DNA target sequence as a dimer. We have found that E2 dimers, performed in solution independent of DNA, exhibit substantial cooperativity of DNA binding as detected by both nitrocellulose filter retention and footprint analysis techniques. If the binding sites are widely spaced, E2 forms stable DNA loops visible by electron microscopy. When three widely separated binding sites reside on te DNA, E2 condenses the molecule into a bow-tie structure. This implies that each E2 dimer has at least two independent surfaces for multimerization. Two naturally occurring shorter forms of the protein, E2C and D8/E2, which function in vivo as repressors of transcription, do not form such loops. Thus, the looping function of E2 maps to the 161-amino acid activation domain. These results support the looping model of transcription activation by enhancers.

Biomimetic Materials and Fabrication Approaches for Bone Tissue Engineering.

PubMed

Kim, Hwan D; Amirthalingam, Sivashanmugam; Kim, Seunghyun L; Lee, Seunghun S; Rangasamy, Jayakumar; Hwang, Nathaniel S

2017-12-01

Various strategies have been explored to overcome critically sized bone defects via bone tissue engineering approaches that incorporate biomimetic scaffolds. Biomimetic scaffolds may provide a novel platform for phenotypically stable tissue formation and stem cell differentiation. In recent years, osteoinductive and inorganic biomimetic scaffold materials have been optimized to offer an osteo-friendly microenvironment for the osteogenic commitment of stem cells. Furthermore, scaffold structures with a microarchitecture design similar to native bone tissue are necessary for successful bone tissue regeneration. For this reason, various methods for fabricating 3D porous structures have been developed. Innovative techniques, such as 3D printing methods, are currently being utilized for optimal host stem cell infiltration, vascularization, nutrient transfer, and stem cell differentiation. In this progress report, biomimetic materials and fabrication approaches that are currently being utilized for biomimetic scaffold design are reviewed. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The role of modifications in codon discrimination by tRNA(Lys)UUU.

PubMed

Murphy, Frank V; Ramakrishnan, Venki; Malkiewicz, Andrzej; Agris, Paul F

2004-12-01

The natural modification of specific nucleosides in many tRNAs is essential during decoding of mRNA by the ribosome. For example, tRNA(Lys)(UUU) requires the modification N6-threonylcarbamoyladenosine at position 37 (t(6)A37), adjacent and 3' to the anticodon, to bind AAA in the A site of the ribosomal 30S subunit. Moreover, it can only bind both AAA and AAG lysine codons when doubly modified with t(6)A37 and either 5-methylaminomethyluridine or 2-thiouridine at the wobble position (mnm(5)U34 or s(2)U34). Here we report crystal structures of modified tRNA anticodon stem-loops bound to the 30S ribosomal subunit with lysine codons in the A site. These structures allow the rationalization of how modifications in the anticodon loop enable decoding of both lysine codons AAA and AAG.

The yeast retrotransposon Ty5 uses the anticodon stem-loop of the initiator methionine tRNA as a primer for reverse transcription.

PubMed Central

Ke, N; Gao, X; Keeney, J B; Boeke, J D; Voytas, D F

1999-01-01

Retrotransposons and retroviruses replicate by reverse transcription of an mRNA intermediate. Most retroelements initiate reverse transcription from a host-encoded tRNA primer. DNA synthesis typically extends from the 3'-OH of the acceptor stem, which is complementary to sequences on the retroelement mRNA (the primer binding site, PBS). However, for some retrotransposons, including the yeast Ty5 elements, sequences in the anticodon stem-loop of the initiator methionine tRNA (IMT) are complementary to the PBS. We took advantage of the genetic tractability of the yeast system to investigate the mechanism of Ty5 priming. We found that transposition frequencies decreased at least 800-fold for mutations in the Ty5 PBS that disrupt complementarity with the IMT. Similarly, transposition was reduced at least 200-fold for IMT mutations in the anticodon stem-loop. Base pairing between the Ty5 PBS and IMT is essential for transposition, as compensatory changes that restored base pairing between the two mutant RNAs restored transposition significantly. An analysis of 12 imt mutants with base changes outside of the region of complementarity failed to identify other tRNA residues important for transposition. In addition, assays carried out with heterologous IMTs from Schizosaccharomyces pombe and Arabidopsis thaliana indicated that residues outside of the anticodon stem-loop have at most a fivefold effect on transposition. Our genetic system should make it possible to further define the components required for priming and to understand the mechanism by which Ty5's novel primer is generated. PMID:10411136

Studying the influence of stem composition in pH-sensitive molecular beacons onto their sensing properties.

PubMed

Dembska, Anna; Kierzek, Elzbieta; Juskowiak, Bernard

2017-10-16

Intracellular sensing using fluorescent molecular beacons is a potentially useful strategy for real-time, in vivo monitoring of important cellular events. This work is focused on evaluation of pyrene excimer signaling molecular beacons (MBs) for the monitoring of pH changes in vitro as well as inside living cells. The recognition element in our MB called pHSO (pH-sensitive oligonucleotide) is the loop enclosing cytosine-rich fragment that is able to form i-motif structure in a specific pH range. However, alteration of a sequence of the 6 base pairs containing stem of MB allowed the design of pHSO probes that exhibited different dynamic pH range and possessed slightly different transition midpoint between i-motif and open loop configuration. Moreover, this conformational transition was accompanied by spectral changes showing developed probes different pyrene excimer-monomer emission ratio triggered by pH changes. The potential of these MBs for intracellular pH sensing is demonstrated on the example of HeLa cells line. Copyright © 2017 Elsevier B.V. All rights reserved.

Fine pointing control for a Next-Generation Space Telescope

NASA Astrophysics Data System (ADS)

Mosier, Gary E.; Femiano, Michael; Ha, Kong; Bely, Pierre Y.; Burg, Richard; Redding, David C.; Kissil, Andrew; Rakoczy, John; Craig, Larry

1998-08-01

The Next Generation Space Telescope will provide at least ten times the collecting area of the Hubble Space Telescope in a package that fits into the shroud of an expendable launch vehicle. The resulting large, flexible structure provides a challenge to the design of a pointing control system for which the requirements are at the milli-arcsecond level. This paper describes a design concept in which pointing stability is achieved by means of a nested-loop design involving an inertial attitude control system (ACS) and a fast steering mirror (FSM). A key to the integrated control design is that the ACS controllers has a bandwidth well below known structural modes and the FSM uses a rotationally balanced mechanism which should not interact with the flexible modes that are within its control bandwidth. The ACS controller provides stable pointing of the spacecraft bus with star trackers and gyros. This low bandwidth loop uses nearly co-located sensors and actuators to slew and acquire faint guide stars in the NIR camera. This controller provides a payload reference stable to the arcsecond level. Low-frequency pointing errors due to sensor noise and dynamic disturbances are suppressed by a 2-axis gimbaled FSM locate din the instrument module. The FSM servo bandwidth of 6 Hz is intended to keep the guide star position stable in the NIR focal plane to the required milli-arcsecond level. The mirror is kept centered in its range of travel by a low-bandwidth loop closed around the ACS. This paper presents the result of parametric trade studies designed to assess the performance of this control design in the presence of modeled reaction wheel disturbances, assumed to be the principle source of vibration for the NGST, and variations in structural dynamics. Additionally, requirements for reaction wheel disturbance levels and potential vibration isolation subsystems were developed.

The chloroplast tRNALys(UUU) gene from mustard (Sinapis alba) contains a class II intron potentially coding for a maturase-related polypeptide.

PubMed

Neuhaus, H; Link, G

1987-01-01

The trnK gene endocing the tRNALys(UUU) has been located on mustard (Sinapis alba) chloroplast DNA, 263 bp upstream of the psbA gene on the same strand. The nucleotide sequence of the trnK gene and its flanking regions as well as the putative transcription start and termination sites are shown. The 5' end of the transcript lies 121 bp upstream of the 5' tRNA coding region and is preceded by procaryotic-type "-10" and "-35" sequence elements, while the 3' end maps 2.77 kb downstream to a DNA region with possible stemloop secondary structure. The anticodon loop of the tRNALys is interrupted by a 2,574 bp intron containing a long open reading frame, which codes for 524 amino acids. Based on conserved stem and loop structures, this intron has characteristic features of a class II intron. A region near the carboxyl terminus of the derived polypeptide appears structurally related to maturases.

Sox2+ Stem Cells Contribute to All Epithelial Lineages of the Tooth via Sfrp5+ Progenitors

PubMed Central

Juuri, Emma; Saito, Kan; Ahtiainen, Laura; Seidel, Kerstin; Tummers, Mark; Hochedlinger, Konrad; Klein, Ophir D.; Thesleff, Irma; Michon, Frederic

2012-01-01

SUMMARY The continuously growing mouse incisor serves as a valuable model to study stem cell regulation during organ renewal. Epithelial stem cells are localized in the proximal end of the incisor in the labial cervical loop. Here, we show that the transcription factor Sox2 is a specific marker for these stem cells. Sox2+ cells became restricted to the labial cervical loop during tooth morphogenesis, and they contributed to the renewal of enamel-producing ameloblasts as well as all other epithelial cell lineages of the tooth. The early progeny of Sox2-positive stem cells transiently expressed the Wnt inhibitor Sfrp5. Sox2 expression was regulated by the tooth initiation marker FGF8 and specific miRNAs, suggesting a fine-tuning to maintain homeostasis of the dental epithelium. The identification of Sox2 as a marker for the dental epithelial stem cells will facilitate further studies on their lineage segregation and differentiation during tooth renewal. PMID:22819339

Structure/cleavage-based insights into helical perturbations at bulge sites within T. thermophilus Argonaute silencing complexes

PubMed Central

Sheng, Gang; Gogakos, Tasos; Wang, Jiuyu; Zhao, Hongtu; Serganov, Artem; Juranek, Stefan

2017-01-01

Abstract We have undertaken a systematic structural study of Thermus thermophilus Argonaute (TtAgo) ternary complexes containing single-base bulges positioned either within the seed segment of the guide or target strands and at the cleavage site. Our studies establish that single-base bulges 7T8, 5A6 and 4A5 on the guide strand are stacked-into the duplex, with conformational changes localized to the bulge site, thereby having minimal impact on the cleavage site. By contrast, single-base bulges 6’U7’ and 6’A7’ on the target strand are looped-out of the duplex, with the resulting conformational transitions shifting the cleavable phosphate by one step. We observe a stable alignment for the looped-out 6’N7’ bulge base, which stacks on the unpaired first base of the guide strand, with the looped-out alignment facilitated by weakened Watson–Crick and reversed non-canonical flanking pairs. These structural studies are complemented by cleavage assays that independently monitor the impact of bulges on TtAgo-mediated cleavage reaction. PMID:28911094

In Vitro Assembly of Human H/ACA Small Nucleolar RNPs Reveals Unique Features of U17 and Telomerase RNAs

PubMed Central

Dragon, François; Pogačić, Vanda; Filipowicz, Witold

2000-01-01

The H/ACA small nucleolar RNAs (snoRNAs) are involved in pseudouridylation of pre-rRNAs. They usually fold into a two-domain hairpin-hinge-hairpin-tail structure, with the conserved motifs H and ACA located in the hinge and tail, respectively. Synthetic RNA transcripts and extracts from HeLa cells were used to reconstitute human U17 and other H/ACA ribonucleoproteins (RNPs) in vitro. Competition and UV cross-linking experiments showed that proteins of about 60, 29, 23, and 14 kDa interact specifically with U17 RNA. Except for U17, RNPs could be reconstituted only with full-length H/ACA snoRNAs. For U17, the 3′-terminal stem-loop followed by box ACA (U17/3′st) was sufficient to form an RNP, and U17/3′st could compete other full-length H/ACA snoRNAs for assembly. The H/ACA-like domain that constitutes the 3′ moiety of human telomerase RNA (hTR), and its 3′-terminal stem-loop (hTR/3′st), also could form an RNP by binding H/ACA proteins. Hence, the 3′-terminal stem-loops of U17 and hTR have some specific features that distinguish them from other H/ACA RNAs. Antibodies that specifically recognize the human GAR1 (hGAR1) protein could immunoprecipitate H/ACA snoRNAs and hTR from HeLa cell extracts, which demonstrates that hGAR1 is a component of H/ACA snoRNPs and telomerase in vivo. Moreover, we show that in vitro-reconstituted RNPs contain hGAR1 and that binding of hGAR1 does not appear to be a prerequisite for the assembly of the other H/ACA proteins. PMID:10757788

Mimicking a p53-MDM2 interaction based on a stable immunoglobulin-like domain scaffold.

PubMed

Jimenez-Sandoval, Pedro; Madrigal-Carrillo, Ezequiel A; Santamaría-Suárez, Hugo A; Maturana, Daniel; Rentería-González, Itzel; Benitez-Cardoza, Claudia G; Torres-Larios, Alfredo; Brieba, Luis G

2018-04-26

Antibodies recognize protein targets with great affinity and specificity. However, posttranslational modifications and the presence of intrinsic disulfide-bonds pose difficulties for their industrial use. The immunoglobulin fold is one of the most ubiquitous folds in nature and it is found in many proteins besides antibodies. An example of a protein family with an immunoglobulin-like fold is the Cysteine Protease Inhibitors (ICP) family I42 of the MEROPs database for protease and protease inhibitors. Members of this protein family are thermostable and do not present internal disulfide bonds. Crystal structures of several ICPs indicate that they resemble the Ig-like domain of the human T cell co-receptor CD8α As ICPs present 2 flexible recognition loops that vary accordingly to their targeted protease, we hypothesize that members of this protein family would be ideal to design peptide aptamers that mimic protein-protein interactions. Herein, we use an ICP variant from Entamoeba histolytica (EhICP1) to mimic the interaction between p53 and MDM2. We found that a 13 amino-acid peptide derived from p53 can be introduced in 2 variable loops (DE, FG) but not the third (BC). Chimeric EhICP1-p53 form a stable complex with MDM2 at a micromolar range. Crystal structure of the EhICP1-p53(FG)-loop variant in complex with MDM2 reveals a swapping subdomain between 2 chimeric molecules, however, the p53 peptide interacts with MDM2 as in previous crystal structures. The structural details of the EhICP1-p53(FG) interaction with MDM2 resemble the interaction between an antibody and MDM2. © 2018 Wiley Periodicals, Inc.

A Small Stem Loop Structure Of The Ebola Virus Trailer Is Essential For Replication And Interacts With Heat Shock Protein A8

DTIC Science & Technology

2016-12-02

agarose gel electrophoresis TR-16-205 Nucleic Acids Research, 2016 3 (Seakem GTG , Sigma-Aldrich) and purified using the QI- Aquick Gel Extraction Kit... gtg +cga 1923-1938 3′ LNA2 +caa+aaa+ga+aa+gaa+gaa 3E-5E eGFP, 3E-5E plasmid containing enhanced green fluorescent protein; aiSHAPE, antisense-interfered

Loss of G-A base pairs is insufficient for achieving a large opening of U4 snRNA K-turn motif.

PubMed

Cojocaru, Vlad; Klement, Reinhard; Jovin, Thomas M

2005-01-01

Upon binding to the 15.5K protein, two tandem-sheared G-A base pairs are formed in the internal loop of the kink-turn motif of U4 snRNA (Kt-U4). We have reported that the folding of Kt-U4 is assisted by protein binding. Unstable interactions that contribute to a large opening of the free RNA ('k-e motion') were identified using locally enhanced sampling molecular dynamics simulations, results that agree with experiments. A detailed analysis of the simulations reveals that the k-e motion in Kt-U4 is triggered both by loss of G-A base pairs in the internal loop and backbone flexibility in the stems. Essential dynamics show that the loss of G-A base pairs is correlated along the first mode but anti-correlated along the third mode with the k-e motion. Moreover, when enhanced sampling was confined to the internal loop, the RNA adopted an alternative conformation characterized by a sharper kink, opening of G-A base pairs and modified stacking interactions. Thus, loss of G-A base pairs is insufficient for achieving a large opening of the free RNA. These findings, supported by previously published RNA structure probing experiments, suggest that G-A base pair formation occurs upon protein binding, thereby stabilizing a selective orientation of the stems.

Molecular dynamics simulations of human tRNA Lys,3 UUU: the role of modified bases in mRNA recognition.

PubMed

McCrate, Nina E; Varner, Mychel E; Kim, Kenneth I; Nagan, Maria C

2006-01-01

Accuracy in translation of the genetic code into proteins depends upon correct tRNA-mRNA recognition in the context of the ribosome. In human tRNA(Lys,3)UUU three modified bases are present in the anticodon stem-loop--2-methylthio-N6-threonylcarbamoyladenosine at position 37 (ms2t6A37), 5-methoxycarbonylmethyl-2-thiouridine at position 34 (mcm5s2U34) and pseudouridine (psi) at position 39--two of which, ms2t6A37 and mcm5s2U34, are required to achieve wild-type binding activity of wild-type human tRNA(Lys,3)UUU [C. Yarian, M. Marszalek, E. Sochacka, A. Malkiewicz, R. Guenther, A. Miskiewicz and P. F. Agris (2000) Biochemistry, 39, 13390-13395]. Molecular dynamics simulations of nine tRNA anticodon stem-loops with different combinations of nonstandard bases were performed. The wild-type simulation exhibited a canonical anticodon stair-stepped conformation. The ms2t6 modification at position 37 is required for maintenance of this structure and reduces solvent accessibility of U36. Ms2t6A37 generally hydrogen bonds across the loop and may prevent U36 from rotating into solution. A water molecule does coordinate to psi39 most of the simulation time but weakly, as most of the residence lifetimes are <40 ps.

Efficient Ligation of the Schistosoma Hammerhead Ribozyme †

PubMed Central

Canny, Marella D.; Jucker, Fiona M.; Pardi, Arthur

2011-01-01

The hammerhead ribozyme from Schistosoma mansoni is the best characterized of the natural hammerhead ribozymes. Biophysical, biochemical, and structural studies have shown that the formation of the loop-loop tertiary interaction between stems I and II alters the global folding, cleavage kinetics, and conformation of the catalytic core of this hammerhead, leading to a ribozyme that is readily cleaved under physiological conditions. This study investigates the ligation kinetics and the internal equilibrium between cleavage and ligation for the Schistosoma hammerhead. Single turnover kinetic studies on a construct where the ribozyme cleaves and ligates substrate(s) in trans showed up to 23% ligation when starting from fully cleaved products. This was achieved by a ~2,000-fold increase in the rate of ligation compared to a minimal hammerhead without the loop-loop tertiary interaction, yielding an internal equilibrium that ranges from 2–3 at physiological Mg2+ ion concentrations (0.1 –1 mM). Thus, the natural Schistosoma hammerhead ribozyme is almost as efficient at ligation as it is at cleavage. The results here are consistent with a model where formation of the loop-loop tertiary interaction leads to a higher population of catalytically active molecules, and where formation of this tertiary interaction has a much larger effect on the ligation than the cleavage activity of the Schistosoma hammerhead ribozyme. PMID:17319693

Evolution of Nucleotide Punctuation Marks: From Structural to Linear Signals.

PubMed

El Houmami, Nawal; Seligmann, Hervé

2017-01-01

We present an evolutionary hypothesis assuming that signals marking nucleotide synthesis (DNA replication and RNA transcription) evolved from multi- to unidimensional structures, and were carried over from transcription to translation. This evolutionary scenario presumes that signals combining secondary and primary nucleotide structures are evolutionary transitions. Mitochondrial replication initiation fits this scenario. Some observations reported in the literature corroborate that several signals for nucleotide synthesis function in translation, and vice versa. (a) Polymerase-induced frameshift mutations occur preferentially at translational termination signals (nucleotide deletion is interpreted as termination of nucleotide polymerization, paralleling the role of stop codons in translation). (b) Stem-loop hairpin presence/absence modulates codon-amino acid assignments, showing that translational signals sometimes combine primary and secondary nucleotide structures (here codon and stem-loop). (c) Homopolymer nucleotide triplets (AAA, CCC, GGG, TTT) cause transcriptional and ribosomal frameshifts. Here we find in recently described human mitochondrial RNAs that systematically lack mono-, dinucleotides after each trinucleotide (delRNAs) that delRNA triplets include 2x more homopolymers than mitogenome regions not covered by delRNA. Further analyses of delRNAs show that the natural circular code X (a little-known group of 20 translational signals enabling ribosomal frame retrieval consisting of 20 codons {AAC, AAT, ACC, ATC, ATT, CAG, CTC, CTG, GAA, GAC, GAG, GAT, GCC, GGC, GGT, GTA, GTC, GTT, TAC, TTC} universally overrepresented in coding versus other frames of gene sequences), regulates frameshift in transcription and translation. This dual transcription and translation role confirms for X the hypothesis that translational signals were carried over from transcriptional signals.

Mammalian DNA enriched for replication origins is enriched for snap-back sequences.

PubMed

Zannis-Hadjopoulos, M; Kaufmann, G; Martin, R G

1984-11-15

Using the instability of replication loops as a method for the isolation of double-stranded nascent DNA, extruded DNA enriched for replication origins was obtained and denatured. Snap-back DNA, single-stranded DNA with inverted repeats (palindromic sequences), reassociates rapidly into stem-loop structures with zero-order kinetics when conditions are changed from denaturing to renaturing, and can be assayed by chromatography on hydroxyapatite. Origin-enriched nascent DNA strands from mouse, rat and monkey cells growing either synchronously or asynchronously were purified and assayed for the presence of snap-back sequences. The results show that origin-enriched DNA is also enriched for snap-back sequences, implying that some origins for mammalian DNA replication contain or lie near palindromic sequences.

Aromatic Ring Currents Illustrated--NMR Spectra of Tin(IV) Porphyrin Complexes. An Advanced Undergraduate Experiment.

ERIC Educational Resources Information Center

Arnold, Dennis P.

1988-01-01

Attempts to show that in the closed loops of cyclic structures the protons situated in conic regions above and below the ring will be shielded. Uses the diamagnetic and air stable octahedral tin(IV) complexes of porphyrins for study. Notes complexes crystallize easily and offer spectacular purple colors. (MVL)

Precise Regulation of miR-210 Is Critical for the Cellular Homeostasis Maintenance and Transplantation Efficacy Enhancement of Mesenchymal Stem Cells in Acute Liver Failure Therapy.

PubMed

Liu, Yingxia; Xiong, Yongjia; Xing, Feiyue; Gao, Hao; Wang, Xiaogang; He, Liumin; Ren, Chaoran; Liu, Lei; So, Kwok-Fai; Xiao, Jia

2017-05-09

Stem cell transplantation is a promising clinical strategy to cure acute liver failure. However, a low cell survival ratio after transplantation significantly impairs its therapeutic efficacy. This is partly due to insufficient resistance of transplanted stem cells to severe oxidative and inflammatory stress at the injury sites. In the current study, we demonstrated that a small molecule zeaxanthin dipalmitate (ZD) could enhance the defensive abilities against adverse stresses of human adipose-derived mesenchymal stem cells (hADMSCs) in vitro and increase their therapeutic outcomes of acute liver failure after transplantation in vivo. Treatment with ZD dramatically improved cell survival and suppressed apoptosis, inflammation, and reactive oxygen species (ROS) production of hADMSCs through the PKC/Raf-1/MAPK/NF-κB pathway to maintain a reasonably high expression level of microRNA-210 (miR-210). The regulation loop between miR-210 and cellular/mitochondrial ROS production was found to be linked by the ROS inhibitor iron-sulfur cluster assembly proteins (ISCU). Pretreatment with ZD and stable knockdown of miR-210 significantly improved and impaired the stem cell transplantation efficacy through the alteration of hepatic cell expansion and injury amelioration, respectively. Vehicle treatment with ZD did not pose any adverse effect on cell homeostasis or healthy animal. In conclusion, elevating endogenous antioxidant level of hADMSCs with ZD significantly enhances their hepatic tissue-repairing capabilities. Maintenance of a physiological level of miR-210 is critical for hADMSC homeostasis.

Mobile Element Evolution Playing Jigsaw - SINEs in Gastropod and Bivalve Mollusks.

PubMed

Matetovici, Irina; Sajgo, Szilard; Ianc, Bianca; Ochis, Cornelia; Bulzu, Paul; Popescu, Octavian; Damert, Annette

2016-01-06

SINEs (Short INterspersed Elements) are widely distributed among eukaryotes. Some SINE families are organized in superfamilies characterized by a shared central domain. These central domains are conserved across species, classes, and even phyla. Here we report the identification of two novel such superfamilies in the genomes of gastropod and bivalve mollusks. The central conserved domain of the first superfamily is present in SINEs in Caenogastropoda and Vetigastropoda as well as in all four subclasses of Bivalvia. We designated the domain MESC (Romanian for MElc-snail and SCoica-mussel) because it appears to be restricted to snails and mussels. The second superfamily is restricted to Caenogastropoda. Its central conserved domain-Snail-is related to the Nin-DC domain. Furthermore, we provide evidence that a 40-bp subdomain of the SINE V-domain is conserved in SINEs in mollusks and arthropods. It is predicted to form a stable stem-loop structure that is preserved in the context of the overall SINE RNA secondary structure in invertebrates. Our analysis also recovered short retrotransposons with a Long INterspersed Element (LINE)-derived 5' end. These share the body and/or the tail with transfer RNA (tRNA)-derived SINEs within and across species. Finally, we identified CORE SINEs in gastropods and bivalves-extending the distribution range of this superfamily. © The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

The complete mitochondrial genome of Plodia interpunctella (Lepidoptera: Pyralidae) and comparison with other Pyraloidea insects.

PubMed

Liu, Qiu-Ning; Chai, Xin-Yue; Bian, Dan-Dan; Zhou, Chun-Lin; Tang, Bo-Ping

2016-01-01

The mitochondrial (mt) genome can provide important information for the understanding of phylogenetic relationships. The complete mt genome of Plodia interpunctella (Lepidoptera: Pyralidae) has been sequenced. The circular genome is 15 287 bp in size, encoding 13 protein-coding genes (PCGs), 2 rRNA genes, 22 tRNA genes, and a control region. The AT skew of this mt genome is slightly negative, and the nucleotide composition is biased toward A+T nucleotides (80.15%). All PCGs start with the typical ATN (ATA, ATC, ATG, and ATT) codons, except for the cox1 gene which may start with the CGA codon. Four of the 13 PCGs harbor the incomplete termination codon T or TA. All the tRNA genes are folded into the typical clover-leaf structure of mitochondrial tRNA, except for trnS1 (AGN) in which the DHU arm fails to form a stable stem-loop structure. The overlapping sequences are 35 bp in total and are found in seven different locations. A total of 240 bp of intergenic spacers are scattered in 16 regions. The control region of the mt genome is 327 bp in length and consisted of several features common to the sequenced lepidopteran insects. Phylogenetic analysis based on 13 PCGs using the Maximum Likelihood method shows that the placement of P. interpunctella was within the Pyralidae.

Mutations in yeast proliferating cell nuclear antigen define distinct sites for interaction with DNA polymerase delta and DNA polymerase epsilon.

PubMed Central

Eissenberg, J C; Ayyagari, R; Gomes, X V; Burgers, P M

1997-01-01

The importance of the interdomain connector loop and of the carboxy-terminal domain of Saccharomyces cerevisiae proliferating cell nuclear antigen (PCNA) for functional interaction with DNA polymerases delta (Poldelta) and epsilon (Pol epsilon) was investigated by site-directed mutagenesis. Two alleles, pol30-79 (IL126,128AA) in the interdomain connector loop and pol30-90 (PK252,253AA) near the carboxy terminus, caused growth defects and elevated sensitivity to DNA-damaging agents. These two mutants also had elevated rates of spontaneous mutations. The mutator phenotype of pol30-90 was due to partially defective mismatch repair in the mutant. In vitro, the mutant PCNAs showed defects in DNA synthesis. Interestingly, the pol30-79 mutant PCNA (pcna-79) was most defective in replication with Poldelta, whereas pcna-90 was defective in replication with Pol epsilon. Protein-protein interaction studies showed that pcna-79 and pcna-90 failed to interact with Pol delta and Pol epsilon, respectively. In addition, pcna-90 was defective in interaction with the FEN-1 endo-exonuclease (RTH1 product). A loss of interaction between pcna-79 and the smallest subunit of Poldelta, the POL32 gene product, implicates this interaction in the observed defect with the polymerase. Neither PCNA mutant showed a defect in the interaction with replication factor C or in loading by this complex. Processivity of DNA synthesis by the mutant holoenzyme containing pcna-79 was unaffected on poly(dA) x oligo(dT) but was dramatically reduced on a natural template with secondary structure. A stem-loop structure with a 20-bp stem formed a virtually complete block for the holoenzyme containing pcna-79 but posed only a minor pause site for wild-type holoenzyme, indicating a function of the POL32 gene product in allowing replication past structural blocks. PMID:9343398

Charged string loops in Reissner-Nordström black hole background

NASA Astrophysics Data System (ADS)

Oteev, Tursinbay; Kološ, Martin; Stuchlík, Zdeněk

2018-03-01

We study the motion of current carrying charged string loops in the Reissner-Nordström black hole background combining the gravitational and electromagnetic field. Introducing new electromagnetic interaction between central charge and charged string loop makes the string loop equations of motion to be non-integrable even in the flat spacetime limit, but it can be governed by an effective potential even in the black hole background. We classify different types of the string loop trajectories using effective potential approach, and we compare the innermost stable string loop positions with loci of the charged particle innermost stable orbits. We examine string loop small oscillations around minima of the string loop effective potential, and we plot radial profiles of the string loop oscillation frequencies for both the radial and vertical modes. We construct charged string loop quasi-periodic oscillations model and we compare it with observed data from microquasars GRO 1655-40, XTE 1550-564, and GRS 1915+105. We also study the acceleration of current carrying string loops along the vertical axis and the string loop ejection from RN black hole neighbourhood, taking also into account the electromagnetic interaction.

Long-Term Culture of Genome-Stable Bipotent Stem Cells from Adult Human Liver

PubMed Central

Huch, Meritxell; Gehart, Helmuth; van Boxtel, Ruben; Hamer, Karien; Blokzijl, Francis; Verstegen, Monique M.A.; Ellis, Ewa; van Wenum, Martien; Fuchs, Sabine A.; de Ligt, Joep; van de Wetering, Marc; Sasaki, Nobuo; Boers, Susanne J.; Kemperman, Hans; de Jonge, Jeroen; Ijzermans, Jan N.M.; Nieuwenhuis, Edward E.S.; Hoekstra, Ruurdtje; Strom, Stephen; Vries, Robert R.G.; van der Laan, Luc J.W.; Cuppen, Edwin; Clevers, Hans

2015-01-01

Summary Despite the enormous replication potential of the human liver, there are currently no culture systems available that sustain hepatocyte replication and/or function in vitro. We have shown previously that single mouse Lgr5+ liver stem cells can be expanded as epithelial organoids in vitro and can be differentiated into functional hepatocytes in vitro and in vivo. We now describe conditions allowing long-term expansion of adult bile duct-derived bipotent progenitor cells from human liver. The expanded cells are highly stable at the chromosome and structural level, while single base changes occur at very low rates. The cells can readily be converted into functional hepatocytes in vitro and upon transplantation in vivo. Organoids from α1-antitrypsin deficiency and Alagille syndrome patients mirror the in vivo pathology. Clonal long-term expansion of primary adult liver stem cells opens up experimental avenues for disease modeling, toxicology studies, regenerative medicine, and gene therapy. PMID:25533785

Glycols modulate terminator stem stability and ligand-dependency of a glycine riboswitch.

PubMed

Hamachi, Kokoro; Hayashi, Hikari; Shimamura, Miyuki; Yamaji, Yuiha; Kaneko, Ai; Fujisawa, Aruma; Umehara, Takuya; Tamura, Koji

2013-08-01

The Bacillus subtilis glycine riboswitch comprises tandem glycine-binding aptamers and a putative terminator stem followed by the gcvT operon. Gene expression is regulated via the sensing of glycine. However, we found that the riboswitch behaves in a "glycine-independent" manner in the presence of polyethylene glycol (PEG) and ethylene glycol. The effect is related to the formation of a terminator stem within the expression platform under such conditions. The results revealed that increasing PEG stabilized the structure of the terminator stem. By contrast, the addition of ethylene glycol destabilized the terminator stem. PEG and ethylene glycol have opposite effects on transcription as well as on stable terminator stem formation. The glycine-independency of the riboswitch and the effects of such glycols might shed light on the evolution of riboswitches. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Replication enhancer elements within the open reading frame of tick-borne encephalitis virus and their evolution within the Flavivirus genus

PubMed Central

Tuplin, A.; Evans, D. J.; Buckley, A.; Jones, I. M.; Gould, E. A.; Gritsun, T. S.

2011-01-01

We provide experimental evidence of a replication enhancer element (REE) within the capsid gene of tick-borne encephalitis virus (TBEV, genus Flavivirus). Thermodynamic and phylogenetic analyses predicted that the REE folds as a long stable stem–loop (designated SL6), conserved among all tick-borne flaviviruses (TBFV). Homologous sequences and potential base pairing were found in the corresponding regions of mosquito-borne flaviviruses, but not in more genetically distant flaviviruses. To investigate the role of SL6, nucleotide substitutions were introduced which changed a conserved hexanucleotide motif, the conformation of the terminal loop and the base-paired dsRNA stacking. Substitutions were made within a TBEV reverse genetic system and recovered mutants were compared for plaque morphology, single-step replication kinetics and cytopathic effect. The greatest phenotypic changes were observed in mutants with a destabilized stem. Point mutations in the conserved hexanucleotide motif of the terminal loop caused moderate virus attenuation. However, all mutants eventually reached the titre of wild-type virus late post-infection. Thus, although not essential for growth in tissue culture, the SL6 REE acts to up-regulate virus replication. We hypothesize that this modulatory role may be important for TBEV survival in nature, where the virus circulates by non-viraemic transmission between infected and non-infected ticks, during co-feeding on local rodents. PMID:21622960

Structural Plasticity and Rapid Evolution in a Viral RNA Revealed by In Vivo Genetic Selection▿ †

PubMed Central

Guo, Rong; Lin, Wai; Zhang, Jiuchun; Simon, Anne E.; Kushner, David B.

2009-01-01

Satellite RNAs usually lack substantial homology with their helper viruses. The 356-nucleotide satC of Turnip crinkle virus (TCV) is unusual in that its 3′-half shares high sequence similarity with the TCV 3′ end. Computer modeling, structure probing, and/or compensatory mutagenesis identified four hairpins and three pseudoknots in this TCV region that participate in replication and/or translation. Two hairpins and two pseudoknots have been confirmed as important for satC replication. One portion of the related 3′ end of satC that remains poorly characterized corresponds to juxtaposed TCV hairpins H4a and H4b and pseudoknot ψ3, which are required for the TCV-specific requirement of translation (V. A. Stupina et al., RNA 14:2379-2393, 2008). Replacement of satC H4a with randomized sequence and scoring for fitness in plants by in vivo genetic selection (SELEX) resulted in winning sequences that contain an H4a-like stem-loop, which can have additional upstream sequence composing a portion of the stem. SELEX of the combined H4a and H4b region in satC generated three distinct groups of winning sequences. One group models into two stem-loops similar to H4a and H4b of TCV. However, the selected sequences in the other two groups model into single hairpins. Evolution of these single-hairpin SELEX winners in plants resulted in satC that can accumulate to wild-type (wt) levels in protoplasts but remain less fit in planta when competed against wt satC. These data indicate that two highly distinct RNA conformations in the H4a and H4b region can mediate satC fitness in protoplasts. PMID:19004956

PNPase autocontrols its expression by degrading a double-stranded structure in the pnp mRNA leader

PubMed Central

Jarrige, Anne-Charlotte; Mathy, Nathalie; Portier, Claude

2001-01-01

Polynucleotide phosphorylase synthesis is autocontrolled at a post-transcriptional level in an RNase III-dependent mechanism. RNase III cleaves a long stem–loop in the pnp leader, which triggers pnp mRNA instability, resulting in a decrease in the synthesis of polynucleotide phosphorylase. The staggered cleavage by RNase III removes the upper part of the stem–loop structure, creating a duplex with a short 3′ extension. Mutations or high temperatures, which destabilize the cleaved stem–loop, decrease expression of pnp, while mutations that stabilize the stem increase expression. We propose that the dangling 3′ end of the duplex created by RNase III constitutes a target for polynucleotide phosphorylase, which binds to and degrades the upstream half of this duplex, hence inducing pnp mRNA instability. Consistent with this interpretation, a pnp mRNA starting at the downstream RNase III processing site exhibits a very low level of expression, regardless of the presence of polynucleotide phosphorylase. Moreover, using an in vitro synthesized pnp leader transcript, it is shown that polynucleotide phosphorylase is able to digest the duplex formed after RNase III cleavage. PMID:11726520

Complete mitochondrial genomes of three crickets (Orthoptera: Gryllidae) and comparative analyses within Ensifera mitogenomes.

PubMed

Yang, Jing; Ren, Qianli; Huang, Yuan

2016-03-17

The complete mitochondrial genomes (mitogenomes) of Velarifictorus hemelytrus, Loxoblemmus equestris and Teleogryllus emma are 16123 bp, 16314 bp and 15697 bp, in size, respectively. All three mitogenomes possess the same gene order of the inversion of the gene cluster trnE-trnS^(AGN)-trnN compared with the ancestral gene order of Orthoptera. The atypical initiation codon for the cox1 gene in three crickets is TTA. Pronounced A skew and T skew have been found in Grylloidea comparing with Gryllotalpoidea and Tettigonioidea. The T-stretch in the minority strand is interrupted by C to form (T)_n(C)₂(T)_n sequences in five species of Gryllinae (V. hemelytrus, L. equestris, T. emma, T. oceanicus, T. commodus). This T-stretch variant with its neighbouring A-stretch variant (A-stretch is interrupted by G), which were discovered in the A+T-rich regions of all taxa from infraorder Gryllidea, could form a conserved stem-loop structure (including 15 ~ 17 base pairs). This potential stem-loop structure is a favorable candidate that may participate in the replication origin of the minority strand of Gryllidea mitogenome. Phylogenetic analysis indicated that within the Gryllinae, genus Teleogryllus and Velarifictorus are closely related, sister to the genus Loxoblemmus. The relationships among the five superfamilies of Ensifera presented here were ((Grylloidea, Gryllotalpoidea) (Tettigonioidea, (Hagloidea, Rhaphidophoroidea))).

Reconstitution of a functional 7SK snRNP

PubMed Central

Brogie, John E.

2017-01-01

Abstract The 7SK small nuclear ribonucleoprotein (snRNP) plays a central role in RNA polymerase II elongation control by regulating the availability of active P-TEFb. We optimized conditions for analyzing 7SK RNA by SHAPE and demonstrated a hysteretic effect of magnesium on 7SK folding dynamics including a 7SK GAUC motif switch. We also found evidence that the 5΄ end pairs alternatively with two different regions of 7SK giving rise to open and closed forms that dictate the state of the 7SK motif. We then used recombinant P-TEFb, HEXIM1, LARP7 and MEPCE to reconstruct a functional 7SK snRNP in vitro. Stably associated P-TEFb was highly inhibited, but could still be released and activated by HIV-1 Tat. Notably, P-TEFb association with both in vitro-reconstituted and cellular snRNPs led to similar changes in SHAPE reactivities, confirming that 7SK undergoes a P-TEFb-dependent structural change. We determined that the xRRM of LARP7 binds to the 3΄ stem loop of 7SK and inhibits the methyltransferase activity of MEPCE through a C-terminal MEPCE interaction domain (MID). Inhibition of MEPCE is dependent on the structure of the 3΄ stem loop and the closed form of 7SK RNA. This study provides important insights into intramolecular interactions within the 7SK snRNP. PMID:28431135

System and circuitry to provide stable transconductance for biasing

NASA Technical Reports Server (NTRS)

Garverick, Steven L. (Inventor); Yu, Xinyu (Inventor)

2012-01-01

An amplifier system can include an input amplifier configured to receive an analog input signal and provide an amplified signal corresponding to the analog input signal. A tracking loop is configured to employ delta modulation for tracking the amplified signal, the tracking loop providing a corresponding output signal. A biasing circuit is configured to adjust a bias current to maintain stable transconductance over temperature variations, the biasing circuit providing at least one bias signal for biasing at least one of the input amplifier and the tracking loop, whereby the circuitry receiving the at least one bias signal exhibits stable performance over the temperature variations. In another embodiment the biasing circuit can be utilized in other applications.

Tilting the balance between canonical and noncanonical conformations for the H1 hypervariable loop of a llama VHH through point mutations.

PubMed

Mahajan, Sai Pooja; Velez-Vega, Camilo; Escobedo, Fernando A

2013-01-10

Nanobodies are single-domain antibodies found in camelids. These are the smallest naturally occurring binding domains and derive functionality via three hypervariable loops (H1-H3) that form the binding surface. They are excellent candidates for antibody engineering because of their favorable characteristics like small size, high solubility, and stability. To rationally engineer antibodies with affinity for a specific target, the hypervariable loops can be tailored to obtain the desired binding surface. As a first step toward such a goal, we consider the design of loops with a desired conformation. In this study, we focus on the H1 loop of the anti-hCG llama nanobody that exhibits a noncanonical conformation. We aim to "tilt" the stability of the H1 loop structure from a noncanonical conformation to a (humanized) type 1 canonical conformation by studying the effect of selected mutations to the amino acid sequence of the H1, H2, and proximal residues. We use all-atomistic, explicit-solvent, biased molecular dynamic simulations to simulate the wild-type and mutant loops in a prefolded framework. We thus find mutants with increasing propensity to form a stable type 1 canonical conformation of the H1 loop. Free energy landscapes reveal the existence of conformational isomers of the canonical conformation that may play a role in binding different antigenic surfaces. We also elucidate the approximate mechanism and kinetics of transitions between such conformational isomers by using a Markovian model. We find that a particular three-point mutant has the strongest thermodynamic propensity to form the H1 type 1 canonical structure but also to exhibit transitions between conformational isomers, while a different, more rigid three-point mutant has the strongest propensity to be kinetically trapped in such a canonical structure.

Nucleocapsid protein-dependent assembly of the RNA packaging signal of Middle East respiratory syndrome coronavirus.

PubMed

Hsin, Wei-Chen; Chang, Chan-Hua; Chang, Chi-You; Peng, Wei-Hao; Chien, Chung-Liang; Chang, Ming-Fu; Chang, Shin C

2018-05-24

Middle East respiratory syndrome coronavirus (MERS-CoV) consists of a positive-sense, single-stranded RNA genome and four structural proteins: the spike, envelope, membrane, and nucleocapsid protein. The assembly of the viral genome into virus particles involves viral structural proteins and is believed to be mediated through recognition of specific sequences and RNA structures of the viral genome. A culture system for the production of MERS coronavirus-like particles (MERS VLPs) was determined and established by electron microscopy and the detection of coexpressed viral structural proteins. Using the VLP system, a 258-nucleotide RNA fragment, which spans nucleotides 19,712 to 19,969 of the MERS-CoV genome (designated PS258(19712-19969) ME ), was identified to function as a packaging signal. Assembly of the RNA packaging signal into MERS VLPs is dependent on the viral nucleocapsid protein. In addition, a 45-nucleotide stable stem-loop substructure of the PS258(19712-19969) ME interacted with both the N-terminal domain and the C-terminal domain of the viral nucleocapsid protein. Furthermore, a functional SARS-CoV RNA packaging signal failed to assemble into the MERS VLPs, which indicated virus-specific assembly of the RNA genome. A MERS-oV RNA packaging signal was identified by the detection of GFP expression following an incubation of MERS VLPs carrying the heterologous mRNA GFP-PS258(19712-19969) ME with virus permissive Huh7 cells. The MERS VLP system could help us in understanding virus infection and morphogenesis.

Radiation resistance of oxide dispersion strengthened alloys: Perspectives from in situ observations and rate theory calculations

DOE PAGES

Liu, Xiang; Miao, Yinbin; Li, Meimei; ...

2018-04-15

Here, in situ ion irradiation and rate theory calculations were employed to directly compare the radiation resistance of an oxide dispersion strengthened alloy with that of a conventional ferritic/martensitic alloy. Compared to the rapid buildup of dislocation loops, loop growth, and formation of network dislocations in the conventional ferritic/martensitic alloy, the superior radiation resistance of the oxide dispersion strengthened alloy is manifested by its stable dislocation structure under the same irradiation conditions. Thus, the results are consistent with rate theory calculations, which show that high-density nanoparticles can significantly reduce freely migrating defects and suppress the buildup of clustered defects.

Radiation resistance of oxide dispersion strengthened alloys: Perspectives from in situ observations and rate theory calculations

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Xiang; Miao, Yinbin; Li, Meimei

Here, in situ ion irradiation and rate theory calculations were employed to directly compare the radiation resistance of an oxide dispersion strengthened alloy with that of a conventional ferritic/martensitic alloy. Compared to the rapid buildup of dislocation loops, loop growth, and formation of network dislocations in the conventional ferritic/martensitic alloy, the superior radiation resistance of the oxide dispersion strengthened alloy is manifested by its stable dislocation structure under the same irradiation conditions. Thus, the results are consistent with rate theory calculations, which show that high-density nanoparticles can significantly reduce freely migrating defects and suppress the buildup of clustered defects.

Diverse binding site structures revealed in homology models of polyreactive immunoglobulins

NASA Astrophysics Data System (ADS)

Ramsland, Paul A.; Guddat, Luke W.; Edmundson, Allen B.; Raison, Robert L.

1997-09-01

We describe here computer-assisted homology models of the combiningsite structure of three polyreactive immunoglobulins. Template-based modelsof Fv (VL-VH) fragments were derived forthe surface IgM expressed by the malignant CD5 positive B cells from threepatients with chronic lymphocytic leukaemia (CLL). The conserved frameworkregions were constructed using crystal coordinates taken from highlyhomologous human variable domain structures (Pot and Hil). Complementaritydetermining regions (CDRs) were predicted by grafting loops, taken fromknown immunoglobulin structures, onto the Fv framework models. The CDRtemplates were chosen, where possible, to be of the same length and of highresidue identity or similarity. LCDR1, 2 and 3 as well as HCDR1 and 2 forthe Fv were constructed using this strategy. For HCDR3 prediction, adatabase containing the Cartesian coordinates of 30 of these loops wascompiled from unliganded antibody X-ray crystallographic structures and anHCDR3 of the same length as that of the B CLL Fv was selected as a template.In one case (Yar), the resulting HCDR3 model gave unfavourable interactionswhen incorporated into the Fv model. This HCDR3 was therefore modelled usingan alternative strategy of construction of the loop stems, using apreviously described HCDR3 conformation (Pot), followed by chain closurewith a β-turn. The template models were subjected to positionalrefinement using energy minimisation and molecular dynamics simulations(X-PLOR). An electrostatic surface description (GRASP) did not reveal acommon structural feature within the binding sites of the three polyreactiveFv. Thus, polyreactive immunoglobulins may recognise similar and multipleantigens through a diverse array of binding site structures.

RNA polymerase II trigger loop residues stabilize and position the incoming nucleotide triphosphate in transcription

PubMed Central

Huang, Xuhui; Wang, Dong; Weiss, Dahlia R.; Bushnell, David A.; Kornberg, Roger D.; Levitt, Michael

2010-01-01

A structurally conserved element, the trigger loop, has been suggested to play a key role in substrate selection and catalysis of RNA polymerase II (pol II) transcription elongation. Recently resolved X-ray structures showed that the trigger loop forms direct interactions with the β-phosphate and base of the matched nucleotide triphosphate (NTP) through residues His1085 and Leu1081, respectively. In order to understand the role of these two critical residues in stabilizing active site conformation in the dynamic complex, we performed all-atom molecular dynamics simulations of the wild-type pol II elongation complex and its mutants in explicit solvent. In the wild-type complex, we found that the trigger loop is stabilized in the “closed” conformation, and His1085 forms a stable interaction with the NTP. Simulations of point mutations of His1085 are shown to affect this interaction; simulations of alternative protonation states, which are inaccessible through experiment, indicate that only the protonated form is able to stabilize the His1085-NTP interaction. Another trigger loop residue, Leu1081, stabilizes the incoming nucleotide position through interaction with the nucleotide base. Our simulations of this Leu mutant suggest a three-component mechanism for correctly positioning the incoming NTP in which (i) hydrophobic contact through Leu1081, (ii) base stacking, and (iii) base pairing work together to minimize the motion of the incoming NTP base. These results complement experimental observations and provide insight into the role of the trigger loop on transcription fidelity. PMID:20798057

Mutation of mapped TIA-1/TIAR binding sites in the 3' terminal stem-loop of West Nile virus minus-strand RNA in an infectious clone negatively affects genomic RNA amplification.

PubMed

Emara, Mohamed M; Liu, Hsuan; Davis, William G; Brinton, Margo A

2008-11-01

Previous data showed that the cellular proteins TIA-1 and TIAR bound specifically to the West Nile virus 3' minus-strand stem-loop [WNV3'(-)SL] RNA (37) and colocalized with flavivirus replication complexes in WNV- and dengue virus-infected cells (21). In the present study, the sites on the WNV3'(-)SL RNA required for efficient in vitro T-cell intracellular antigen-related (TIAR) and T-cell intracellular antigen-1 (TIA-1) protein binding were mapped to short AU sequences (UAAUU) located in two internal loops of the WNV3'(-)SL RNA structure. Infectious clone RNAs with all or most of the binding site nucleotides in one of the 3' (-)SL loops deleted or substituted did not produce detectable virus after transfection or subsequent passage. With one exception, deletion/mutation of a single terminal nucleotide in one of the binding sequences had little effect on the efficiency of protein binding or virus production, but mutation of a nucleotide in the middle of a binding sequence reduced both the in vitro protein binding efficiency and virus production. Plaque size, intracellular genomic RNA levels, and virus production progressively decreased with decreasing in vitro TIAR/TIA-1 binding activity, but the translation efficiency of the various mutant RNAs was similar to that of the parental RNA. Several of the mutant RNAs that inefficiently interacted with TIAR/TIA-1 in vitro rapidly reverted in vivo, indicating that they could replicate at a low level and suggesting that an interaction between TIAR/TIA-1 and the viral 3'(-)SL RNA is not required for initial low-level symmetric RNA replication but instead facilitates the subsequent asymmetric amplification of genome RNA from the minus-strand template.

Refined molecular hinge between allosteric and catalytic domain determines allosteric regulation and stability of fungal chorismate mutase

PubMed Central

Helmstaedt, Kerstin; Heinrich, Gabriele; Lipscomb, William N.; Braus, Gerhard H.

2002-01-01

The yeast chorismate mutase is regulated by tyrosine as feedback inhibitor and tryptophan as crosspathway activator. The monomer consists of a catalytic and a regulatory domain covalently linked by the loop L220s (212–226), which functions as a molecular hinge. Two monomers form the active dimeric enzyme stabilized by hydrophobic interactions in the vicinity of loop L220s. The role of loop L220s and its environment for enzyme regulation, dimerization, and stability was analyzed. Substitution of yeast loop L220s in place of the homologous loop from the corresponding and similarly regulated Aspergillus enzyme (and the reverse substitution) changed tyrosine inhibition to activation. Yeast loop L220s substituted into the Aspergillus enzyme resulted in a tryptophan-inhibitable enzyme. Monomeric yeast chorismate mutases could be generated by substituting two hydrophobic residues in and near the hinge region. The resulting Thr-212→Asp–Phe-28→Asp enzyme was as stable as wild type, but lost allosteric regulation and showed reduced catalytic activity. These results underline the crucial role of this molecular hinge for inhibition, activation, quaternary structure, and stability of yeast chorismate mutase. PMID:11997452

Probing the Self-Assembly and the Accompanying Structural Changes of Hydrophobin SC3 on a Hydrophobic Surface by Mass Spectrometry

PubMed Central

Wang, X.; Permentier, H. P.; Rink, R.; Kruijtzer, J. A. W.; Liskamp, R. M. J.; Wösten, H. A. B.; Poolman, B.; Robillard, G. T.

2004-01-01

The fungal class I hydrophobin SC3 self-assembles into an amphipathic membrane at hydrophilic-hydrophobic interfaces such as the water-air and water-Teflon interface. During self-assembly, the water-soluble state of SC3 proceeds via the intermediate α-helical state to the stable end form called the β-sheet state. Self-assembly of the hydrophobin at the Teflon surface is arrested in the α-helical state. The β-sheet state can be induced at elevated temperature in the presence of detergent. The structural changes of SC3 were monitored by various mass spectrometry techniques. We show that the so-called second loop of SC3 (C39–S72) has a high affinity for Teflon. Binding of this part of SC3 to Teflon was accompanied by the formation of α-helical structure and resulted in low solvent accessibility. The solvent-protected region of the second loop extended upon conversion to the β-sheet state. In contrast, the C-terminal part of SC3 became more exposed to the solvent. The results indicate that the second loop of class I hydrophobins plays a pivotal role in self-assembly at the hydrophilic-hydrophobic interface. Of interest, this loop is much smaller in case of class II hydrophobins, which may explain the differences in their assembly. PMID:15345568

Cell-cycle dynamics of chromosomal organisation at single-cell resolution

PubMed Central

Nagano, Takashi; Lubling, Yaniv; Várnai, Csilla; Dudley, Carmel; Leung, Wing; Baran, Yael; Mendelson-Cohen, Netta; Wingett, Steven; Fraser, Peter; Tanay, Amos

2017-01-01

Summary Chromosomes in proliferating metazoan cells undergo dramatic structural metamorphoses every cell cycle, alternating between highly condensed mitotic structures facilitating chromosome segregation, and decondensed interphase structures accommodating transcription, gene silencing and DNA replication. Here we use single-cell Hi-C to study chromosome conformations in thousands of individual cells, and discover a continuum of cis-interaction profiles that finely position individual cells along the cell cycle. We show that chromosomal compartments, topological associated domains (TADs), contact insulation and long-range loops, all defined by bulk Hi-C maps, are governed by distinct cell-cycle dynamics. In particular, DNA replication correlates with build-up of compartments and reduction in TAD insulation, while loops are generally stable from G1 through S and G2. Whole-genome 3D structural models reveal a radial architecture of chromosomal compartments with distinct epigenomic signatures. Our single-cell data thereby allow for re-interpretation of chromosome conformation maps through the prism of the cell cycle. PMID:28682332

Structure of the β-form of human MK2 in complex with the non-selective kinase inhibitor TEI-L03090

PubMed Central

Fujino, Aiko; Fukushima, Kei; Kubota, Takaharu; Matsumoto, Yoshiyuki; Takimoto-Kamimura, Midori

2013-01-01

Mitogen-activated protein kinase-activated protein kinase 2 (MK2 or MAPKAP-K2), a serine/threonine kinase from the p38 mitogen-activated protein kinase signalling pathway, plays an important role in the production of TNF-α and other cytokines. In a previous report, it was shown that MK2 in complex with the selective inhibitor TEI-I01800 adopts an α-helical glycine-rich loop that is induced by the stable nonplanar conformer of TEI-I01800. To understand the mechanism of the structural change, the structure of MK2 bound to TEI-L03090, which lacks the key substituent found in TEI-I01800, was determined. MK2–TEI-L03090 has a β-sheet glycine-rich loop in common with other kinases, as predicted. This result suggests that a small compound can induce a drastic conformational change in the target protein structure and can be used to design potent and selective inhibitors. PMID:24316826

Real-time flutter analysis of an active flutter-suppression system on a remotely piloted research aircraft

NASA Technical Reports Server (NTRS)

Gilyard, G. B.; Edwards, J. W.

1983-01-01

Flight flutter-test results of the first aeroelastic research wing (ARW-1) of NASA's drones for aerodynamic and structural testing program are presented. The flight-test operation and the implementation of the active flutter-suppression system are described as well as the software techniques used to obtain real-time damping estimates and the actual flutter testing procedure. Real-time analysis of fast-frequency aileron excitation sweeps provided reliable damping estimates. The open-loop flutter boundary was well defined at two altitudes; a maximum Mach number of 0.91 was obtained. Both open-loop and closed-loop data were of exceptionally high quality. Although the flutter-suppression system provided augmented damping at speeds below the flutter boundary, an error in the implementation of the system resulted in the system being less stable than predicted. The vehicle encountered system-on flutter shortly after crossing the open-loop flutter boundary on the third flight and was lost. The aircraft was rebuilt. Changes made in real-time test techniques are included.

Simple and Flexible Self-Reproducing Structures in Asynchronous Cellular Automata and Their Dynamics

NASA Astrophysics Data System (ADS)

Huang, Xin; Lee, Jia; Yang, Rui-Long; Zhu, Qing-Sheng

2013-03-01

Self-reproduction on asynchronous cellular automata (ACAs) has attracted wide attention due to the evident artifacts induced by synchronous updating. Asynchronous updating, which allows cells to undergo transitions independently at random times, might be more compatible with the natural processes occurring at micro-scale, but the dark side of the coin is the increment in the complexity of an ACA in order to accomplish stable self-reproduction. This paper proposes a novel model of self-timed cellular automata (STCAs), a special type of ACAs, where unsheathed loops are able to duplicate themselves reliably in parallel. The removal of sheath cannot only allow various loops with more flexible and compact structures to replicate themselves, but also reduce the number of cell states of the STCA as compared to the previous model adopting sheathed loops [Y. Takada, T. Isokawa, F. Peper and N. Matsui, Physica D227, 26 (2007)]. The lack of sheath, on the other hand, often tends to cause much more complicated interactions among loops, when all of them struggle independently to stretch out their constructing arms at the same time. In particular, such intense collisions may even cause the emergence of a mess of twisted constructing arms in the cellular space. By using a simple and natural method, our self-reproducing loops (SRLs) are able to retract their arms successively, thereby disentangling from the mess successfully.

Studies of uranium-sodium suspensions. Part I. Construction and operation of experimental loop

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bett, F L; Hilditch, R J; Mepham, R G

1961-08-01

An experimnental uranium- sodium suspension loop was operated for 4320 hr. The design, construction, commissioning, and operation of the loop to the point where a comnplete stable suspension was obtained is described.

Closed-Loop Control of Vortex Formation in Separated Flows

NASA Technical Reports Server (NTRS)

Colonius, Tim; Joe, Won Tae; MacMynowski, Doug; Rowley, Clancy; Taira, Sam; Ahuja, Sunil

2010-01-01

In order to phase lock the flow at the desired shedding cycle, particularly at Phi,best, We designed a feedback compensator. (Even though the open-loop forcing at Wf below Wn can lead to phase-locked limit cycles with a high average lift,) This feedback controller resulted in the phase-locked limit cycles that the open-loop control could not achieve for alpha=30 and 40 Particularly for alpha=40, the feedback was able to stabilize the limit cycle that was not stable with any of the open-loop periodic forcing. This results in stable phase-locked limit cycles for a larger range of forcing frequencies than the open-loop control. Also, it was shown that the feedback achieved the high-lift unsteady flow states that open-loop control could not sustain even after the states have been achieved for a long period of time.

Reduced modeling of flexible structures for decentralized control

NASA Technical Reports Server (NTRS)

Yousuff, A.; Tan, T. M.; Bahar, L. Y.; Konstantinidis, M. F.

1986-01-01

Based upon the modified finite element-transfer matrix method, this paper presents a technique for reduced modeling of flexible structures for decentralized control. The modeling decisions are carried out at (finite-) element level, and are dictated by control objectives. A simply supported beam with two sets of actuators and sensors (linear force actuator and linear position and velocity sensors) is considered for illustration. In this case, it is conjectured that the decentrally controlled closed loop system is guaranteed to be at least marginally stable.

Conformational Flexibility of a Short Loop near the Active Site of the SARS-3CLpro is Essential to Maintain Catalytic Activity

NASA Astrophysics Data System (ADS)

Li, Chunmei; Teng, Xin; Qi, Yifei; Tang, Bo; Shi, Hailing; Ma, Xiaomin; Lai, Luhua

2016-02-01

The SARS 3C-like proteinase (SARS-3CLpro), which is the main proteinase of the SARS coronavirus, is essential to the virus life cycle. This enzyme has been shown to be active as a dimer in which only one protomer is active. However, it remains unknown how the dimer structure maintains an active monomer conformation. It has been observed that the Ser139-Leu141 loop forms a short 310-helix that disrupts the catalytic machinery in the inactive monomer structure. We have tried to disrupt this helical conformation by mutating L141 to T in the stable inactive monomer G11A/R298A/Q299A. The resulting tetra-mutant G11A/L141T/R298A/Q299A is indeed enzymatically active as a monomer. Molecular dynamics simulations revealed that the L141T mutation disrupts the 310-helix and helps to stabilize the active conformation. The coil-310-helix conformational transition of the Ser139-Leu141 loop serves as an enzyme activity switch. Our study therefore indicates that the dimer structure can stabilize the active conformation but is not a required structure in the evolution of the active enzyme, which can also arise through simple mutations.

A short autocomplementary sequence plays an essential role in avian sarcoma-leukosis virus RNA dimerization.

PubMed

Fossé, P; Motté, N; Roumier, A; Gabus, C; Muriaux, D; Darlix, J L; Paoletti, J

1996-12-24

Retroviral genomes consist of two identical RNA molecules joined noncovalently near their 5'-ends. Recently, two models have been proposed for RNA dimer formation on the basis of results obtained in vitro with human immunodeficiency virus type 1 RNA and Moloney murine leukemia virus RNA. It was first proposed that viral RNA dimerizes by forming an interstrand quadruple helix with purine tetrads. The second model postulates that RNA dimerization is initiated by a loop-loop interaction between the two RNA molecules. In order to better characterize the dimerization process of retroviral genomic RNA, we analyzed the in vitro dimerization of avian sarcoma-leukosis virus (ASLV) RNA using different transcripts. We determined the requirements for heterodimer formation, the thermal dissociation of RNA dimers, and the influence of antisense DNA oligonucleotides on dimer formation. Our results strongly suggest that purine tetrads are not involved in dimer formation. Data show that an autocomplementary sequence located upstream from the splice donor site and within a major packaging signal plays a crucial role in ASLV RNA dimer formation in vitro. This sequence is able to form a stem-loop structure, and phylogenetic analysis reveals that it is conserved in 28 different avian sarcoma and leukosis viruses. These results suggest that dimerization of ASLV RNA is initiated by a loop-loop interaction between two RNA molecules and provide an additional argument for the ubiquity of the dimerization process via loop-loop interaction.

Double stranded nucleic acid biochips

DOEpatents

Chernov, Boris; Golova, Julia

2006-05-23

This invention describes a new method of constructing double-stranded DNA (dsDNA) microarrays based on the use of pre-synthesized or natural DNA duplexes without a stem-loop structure. The complementary oligonucleotide chains are bonded together by a novel connector that includes a linker for immobilization on a matrix. A non-enzymatic method for synthesizing double-stranded nucleic acids with this novel connector enables the construction of inexpensive and robust dsDNA/dsRNA microarrays. DNA-DNA and DNA-protein interactions are investigated using the microarrays.

Loss of a single N-linked glycan allows CD4-independent human immunodeficiency virus type 1 infection by altering the position of the gp120 V1/V2 variable loops.

PubMed

Kolchinsky, P; Kiprilov, E; Bartley, P; Rubinstein, R; Sodroski, J

2001-04-01

The gp120 envelope glycoprotein of primary human immunodeficiency virus type 1 (HIV-1) promotes virus entry by sequentially binding CD4 and the CCR5 chemokine receptor on the target cell. Previously, we adapted a primary HIV-1 isolate, ADA, to replicate in CD4-negative canine cells expressing human CCR5. The gp120 changes responsible for CD4-independent replication were limited to the V2 loop-V1/V2 stem. Here we show that elimination of a single glycosylation site at asparagine 197 in the V1/V2 stem is sufficient for CD4-independent gp120 binding to CCR5 and for HIV-1 entry into CD4-negative cells expressing CCR5. Deletion of the V1/V2 loops also allowed CD4-independent viral entry and gp120 binding to CCR5. The binding of the wild-type ADA gp120 to CCR5 was less dependent upon CD4 at 4 degrees C than at 37 degrees C. In the absence of the V1/V2 loops, neither removal of the N-linked carbohydrate at asparagine 197 nor lowering of the temperature increased the CD4-independent phenotypes. A CCR5-binding conformation of gp120, achieved by CD4 interaction or by modification of temperature, glycosylation, or variable loops, was preferentially recognized by the monoclonal antibody 48d. These results suggest that the CCR5-binding region of gp120 is occluded by the V1/V2 variable loops, the position of which can be modulated by temperature, CD4 binding, or an N-linked glycan in the V1/V2 stem.

High-throughput determination of RNA structure by proximity ligation.

PubMed

Ramani, Vijay; Qiu, Ruolan; Shendure, Jay

2015-09-01

We present an unbiased method to globally resolve RNA structures through pairwise contact measurements between interacting regions. RNA proximity ligation (RPL) uses proximity ligation of native RNA followed by deep sequencing to yield chimeric reads with ligation junctions in the vicinity of structurally proximate bases. We apply RPL in both baker's yeast (Saccharomyces cerevisiae) and human cells and generate contact probability maps for ribosomal and other abundant RNAs, including yeast snoRNAs, the RNA subunit of the signal recognition particle and the yeast U2 spliceosomal RNA homolog. RPL measurements correlate with established secondary structures for these RNA molecules, including stem-loop structures and long-range pseudoknots. We anticipate that RPL will complement the current repertoire of computational and experimental approaches in enabling the high-throughput determination of secondary and tertiary RNA structures.

Identification of antisense nucleic acid hybridization sites in mRNA molecules with self-quenching fluorescent reporter molecules

PubMed Central

Gifford, Lida K.; Opalinska, Joanna B.; Jordan, David; Pattanayak, Vikram; Greenham, Paul; Kalota, Anna; Robbins, Michelle; Vernovsky, Kathy; Rodriguez, Lesbeth C.; Do, Bao T.; Lu, Ponzy; Gewirtz, Alan M.

2005-01-01

We describe a physical mRNA mapping strategy employing fluorescent self-quenching reporter molecules (SQRMs) that facilitates the identification of mRNA sequence accessible for hybridization with antisense nucleic acids in vitro and in vivo, real time. SQRMs are 20–30 base oligodeoxynucleotides with 5–6 bp complementary ends to which a 5′ fluorophore and 3′ quenching group are attached. Alone, the SQRM complementary ends form a stem that holds the fluorophore and quencher in contact. When the SQRM forms base pairs with its target, the structure separates the fluorophore from the quencher. This event can be reported by fluorescence emission when the fluorophore is excited. The stem–loop of the SQRM suggests that SQRM be made to target natural stem–loop structures formed during mRNA synthesis. The general utility of this method is demonstrated by SQRM identification of targetable sequence within c-myb and bcl-6 mRNA. Corresponding antisense oligonucleotides reduce these gene products in cells. PMID:15718294

Complete Sequence of the mitochondrial genome of the tapeworm Hymenolepis diminuta: Gene arrangements indicate that platyhelminths are eutrochozoans

DOE Office of Scientific and Technical Information (OSTI.GOV)

von Nickisch-Rosenegk, Markus; Brown, Wesley M.; Boore, Jeffrey L.

2001-01-01

Using ''long-PCR'' we have amplified in overlapping fragments the complete mitochondrial genome of the tapeworm Hymenolepis diminuta (Platyhelminthes: Cestoda) and determined its 13,900 nucleotide sequence. The gene content is the same as that typically found for animal mitochondrial DNA (mtDNA) except that atp8 appears to be lacking, a condition found previously for several other animals. Despite the small size of this mtDNA, there are two large non-coding regions, one of which contains 13 repeats of a 31 nucleotide sequence and a potential stem-loop structure of 25 base pairs with an 11-member loop. Large potential secondary structures are identified also formore » the non-coding regions of two other cestode mtDNAs. Comparison of the mitochondrial gene arrangement of H. diminuta with those previously published supports a phylogenetic position of flatworms as members of the Eutrochozoa, rather than being basal to either a clade of protostomes or a clade of coelomates.« less

The mitochondrial genome of Pocillopora (Cnidaria: Scleractinia) contains two variable regions: the putative D-loop and a novel ORF of unknown function.

PubMed

Flot, Jean-François; Tillier, Simon

2007-10-15

The complete mitochondrial genomes of two individuals attributed to different morphospecies of the scleractinian coral genus Pocillopora have been sequenced. Both genomes, respectively 17,415 and 17,422 nt long, share the presence of a previously undescribed ORF encoding a putative protein made up of 302 amino acids and of unknown function. Surprisingly, this ORF turns out to be the second most variable region of the mitochondrial genome (1% nucleotide sequence difference between the two individuals) after the putative control region (1.5% sequence difference). Except for the presence of this ORF and for the location of the putative control region, the mitochondrial genome of Pocillopora is organized in a fashion similar to the other scleractinian coral genomes published to date. For the first time in a cnidarian, a putative second origin of replication is described based on its secondary structure similar to the stem-loop structure of O(L), the origin of L-strand replication in vertebrates.

New tRNA contacts facilitate ligand binding in a Mycobacterium smegmatis T box riboswitch.

PubMed

Sherwood, Anna V; Frandsen, Jane K; Grundy, Frank J; Henkin, Tina M

2018-04-10

T box riboswitches are RNA regulatory elements widely used by organisms in the phyla Firmicutes and Actinobacteria to regulate expression of amino acid-related genes. Expression of T box family genes is down-regulated by transcription attenuation or inhibition of translation initiation in response to increased charging of the cognate tRNA. Three direct contacts with tRNA have been described; however, one of these contacts is absent in a subclass of T box RNAs and the roles of several structural domains conserved in most T box RNAs are unknown. In this study, structural elements of a Mycobacterium smegmatis ileS T box riboswitch variant with an Ultrashort (US) Stem I were sequentially deleted, which resulted in a progressive decrease in binding affinity for the tRNA Ile ligand. Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) revealed structural changes in conserved riboswitch domains upon interaction with the tRNA ligand. Cross-linking and mutational analyses identified two interaction sites, one between the S-turn element in Stem II and the T arm of tRNA Ile and the other between the Stem IIA/B pseudoknot and the D loop of tRNA Ile These newly identified RNA contacts add information about tRNA recognition by the T box riboswitch and demonstrate a role for the S-turn and pseudoknot elements, which resemble structural elements that are common in many cellular RNAs.

Dissecting the hybridization of oligonucleotides to structured complementary sequences.

PubMed

Peracchi, Alessio

2016-06-01

When oligonucleotides hybridize to long target molecules, the process is slowed by the secondary structure in the targets. The phenomenon has been analyzed in several previous studies, but many details remain poorly understood. I used a spectrofluorometric strategy, focusing on the formation/breaking of individual base pairs, to study the kinetics of association between a DNA hairpin and >20 complementary oligonucleotides ('antisenses'). Hybridization rates differed by over three orders of magnitude. Association was toehold-mediated, both for antisenses binding to the target's ends and for those designed to interact with the loop. Binding of these latter, besides being consistently slower, was affected to variable, non-uniform extents by the asymmetric loop structure. Divalent metal ions accelerated hybridization, more pronouncedly when nucleation occurred at the loop. Incorporation of locked nucleic acid (LNA) residues in the antisenses substantially improved the kinetics only when LNAs participated to the earliest hybridization steps. The effects of individual LNAs placed along the antisense indicated that the reaction transition state occurred after invading at least the first base pair of the stem. The experimental approach helps dissect hybridization reactions involving structured nucleic acids. Toehold-dependent, nucleation-invasion models appear fully appropriate for describing such reactions. Estimating the stability of nucleation complexes formed at internal toeholds is the major hurdle for the quantitative prediction of hybridization rates. While analyzing the mechanisms of a fundamental biochemical process (hybridization), this work also provides suggestions for the improvement of technologies that rely on such process. Copyright © 2016 Elsevier B.V. All rights reserved.

pH Dependence of a 310-Helix versus a Turn in the M-Loop Region of PDE4: Observations on PDB Entries and an Electronic Structure Study.

PubMed

Usharani, Dandamudi; Srivani, Palakuri; Sastry, G Narahari; Jemmis, Eluvathingal D

2008-06-01

Available X-ray crystal structures of phosphodiesterase 4 (PDE 4) are classified into two groups based on a secondary structure difference of a 310-helix versus a turn in the M-loop region. The only variable that was discernible between these two sets is the pH at the crystallization conditions. Assuming that at lower pH there is a possibility of protonation, thermodynamics of protonation and deprotonation of the aspartic acid, cysteine side chains, and amide bonds are calculated. The models in the gas phase and in the explicit solvent using the ONIOM method are calculated at the B3LYP/6-31+G* and B3LYP/6-31+G*:UFF levels of theory, respectively. The molecular dynamics (MD) simulations are also performed on the M-loop region of a 310-helix and a turn with explicit water for 10 ns under NPT conditions. The isodesmic equations of the various protonation states show that the turn containing structure is thermodynamically more stable when proline or cysteine is protonated. The preference for the turn structure on protonation (pH = 6.5-7.5) is due to an increase in the number of the hydrogen bonding and electrostatic interactions gained by the surrounding environment such as adjacent residues and solvent molecules.

Fe-rich and Si-rich ejecta distribution in the Cygnus Loop

NASA Astrophysics Data System (ADS)

Nakajima, Hiroshi; Tsunemi, Hiroshi; Kimura, Masashi; Uchida, Hiroyuki; Katsuda, Satoru

2012-03-01

We report on the results of Suzaku observations of central region of the proto-typical middle-aged Supernova remnant (SNR) Cygnus Loop. The southeastern region of the SNR with the distance from the geometric center ranging from 10' to 55' is observed using XIS. Thanks to the low and stable background of Suzaku, the distribution maps of heavy species such as Fe, S, Si, Mg, Ne and O arereliably obtained. We found that the relatively heavier four species are confined to the small centermost region while the lighter species show uniform distribution or depressed center region. These results exhibit an onion-like metallicity structure in this SNR, which reflects the metal distribution of the progenitor.

Beyond R0 Maximisation: On Pathogen Evolution and Environmental Dimensions.

PubMed

Lion, Sébastien; Metz, Johan A J

2018-06-01

A widespread tenet is that evolution of pathogens maximises their basic reproduction ratio, R 0 . The breakdown of this principle is typically discussed as exception. Here, we argue that a radically different stance is needed, based on evolutionarily stable strategy (ESS) arguments that take account of the 'dimension of the environmental feedback loop'. The R 0 maximisation paradigm requires this feedback loop to be one-dimensional, which notably excludes pathogen diversification. By contrast, almost all realistic ecological ingredients of host-pathogen interactions (density-dependent mortality, multiple infections, limited cross-immunity, multiple transmission routes, host heterogeneity, and spatial structure) will lead to multidimensional feedbacks. Copyright © 2018 Elsevier Ltd. All rights reserved.

Modelling, design and stability analysis of an improved SEPIC converter for renewable energy systems

NASA Astrophysics Data System (ADS)

G, Dileep; Singh, S. N.; Singh, G. K.

2017-09-01

In this paper, a detailed modelling and analysis of a switched inductor (SI)-based improved single-ended primary inductor converter (SEPIC) has been presented. To increase the gain of conventional SEPIC converter, input and output side inductors are replaced with SI structures. Design and stability analysis for continuous conduction mode operation of the proposed SI-SEPIC converter has also been presented in this paper. State space averaging technique is used to model the converter and carry out the stability analysis. Performance and stability analysis of closed loop configuration is predicted by observing the open loop behaviour using Nyquist diagram and Nichols chart. System was found to stable and critically damped.

miR-137 forms a regulatory loop with nuclear receptor TLX and LSD1 in neural stem cells

PubMed Central

Sun, GuoQiang; Ye, Peng; Murai, Kiyohito; Lang, Ming-Fei; Li, Shengxiu; Zhang, Heying; Li, Wendong; Fu, Chelsea; Yin, Jason; Wang, Allen; Ma, Xiaoxiao; Shi, Yanhong

2012-01-01

miR-137 is a brain-enriched microRNA. Its role in neural development remains unknown. Here we show that miR-137 plays an essential role in controlling embryonic neural stem cell fate determination. miR-137 negatively regulates cell proliferation and accelerates neural differentiation of embryonic neural stem cells. In addition, we show that histone demethylase LSD1, a transcriptional co-repressor of nuclear receptor TLX, is a downstream target of miR-137. In utero electroporation of miR-137 in embryonic mouse brains led to premature differentiation and outward migration of the transfected cells. Introducing a LSD1 expression vector lacking the miR-137 recognition site rescued miR-137-induced precocious differentiation. Furthermore, we demonstrate that TLX, an essential regulator of neural stem cell self-renewal, represses the expression of miR-137 by recruiting LSD1 to the genomic regions of miR-137. Thus, miR-137 forms a feedback regulatory loop with TLX and LSD1 to control the dynamics between neural stem cell proliferation and differentiation during neural development. PMID:22068596

Structure-function analysis of the DNA translocating portal of the bacteriophage T4 packaging machine.

PubMed

Padilla-Sanchez, Victor; Gao, Song; Kim, Hyung Rae; Kihara, Daisuke; Sun, Lei; Rossmann, Michael G; Rao, Venigalla B

2014-03-06

Tailed bacteriophages and herpesviruses consist of a structurally well conserved dodecameric portal at a special 5-fold vertex of the capsid. The portal plays critical roles in head assembly, genome packaging, neck/tail attachment, and genome ejection. Although the structures of portals from phages φ29, SPP1, and P22 have been determined, their mechanistic roles have not been well understood. Structural analysis of phage T4 portal (gp20) has been hampered because of its unusual interaction with the Escherichia coli inner membrane. Here, we predict atomic models for the T4 portal monomer and dodecamer, and we fit the dodecamer into the cryo-electron microscopy density of the phage portal vertex. The core structure, like that from other phages, is cone shaped with the wider end containing the "wing" and "crown" domains inside the phage head. A long "stem" encloses a central channel, and a narrow "stalk" protrudes outside the capsid. A biochemical approach was developed to analyze portal function by incorporating plasmid-expressed portal protein into phage heads and determining the effect of mutations on head assembly, DNA translocation, and virion production. We found that the protruding loops of the stalk domain are involved in assembling the DNA packaging motor. A loop that connects the stalk to the channel might be required for communication between the motor and the portal. The "tunnel" loops that project into the channel are essential for sealing the packaged head. These studies established that the portal is required throughout the DNA packaging process, with different domains participating at different stages of genome packaging. © 2013.

The 5′-tail of antisense RNAII of pMV158 plays a critical role in binding to the target mRNA and in translation inhibition of repB

PubMed Central

López-Aguilar, Celeste; Romero-López, Cristina; Espinosa, Manuel; Berzal-Herranz, Alfredo; del Solar, Gloria

2015-01-01

Rolling-circle replication of streptococcal plasmid pMV158 is controlled by the concerted action of two trans-acting elements, namely transcriptional repressor CopG and antisense RNAII, which inhibit expression of the repB gene encoding the replication initiator protein. The pMV158-encoded antisense RNAII exerts its activity of replication control by inhibiting translation of the essential repB gene. RNAII is the smallest and simplest among the characterized antisense RNAs involved in control of plasmid replication. Structure analysis of RNAII revealed that it folds into an 8-bp-long stem containing a 1-nt bulge and closed by a 6-nt apical loop. This hairpin is flanked by a 17-nt-long single-stranded 5′-tail and an 8-nt-long 3′-terminal U-rich stretch. Here, the 3′ and 5′ regions of the 5′-tail of RNAII are shown to play a critical role in the binding to the target mRNA and in the inhibition of repB translation, respectively. In contrast, the apical loop of the single hairpin of RNAII plays a rather secondary role and the upper stem region hardly contributes to the binding or inhibition processes. The entire 5′-tail is required for efficient inhibition of repB translation, though only the 8-nt-long region adjacent to the hairpin seems to be essential for rapid binding to the mRNA. These results show that a “kissing” interaction involving base-pairing between complementary hairpin loops in RNAII and mRNA is not critical for efficient RNA/RNA binding or repB translation inhibition. A singular binding mechanism is envisaged whereby initial pairing between complementary single-stranded regions in the antisense and sense RNAs progresses upwards into the corresponding hairpin stems to form the intermolecular duplex. PMID:26175752

Highly Stable Bimetallic AuIr/TiO₂ Catalyst: Physical Origins of the Intrinsic High Stability against Sintering.

PubMed

Han, Chang Wan; Majumdar, Paulami; Marinero, Ernesto E; Aguilar-Tapia, Antonio; Zanella, Rodolfo; Greeley, Jeffrey; Ortalan, Volkan

2015-12-09

It has been a long-lived research topic in the field of heterogeneous catalysts to find a way of stabilizing supported gold catalyst against sintering. Herein, we report highly stable AuIr bimetallic nanoparticles on TiO2 synthesized by sequential deposition-precipitation. To reveal the physical origin of the high stability of AuIr/TiO2, we used aberration-corrected scanning transmission electron microscopy (STEM), STEM-tomography, and density functional theory (DFT) calculations. Three-dimensional structures of AuIr/TiO2 obtained by STEM-tomography indicate that AuIr nanoparticles on TiO2 have intrinsically lower free energy and less driving force for sintering than Au nanoparticles. DFT calculations on segregation behavior of AuIr slabs on TiO2 showed that the presence of Ir near the TiO2 surface increases the adhesion energy of the bimetallic slabs to the TiO2 and the attractive interactions between Ir and TiO2 lead to higher stability of AuIr nanoparticles as compared to Au nanoparticles.

Intracellular modifications induced by poliovirus reduce the requirement for structural motifs in the 5' noncoding region of the genome involved in internal initiation of protein synthesis.

PubMed Central

Percy, N; Belsham, G J; Brangwyn, J K; Sullivan, M; Stone, D M; Almond, J W

1992-01-01

A series of genetic deletions based partly on two RNA secondary structure models (M. A. Skinner, V. R. Racaniello, G. Dunn, J. Cooper, P. D. Minor, and J. W. Almond, J. Mol. Biol. 207:379-392, 1989; E. V. Pilipenko, V. M. Blinov, L. I. Romanova, A. N. Sinyakov, S. V. Maslova, and V. I. Agol, Virology 168:201-209, 1989) was made in the cDNA encoding the 5' noncoding region (5' NCR) of the poliovirus genome in order to study the sequences that direct the internal entry of ribosomes. The modified cDNAs were placed between two open reading frames in a single transcriptional unit and used to transfect cells in culture. Internal entry of ribosomes was detected by measuring translation from the second open reading frame in the bicistronic mRNA. When assayed alone, a large proportion of the poliovirus 5' NCR superstructure including several well-defined stem-loops was required for ribosome entry and efficient translation. However, in cells cotransfected with a complete infectious poliovirus cDNA, the requirement for the stem-loops in this large superstructure was reduced. The results suggest that virus infection modifies the cellular translational machinery, so that shortened forms of the 5' NCR are sufficient for cap-independent translation, and that the internal entry of ribosomes occurs by two distinct modes during the virus replication cycle. Images PMID:1310772

Regulation of a maize HD-ZIP IV transcription factor by a non-conventional RDR2-dependent small RNA.

PubMed

Klein-Cosson, Catherine; Chambrier, Pierre; Rogowsky, Peter M; Vernoud, Vanessa

2015-03-01

Small non-coding RNAs are versatile riboregulators that control gene expression at the transcriptional or post-transcriptional level, governing many facets of plant development. Here we present evidence for the existence of a 24 nt small RNA (named small1) that is complementary to the 3' UTR of OCL1 (Outer Cell Layer1), the founding member of the maize HD-ZIP IV gene family encoding plant-specific transcription factors that are mainly involved in epidermis differentiation and specialization. The biogenesis of small1 depends on DICER-like 3 (DCL3), RNA-dependent RNA polymerase 2 (RDR2) and RNA polymerase IV, components that are usually required for RNA-dependent DNA-methylation. Unexpectedly, GFP sensor experiments in transient and stable transformation systems revealed that small1 may regulate its target at the post-transcriptional level, mainly through translational repression. This translational repression is attenuated in an rdr2 mutant background in which small1 does not accumulate. Our experiments further showed the possible involvement of a secondary stem-loop structure present in the 3' UTR of OCL1 for efficient target repression, suggesting the existence of several regulatory mechanisms affecting OCL1 mRNA stability and translation. © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.

Ribosomal frameshifting and transcriptional slippage: From genetic steganography and cryptography to adventitious use

PubMed Central

Atkins, John F.; Loughran, Gary; Bhatt, Pramod R.; Firth, Andrew E.; Baranov, Pavel V.

2016-01-01

Genetic decoding is not ‘frozen’ as was earlier thought, but dynamic. One facet of this is frameshifting that often results in synthesis of a C-terminal region encoded by a new frame. Ribosomal frameshifting is utilized for the synthesis of additional products, for regulatory purposes and for translational ‘correction’ of problem or ‘savior’ indels. Utilization for synthesis of additional products occurs prominently in the decoding of mobile chromosomal element and viral genomes. One class of regulatory frameshifting of stable chromosomal genes governs cellular polyamine levels from yeasts to humans. In many cases of productively utilized frameshifting, the proportion of ribosomes that frameshift at a shift-prone site is enhanced by specific nascent peptide or mRNA context features. Such mRNA signals, which can be 5′ or 3′ of the shift site or both, can act by pairing with ribosomal RNA or as stem loops or pseudoknots even with one component being 4 kb 3′ from the shift site. Transcriptional realignment at slippage-prone sequences also generates productively utilized products encoded trans-frame with respect to the genomic sequence. This too can be enhanced by nucleic acid structure. Together with dynamic codon redefinition, frameshifting is one of the forms of recoding that enriches gene expression. PMID:27436286

Mechanism of the Intramolecular Claisen Condensation Reaction Catalyzed by MenB, a Crotonase Superfamily Member†

PubMed Central

Li, Huei-Jiun; Li, Xiaokai; Liu, Nina; Zhang, Huaning; Truglio, James J.; Mishra, Shambhavi; Kisker, Caroline; Garcia-Diaz, Miguel; Tonge, Peter J.

2014-01-01

MenB, the 1,4-dihydroxy-2-naphthoyl-CoA synthase from the bacterial menaquinone biosynthesis pathway, catalyzes an intramolecular Claisen condensation (Dieckmann reaction) in which the electrophile is an unactivated carboxylic acid. Mechanistic studies on this crotonase family member have been hindered by partial active site disorder in existing MenB X-ray structures. In the current work the 2.0 Å structure of O-succinylbenzoyl-aminoCoA (OSB-NCoA) bound to the MenB from Escherichia coli provides important insight into the catalytic mechanism by revealing the position of all active site residues. This has been accomplished by the use of a stable analogue of the O-succinylbenzoyl-CoA (OSB-CoA) substrate in which the CoA thiol has been replaced by an amine. The resulting OSB-NCoA is stable and the X-ray structure of this molecule bound to MenB reveals the structure of the enzyme-substrate complex poised for carbon-carbon bond formation. The structural data support a mechanism in which two conserved active site Tyr residues, Y97 and Y258, participate directly in the intramolecular transfer of the substrate α-proton to the benzylic carboxylate of the substrate, leading to protonation of the electrophile and formation of the required carbanion. Y97 and Y258 are also ideally positioned to function as the second oxyanion hole required for stabilization of the tetrahedral intermediate formed during carbon-carbon bond formation. In contrast, D163, which is structurally homologous to the acid-base catalyst E144 in crotonase, is not directly involved in carbanion formation and may instead play a structural role by stabilizing the loop that carries Y97. When similar studies were performed on the MenB from Mycobacterium tuberculosis, a twisted hexamer was unexpectedly observed, demonstrating the flexibility of the interfacial loops that are involved in the generation of the novel tertiary and quaternary structures found in the crotonase superfamily. This work reinforces the utility of using a stable substrate analogue as a mechanistic probe in which only one atom has been altered leading to a decrease in α-proton acidity. PMID:21830810

Mechanism of the intramolecular Claisen condensation reaction catalyzed by MenB, a crotonase superfamily member.

PubMed

Li, Huei-Jiun; Li, Xiaokai; Liu, Nina; Zhang, Huaning; Truglio, James J; Mishra, Shambhavi; Kisker, Caroline; Garcia-Diaz, Miguel; Tonge, Peter J

2011-11-08

MenB, the 1,4-dihydroxy-2-naphthoyl-CoA synthase from the bacterial menaquinone biosynthesis pathway, catalyzes an intramolecular Claisen condensation (Dieckmann reaction) in which the electrophile is an unactivated carboxylic acid. Mechanistic studies on this crotonase family member have been hindered by partial active site disorder in existing MenB X-ray structures. In the current work the 2.0 Å structure of O-succinylbenzoyl-aminoCoA (OSB-NCoA) bound to the MenB from Escherichia coli provides important insight into the catalytic mechanism by revealing the position of all active site residues. This has been accomplished by the use of a stable analogue of the O-succinylbenzoyl-CoA (OSB-CoA) substrate in which the CoA thiol has been replaced by an amine. The resulting OSB-NCoA is stable, and the X-ray structure of this molecule bound to MenB reveals the structure of the enzyme-substrate complex poised for carbon-carbon bond formation. The structural data support a mechanism in which two conserved active site Tyr residues, Y97 and Y258, participate directly in the intramolecular transfer of the substrate α-proton to the benzylic carboxylate of the substrate, leading to protonation of the electrophile and formation of the required carbanion. Y97 and Y258 are also ideally positioned to function as the second oxyanion hole required for stabilization of the tetrahedral intermediate formed during carbon-carbon bond formation. In contrast, D163, which is structurally homologous to the acid-base catalyst E144 in crotonase (enoyl-CoA hydratase), is not directly involved in carbanion formation and may instead play a structural role by stabilizing the loop that carries Y97. When similar studies were performed on the MenB from Mycobacterium tuberculosis, a twisted hexamer was unexpectedly observed, demonstrating the flexibility of the interfacial loops that are involved in the generation of the novel tertiary and quaternary structures found in the crotonase superfamily. This work reinforces the utility of using a stable substrate analogue as a mechanistic probe in which only one atom has been altered leading to a decrease in α-proton acidity.

A Temporal Chromatin Signature in Human Embryonic Stem Cells Identifies Regulators of Cardiac Development

PubMed Central

Paige, Sharon L.; Thomas, Sean; Stoick-Cooper, Cristi L.; Wang, Hao; Maves, Lisa; Sandstrom, Richard; Pabon, Lil; Reinecke, Hans; Pratt, Gabriel; Keller, Gordon; Moon, Randall T.; Stamatoyannopoulos, John; Murry, Charles E.

2012-01-01

Summary Directed differentiation of human embryonic stem cells (ESCs) into cardiovascular cells provides a model for studying molecular mechanisms of human cardiovascular development. Though it is known that chromatin modification patterns in ESCs differ markedly from those in lineage-committed progenitors and differentiated cells, the temporal dynamics of chromatin alterations during differentiation along a defined lineage have not been studied. We show that differentiation of human ESCs into cardiovascular cells is accompanied by programmed temporal alterations in chromatin structure that distinguish key regulators of cardiovascular development from other genes. We used this temporal chromatin signature to identify regulators of cardiac development, including the homeobox gene MEIS2. We demonstrate using the zebrafish model that MEIS2 is critical for proper heart tube formation and subsequent cardiac looping. Temporal chromatin signatures should be broadly applicable to other models of stem cell differentiation to identify regulators and provide key insights into major developmental decisions. PMID:22981225

Thermal stability of DNA quadruplex-duplex hybrids.

PubMed

Lim, Kah Wai; Khong, Zi Jian; Phan, Anh Tuân

2014-01-14

DNA has the capacity to adopt several distinct structural forms, such as duplex and quadruplex helices, which have been implicated in cellular processes and shown to exhibit important functional properties. Quadruplex-duplex hybrids, generated from the juxtaposition of these two structural elements, could find applications in therapeutics and nanotechnology. Here we used NMR and CD spectroscopy to investigate the thermal stability of two classes of quadruplex-duplex hybrids comprising fundamentally distinct modes of duplex and quadruplex connectivity: Construct I involves the coaxial orientation of the duplex and quadruplex helices with continual base stacking across the two components; Construct II involves the orthogonal orientation of the duplex and quadruplex helices with no base stacking between the two components. We have found that for both constructs, the stability of the quadruplex generally increases with the length of the stem-loop incorporated, with respect to quadruplexes comprising nonstructured loops of the same length, which showed a continuous drop in stability with increasing loop length. The stability of these complexes, particularly Construct I, can be substantially influenced by the base-pair steps proximal to the quadruplex-duplex junction. Bulges at the junction are largely detrimental to the adoption of the desired G-quadruplex topology for Construct I but not for Construct II. These findings should facilitate future design and prediction of quadruplex-duplex hybrids.

Exploring TAR–RNA aptamer loop–loop interaction by X-ray crystallography, UV spectroscopy and surface plasmon resonance

PubMed Central

Lebars, Isabelle; Legrand, Pierre; Aimé, Ahissan; Pinaud, Noël; Fribourg, Sébastien; Di Primo, Carmelo

2008-01-01

In HIV-1, trans-activation of transcription of the viral genome is regulated by an imperfect hairpin, the trans-activating responsive (TAR) RNA element, located at the 5′ untranslated end of all viral transcripts. TAR acts as a binding site for viral and cellular proteins. In an attempt to identify RNA ligands that would interfere with the virus life-cycle by interacting with TAR, an in vitro selection was previously carried out. RNA hairpins that formed kissing-loop dimers with TAR were selected [Ducongé F. and Toulmé JJ (1999) RNA, 5:1605–1614]. We describe here the crystal structure of TAR bound to a high-affinity RNA aptamer. The two hairpins form a kissing complex and interact through six Watson–Crick base pairs. The complex adopts an overall conformation with an inter-helix angle of 28.1°, thus contrasting with previously reported solution and modelling studies. Structural analysis reveals that inter-backbone hydrogen bonds between ribose 2′ hydroxyl and phosphate oxygens at the stem-loop junctions can be formed. Thermal denaturation and surface plasmon resonance experiments with chemically modified 2′-O-methyl incorporated into both hairpins at key positions, clearly demonstrate the involvement of this intermolecular network of hydrogen bonds in complex stability. PMID:18996893

Irradiation effects in UO2 and CeO2

NASA Astrophysics Data System (ADS)

Ye, Bei; Oaks, Aaron; Kirk, Mark; Yun, Di; Chen, Wei-Ying; Holtzman, Benjamin; Stubbins, James F.

2013-10-01

Single crystal CeO2, as a surrogate material to UO2, was irradiated with 500 keV xenon ions at 800 °C while being observed using in situ transmission electron microscopy (TEM). Experimental results show the formation and growth of defect clusters including dislocation loops and cavities as a function of increasing atomic displacement dose. At high dose, the dislocation loop structure evolves into an extended dislocation line structure, which appears to remain stable to the high dose levels examined in this study. A high concentration of cavities was also present in the microstructure. Despite high atomic displacement doses, the specimen remained crystalline to a cumulated dose of 5 × 1015 ions/cm2, which is consistent with the known stability of the fluorite structure under high dose irradiation. Kinetic Monte Carlo calculations show that oxygen mobility is substantially higher in hypo-stoichiometric UO2/CeO2 than hyper-stoichiometric systems. This result is consistent with the ability of irradiation damage to recover even at intermediate irradiation temperatures.

Self-organized centripetal movement of corneal epithelium in the absence of external cues

NASA Astrophysics Data System (ADS)

Lobo, Erwin P.; Delic, Naomi C.; Richardson, Alex; Raviraj, Vanisri; Halliday, Gary M.; di Girolamo, Nick; Myerscough, Mary R.; Lyons, J. Guy

2016-08-01

Maintaining the structure of the cornea is essential for high-quality vision. In adult mammals, corneal epithelial cells emanate from stem cells in the limbus, driven by an unknown mechanism towards the centre of the cornea as cohesive clonal groups. Here we use complementary mathematical and biological models to show that corneal epithelial cells can self-organize into a cohesive, centripetal growth pattern in the absence of external physiological cues. Three conditions are required: a circumferential location of stem cells, a limited number of cell divisions and mobility in response to population pressure. We have used these complementary models to provide explanations for the increased rate of centripetal migration caused by wounding and the potential for stem cell leakage to account for stable transplants derived from central corneal tissue, despite the predominantly limbal location of stem cells.

In vitro optimization of truncated stem-loop II variants of the hammerhead ribozyme for cleavage in low concentrations of magnesium under non-turnover conditions.

PubMed Central

Zillmann, M; Limauro, S E; Goodchild, J

1997-01-01

By truncating helix II to two base pairs in a hammerhead ribozyme having long flanking sequences (greater than 30 bases), the rate of cleavage in 1 mM magnesium can be increased roughly 100-fold. Replacing most of the nucleotides in a typical stem-loop II with 1-4 randomized nucleotides gave an RNA library that, even before selection, was more active in 1 mM magnesium than the parent ribozyme, but considerably less active than the truncated stem-loop II ribozyme. A novel, multiround selection for intermolecular cleavage was exploited to optimize this library for cleavage in low concentrations of magnesium. After three rounds of selection at sequentially lower concentrations of magnesium, the library cleaved substrate RNA 20-fold faster than the initial pool and was cloned. This pool was heavily enriched for one particular sequence (5'-CGUG-3') that represented 16 of 52 isolates (the next most common sequence was represented only six times). This sequence also represented the most active sequence, exceeding the activity of the short helix II variant under the conditions of the selection, thereby demonstrating the effectiveness of the selection technique. Analysis of the cleavage rates of RNAs made from eight isolates having different four-base insert sequences allowed assignment of highly preferred bases at each position in the insert. Analysis of pool clones having insert of differing lengths showed that, in general, activity decreased as the length of the insert decreased from 4 to 1. This supports the suggested role of stem-loop II in stabilizing the non-Watson-Crick interactions between the conserved bases of the catalytic core. PMID:9214657

Dual-band frequency selective surface with large band separation and stable performance

NASA Astrophysics Data System (ADS)

Zhou, Hang; Qu, Shao-Bo; Peng, Wei-Dong; Lin, Bao-Qin; Wang, Jia-Fu; Ma, Hua; Zhang, Jie-Qiu; Bai, Peng; Wang, Xu-Hua; Xu, Zhuo

2012-05-01

A new technique of designing a dual-band frequency selective surface with large band separation is presented. This technique is based on a delicately designed topology of L- and Ku-band microwave filters. The two band-pass responses are generated by a capacitively-loaded square-loop frequency selective surface and an aperture-coupled frequency selective surface, respectively. A Faraday cage is located between the two frequency selective surface structures to eliminate undesired couplings. Based on this technique, a dual-band frequency selective surface with large band separation is designed, which possesses large band separation, high selectivity, and stable performance under various incident angles and different polarizations.

Proline Restricts Loop I Conformation of the High Affinity WW Domain from Human Nedd4-1 to a Ligand Binding-Competent Type I β-Turn.

PubMed

Schulte, Marianne; Panwalkar, Vineet; Freischem, Stefan; Willbold, Dieter; Dingley, Andrew J

2018-04-19

Sequence alignment of the four WW domains from human Nedd4-1 (neuronal precursor cell expressed developmentally down-regulated gene 4-1) reveals that the highest sequence diversity exists in loop I. Three residues in this type I β-turn interact with the PPxY motif of the human epithelial Na + channel (hENaC) subunits, indicating that peptide affinity is defined by the loop I sequence. The third WW domain (WW3*) has the highest ligand affinity and unlike the other three hNedd4-1 WW domains or other WW domains studied contains the highly statistically preferred proline at the ( i + 1) position found in β-turns. In this report, molecular dynamics simulations and experimental data were combined to characterize loop I stability and dynamics. Exchange of the proline to the equivalent residue in WW4 (Thr) results in the presence of a predominantly open seven residue Ω loop rather than the type I β-turn conformation for the wild-type apo-WW3*. In the presence of the ligand, the structure of the mutated loop I is locked into a type I β-turn. Thus, proline in loop I ensures a stable peptide binding-competent β-turn conformation, indicating that amino acid sequence modulates local flexibility to tune binding preferences and stability of dynamic interaction motifs.

Identification of novel RNA secondary structures within the hepatitis C virus genome reveals a cooperative involvement in genome packaging

PubMed Central

Stewart, H.; Bingham, R.J.; White, S. J.; Dykeman, E. C.; Zothner, C.; Tuplin, A. K.; Stockley, P. G.; Twarock, R.; Harris, M.

2016-01-01

The specific packaging of the hepatitis C virus (HCV) genome is hypothesised to be driven by Core-RNA interactions. To identify the regions of the viral genome involved in this process, we used SELEX (systematic evolution of ligands by exponential enrichment) to identify RNA aptamers which bind specifically to Core in vitro. Comparison of these aptamers to multiple HCV genomes revealed the presence of a conserved terminal loop motif within short RNA stem-loop structures. We postulated that interactions of these motifs, as well as sub-motifs which were present in HCV genomes at statistically significant levels, with the Core protein may drive virion assembly. We mutated 8 of these predicted motifs within the HCV infectious molecular clone JFH-1, thereby producing a range of mutant viruses predicted to possess altered RNA secondary structures. RNA replication and viral titre were unaltered in viruses possessing only one mutated structure. However, infectivity titres were decreased in viruses possessing a higher number of mutated regions. This work thus identified multiple novel RNA motifs which appear to contribute to genome packaging. We suggest that these structures act as cooperative packaging signals to drive specific RNA encapsidation during HCV assembly. PMID:26972799

Adaptive identification and control of structural dynamics systems using recursive lattice filters

NASA Technical Reports Server (NTRS)

Sundararajan, N.; Montgomery, R. C.; Williams, J. P.

1985-01-01

A new approach for adaptive identification and control of structural dynamic systems by using least squares lattice filters thar are widely used in the signal processing area is presented. Testing procedures for interfacing the lattice filter identification methods and modal control method for stable closed loop adaptive control are presented. The methods are illustrated for a free-free beam and for a complex flexible grid, with the basic control objective being vibration suppression. The approach is validated by using both simulations and experimental facilities available at the Langley Research Center.

A Devonian tetrapod-like fish reveals substantial parallelism in stem tetrapod evolution.

PubMed

Zhu, Min; Ahlberg, Per E; Zhao, Wen-Jin; Jia, Lian-Tao

2017-10-01

The fossils assigned to the tetrapod stem group document the evolution of terrestrial vertebrates from lobe-finned fishes. During the past 18 years the phylogenetic structure of this stem group has remained remarkably stable, even when accommodating new discoveries such as the earliest known stem tetrapod Tungsenia and the elpistostegid (fish-tetrapod intermediate) Tiktaalik. Here we present a large lobe-finned fish from the Late Devonian period of China that disrupts this stability. It combines characteristics of rhizodont fishes (supposedly a basal branch in the stem group, distant from tetrapods) with derived elpistostegid-like and tetrapod-like characters. This mélange of characters may reflect either detailed convergence between rhizodonts and elpistostegids plus tetrapods, under a phylogenetic scenario deduced from Bayesian inference analysis, or a previously unrecognized close relationship between these groups, as supported by maximum parsimony analysis. In either case, the overall result reveals a substantial increase in homoplasy in the tetrapod stem group. It also suggests that ecological diversity and biogeographical provinciality in the tetrapod stem group have been underestimated.

Solution NMR analyses of the anticodon arms of proteinogenic and non-proteinogenic tRNAGly

PubMed Central

Chang, Andrew T.; Nikonowicz, Edward P.

2012-01-01

Although the fate of most tRNA molecules in the cell is aminoacylation and delivery to the ribosome, some tRNAs are destined to fulfill other functional roles. In addition to their central role in translation, tRNA molecules participate in processes such as regulation of gene expression, bacterial cell wall biosynthesis, viral replication, antibiotic biosynthesis, and suppression of alternative splicing. In bacteria, glycyl-tRNA molecules with anticodon sequences GCC and UCC exhibit multiple extra-translational functions including transcriptional regulation and cell wall biosynthesis. We have determined the high-resolution structures of three glycyl-tRNA anticodon arms with anticodon sequences GCC and UCC. Two of the tRNA molecules are proteinogenic (tRNAGly,GCC and tRNAGly,UCC) and the third is non-proteinogenic (np-tRNAGly,UCC) and participates in cell wall biosynthesis. The UV-monitored thermal melting curves show that the anticodon arm of tRNAGly,UCC with a loop-closing C-A+ base pair melts at a 10 °C lower temperature than those of tRNAGly,GCC or np-tRNAGly,UCC. U-A and C-G pairs close the loops of the later two molecules and enhance stem stability. Mg2+ stabilizes the tRNAGly,UCC anticodon arm and lessens the Tm differential. The structures of the three tRNAGly anticodon arms exhibit small differences between one another, but none of them form the classical U-turn motif. The anticodon loop of tRNAGly,GCC becomes more dynamic and disordered in the presence of multivalent cations, whereas metal ion coordination in the anticodon loops of tRNAGly,UCC and np-tRNAGly,UCC establishes conformational homogeneity. The conformational similarity of the molecules is greater than their functional differences might suggest. Because aminoacylation of the full-length tRNA molecules is accomplished by one tRNA synthetase, the similar structural context of the loop may facilitate efficient recognition of each of the anticodon sequences. PMID:22468768

Molecular basis of thermal stability in truncated (2/2) hemoglobins.

PubMed

Bustamante, Juan P; Bonamore, Alessandra; Nadra, Alejandro D; Sciamanna, Natascia; Boffi, Alberto; Estrin, Darío A; Boechi, Leonardo

2014-07-01

Understanding the molecular mechanism through which proteins are functional at extreme high and low temperatures is one of the key issues in structural biology. To investigate this phenomenon, we have focused on two instructive truncated hemoglobins from Thermobifida fusca (Tf-trHbO) and Mycobacterium tuberculosis (Mt-trHbO); although the two proteins are structurally nearly identical, only the former is stable at high temperatures. We used molecular dynamics simulations at different temperatures as well as thermal melting profile measurements of both wild type proteins and two mutants designed to interchange the amino acid residue, either Pro or Gly, at E3 position. The results show that the presence of a Pro at the E3 position is able to increase (by 8°) or decrease (by 4°) the melting temperature of Mt-trHbO and Tf-trHbO, respectively. We observed that the ProE3 alters the structure of the CD loop, making it more flexible. This gain in flexibility allows the protein to concentrate its fluctuations in this single loop and avoid unfolding. The alternate conformations of the CD loop also favor the formation of more salt-bridge interactions, together augmenting the protein's thermostability. These results indicate a clear structural and dynamical role of a key residue for thermal stability in truncated hemoglobins. Copyright © 2014 Elsevier B.V. All rights reserved.

Modular high-voltage bias generator powered by dual-looped self-adaptive wireless power transmission.

PubMed

Xie, Kai; Huang, An-Feng; Li, Xiao-Ping; Guo, Shi-Zhong; Zhang, Han-Lu

2015-04-01

We proposed a modular high-voltage (HV) bias generator powered by a novel transmitter-sharing inductive coupled wireless power transmission technology, aimed to extend the generator's flexibility and configurability. To solve the problems caused through an uncertain number of modules, a dual-looped self-adaptive control method is proposed that is capable of tracking resonance frequency while maintaining a relatively stable induction voltage for each HV module. The method combines a phase-locked loop and a current feedback loop, which ensures an accurate resonance state and a relatively constant boost ratio for each module, simplifying the architecture of the boost stage and improving the total efficiency. The prototype was built and tested. The input voltage drop of each module is less than 14% if the module number varies from 3 to 10; resonance tracking is completed within 60 ms. The efficiency of the coupling structure reaches up to 95%, whereas the total efficiency approaches 73% for a rated output. Furthermore, this technology can be used in various multi-load wireless power supply applications.

MicroRNA-like viral small RNA from porcine reproductive and respiratory syndrome virus negatively regulates viral replication by targeting the viral nonstructural protein 2.

PubMed

Li, Na; Yan, Yunhuan; Zhang, Angke; Gao, Jiming; Zhang, Chong; Wang, Xue; Hou, Gaopeng; Zhang, Gaiping; Jia, Jinbu; Zhou, En-Min; Xiao, Shuqi

2016-12-13

Many viruses encode microRNAs (miRNAs) that are small non-coding single-stranded RNAs which play critical roles in virus-host interactions. Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically impactful viruses in the swine industry. The present study sought to determine whether PRRSV encodes miRNAs that could regulate PRRSV replication. Four viral small RNAs (vsRNAs) were mapped to the stem-loop structures in the ORF1a, ORF1b and GP2a regions of the PRRSV genome by bioinformatics prediction and experimental verification. Of these, the structures with the lowest minimum free energy (MFE) values predicted for PRRSV-vsRNA1 corresponded to typical stem-loop, hairpin structures. Inhibition of PRRSV-vsRNA1 function led to significant increases in viral replication. Transfection with PRRSV-vsRNA1 mimics significantly inhibited PRRSV replication in primary porcine alveolar macrophages (PAMs). The time-dependent increase in the abundance of PRRSV-vsRNA1 mirrored the gradual upregulation of PRRSV RNA expression. Knockdown of proteins associated with cellular miRNA biogenesis demonstrated that Drosha and Argonaute (Ago2) are involved in PRRSV-vsRNA1 biogenesis. Moreover, PRRSV-vsRNA1 bound specifically to the nonstructural protein 2 (NSP2)-coding sequence of PRRSV genome RNA. Collectively, the results reveal that PRRSV encodes a functional PRRSV-vsRNA1 which auto-regulates PRRSV replication by directly targeting and suppressing viral NSP2 gene expression. These findings not only provide new insights into the mechanism of the pathogenesis of PRRSV, but also explore a potential avenue for controlling PRRSV infection using viral small RNAs.

Mechanism of inhibition of HIV-1 integrase by G-tetrad-forming oligonucleotides in Vitro.

PubMed

Jing, N; Marchand, C; Liu, J; Mitra, R; Hogan, M E; Pommier, Y

2000-07-14

The G-tetrad-forming oligonucleotides and have been identified as potent inhibitors of human immunodeficiency virus type 1 integrase (HIV-1 IN) activity (Rando, R. F., Ojwang, J., Elbaggari, A., Reyes, G. R., Tinder, R., McGrath, M. S., and Hogan, M. E. (1995) J. Biol. Chem. 270, 1754-1760; Mazumder, A., Neamati, N., Ojwang, J. O., Sunder, S., Rando, R. F., and Pommier, Y. (1996) Biochemistry 35, 13762-13771; Jing, N., and Hogan, M. E. (1998) J. Biol. Chem. 273, 34992-34999). To understand the inhibition of HIV-1 IN activity by the G-quartet inhibitors, we have designed the oligonucleotides and, composed of three and four G-quartets with stem lengths of 19 and 24 A, respectively. The fact that increasing the G-quartet stem length from 15 to 24 A kept inhibition of HIV-1 IN activity unchanged suggests that the binding interaction occurs between a GTGT loop domain of the G-quartet inhibitors and a catalytic site of HIV-1 IN, referred to as a face-to-face interaction. Docking the NMR structure of (Jing and Hogan (1998)) into the x-ray structure of the core domain of HIV-1 IN, HIV-1 IN-(51-209) (Maignan, S., Guilloteau, J.-P. , Qing, Z.-L., Clement-Mella, C., and Mikol, V. (1998) J. Mol. Biol. 282, 359-368), was performed using the GRAMM program. The statistical distributions of hydrogen bonding between HIV-1 IN and were obtained from the analyses of 1000 random docking structures. The docking results show a high probability of interaction between the GTGT loop residues of the G-quartet inhibitors and the catalytic site of HIV-1 IN, in agreement with the experimental observation.

Characterization of host-dependent mutations of apple fruit crinkle viroid replicating in newly identified experimental hosts suggests maintenance of stem-loop structures in the left-hand half of the molecule is important for replication.

PubMed

Suzuki, Takahiro; Fujibayashi, Misato; Hataya, Tatsuji; Taneda, Akito; He, Ying-Hong; Tsushima, Taro; Duraisamy, Ganesh Selvaraj; Siglová, Kristyna; Matoušek, Jaroslav; Sano, Teruo

2017-03-01

Apple fruit crinkle viroid (AFCVd) is a tentative member of the genus Apscaviroid, family Pospiviroidae. AFCVd has a narrow host range and is known to infect apple, hop and persimmon as natural hosts. In this study, tomato, cucumber and wild hop have been identified as new experimental herbaceous hosts. Foliar symptoms were very mild or virtually undetectable, but fruits of infected tomato were small, cracked and distorted. These symptoms resemble those observed on some AFCVd-sensitive apple cultivars. After transfer to tomato, cucumber and wild hop, sequence changes were detected in a natural AFCVd isolate from hop, and major variants in tomato, cucumber and wild hop differed in 10, 8 or 2 nucleotides, respectively, from the predominant one in the inoculum. The major variants in tomato and cucumber were almost identical, and the one in wild hop was very similar to the one in cultivated hop. Detailed analyses of the host-dependent sequence changes that appear in a naturally occurring AFCVd isolate from hop after transfer to tomato using small RNA deep sequence data and infectivity studies with dimeric RNA transcripts followed by progeny analysis indicate that the major AFCVd variant in tomato emerged by selection of a minor variant present in the inoculum (i.e. hop) followed by one to two host-dependent de novo mutations. Comparison of the secondary structures of major variants in hop, tomato and persimmon after transfer to tomato suggested that maintenance of stem-loop structures in the left-hand half of the molecule is critical for infection.

Predicting RNA 3D structure using a coarse-grain helix-centered model

PubMed Central

Kerpedjiev, Peter; Höner zu Siederdissen, Christian; Hofacker, Ivo L.

2015-01-01

A 3D model of RNA structure can provide information about its function and regulation that is not possible with just the sequence or secondary structure. Current models suffer from low accuracy and long running times and either neglect or presume knowledge of the long-range interactions which stabilize the tertiary structure. Our coarse-grained, helix-based, tertiary structure model operates with only a few degrees of freedom compared with all-atom models while preserving the ability to sample tertiary structures given a secondary structure. It strikes a balance between the precision of an all-atom tertiary structure model and the simplicity and effectiveness of a secondary structure representation. It provides a simplified tool for exploring global arrangements of helices and loops within RNA structures. We provide an example of a novel energy function relying only on the positions of stems and loops. We show that coupling our model to this energy function produces predictions as good as or better than the current state of the art tools. We propose that given the wide range of conformational space that needs to be explored, a coarse-grain approach can explore more conformations in less iterations than an all-atom model coupled to a fine-grain energy function. Finally, we emphasize the overarching theme of providing an ensemble of predicted structures, something which our tool excels at, rather than providing a handful of the lowest energy structures. PMID:25904133

Biomechanical evaluation of adjunctive cerclage wire fixation for the prevention of periprosthetic femur fractures using cementless press-fit total hip replacement.

PubMed

Christopher, Scott A; Kim, Stanley E; Roe, Simon; Pozzi, Antonio

2016-08-01

Periprosthetic femoral fractures are a common complication associated with cementless press-fit total hip arthroplasty. The use of prophylactic cerclage wire fixation has been advocated to reduce this complication. The objective of this study was to evaluate whether a double loop cerclage wire, used as adjunctive fixation, increased the peak torsional load to failure in femora implanted with press-fit cementless stems. Peak torsional load to failure was compared between femora without adjunctive fixation and femora receiving a 1 mm double loop cerclage wire placed proximally to the lesser trochanter. Femora treated with adjunctive cerclage wire fixation failed at 20% greater peak torque (P = 0.0001). In conclusion, a double loop cerclage wire may aid in the prevention of periprosthetic fractures associated with press-fit cementless femoral stems. Copyright © 2016. Published by Elsevier Ltd.

Female mice lack adult germ-line stem cells but sustain oogenesis using stable primordial follicles.

PubMed

Lei, Lei; Spradling, Allan C

2013-05-21

Whether or not mammalian females generate new oocytes during adulthood from germ-line stem cells to sustain the ovarian follicle pool has recently generated controversy. We used a sensitive lineage-labeling system to determine whether stem cells are needed in female adult mice to compensate for follicular losses and to directly identify active germ-line stem cells. Primordial follicles generated during fetal life are highly stable, with a half-life during adulthood of 10 mo, and thus are sufficient to sustain adult oogenesis without a source of renewal. Moreover, in normal mice or following germ-cell depletion with Busulfan, only stable, single oocytes are lineage-labeled, rather than cell clusters indicative of new oocyte formation. Even one germ-line stem cell division per 2 wk would have been detected by our method, based on the kinetics of fetal follicle formation. Thus, adult female mice neither require nor contain active germ-line stem cells or produce new oocytes in vivo.

Investigating the mincing method for isolation of adipose-derived stem cells from pregnant women fat.

PubMed

Li, Yuan-Sheng; Chen, Pao-Jen; Wu, Li-Wei; Chou, Pei-Wen; Sun, Li-Yi; Chiou, Tzyy-Wen

2018-02-01

The success of stem cell application in regenerative medicine, usually require a stable source of stem or progenitor cells. Fat tissue represents a good source of stem cells because it is rich in stem cells and there are fewer ethical issues related to the use of such stem cells, unlike embryonic stem cells. Therefore, there has been increased interest in adipose-derived stem cells (ADSCs) for tissue engineering applications. Here, we aim to provide an easy processing method for isolating adult stem cells from human adipose tissue harvested from the subcutaneous fat of the abdominal wall during gynecologic surgery. We used a homogenizer to mince fat and compared the results with those obtained from the traditional cut method involving a sterile scalpel and forceps. Our results showed that our method provides another stable and quality source of stem cells that could be used in cases with a large quantity of fat. Furthermore, we found that pregnancy adipose-derived stem cells (P-ADSCs) could be maintained in vitro for extended periods with a stable population doubling and low senescence levels. P-ADSCs could also differentiate in vitro into adipogenic, osteogenic, chondrogenic, and insulin-producing cells in the presence of lineage-specific induction factors. In conclusion, like human lipoaspirates, adipose tissues obtained from pregnant women contain multipotent cells with better proliferation and showed great promise for use in both stem cell banking studies as well as in stem cell therapy.

The architecture of the spliceosomal U4/U6.U5 tri-snRNP

PubMed Central

Nguyen, Thi Hoang Duong; Galej, Wojciech P.; Bai, Xiao-chen; Savva, Christos G.; Newman, Andrew J.; Scheres, Sjors H. W.; Nagai, Kiyoshi

2015-01-01

U4/U6.U5 tri-snRNP is a 1.5 MDa pre-assembled spliceosomal complex comprising U5 snRNA, extensively base-paired U4/U6 snRNAs and >30 proteins, including the key components Prp8, Brr2 and Snu114. The tri-snRNP combines with a pre-mRNA substrate bound to U1 and U2 snRNPs and transforms into a catalytically active spliceosome following extensive compositional and conformational changes triggered by unwinding of the U4/U6 snRNAs. CryoEM single-particle reconstruction of yeast tri-snRNP at 5.9Å resolution reveals the essentially complete organization of its RNA and protein components. The single-stranded region of U4 snRNA between its 3′-stem-loop and the U4/U6 snRNA stem I is loaded into the Brr2 helicase active site ready for unwinding. Snu114 and the N-terminal domain of Prp8 position U5 snRNA to insert its Loop I, which aligns the exons for splicing, into the Prp8 active site cavity. The structure provides crucial insights into the activation process and the active site of the spliceosome. PMID:26106855

Neurosurgery of the future: Deep brain stimulations and manipulations.

PubMed

Nicolaidis, Stylianos

2017-04-01

Important advances are afoot in the field of neurosurgery-particularly in the realms of deep brain stimulation (DBS), deep brain manipulation (DBM), and the newly introduced refinement "closed-loop" deep brain stimulation (CLDBS). Use of closed-loop technology will make both DBS and DBM more precise as procedures and will broaden their indications. CLDBS utilizes as feedback a variety of sources of electrophysiological and neurochemical afferent information about the function of the brain structures to be treated or studied. The efferent actions will be either electric, i.e. the classic excitatory or inhibitory ones, or micro-injection of such things as neural proteins and transmitters, neural grafts, implants of pluripotent stem cells or mesenchymal stem cells, and some variants of gene therapy. The pathologies to be treated, beside Parkinson's disease and movement disorders, include repair of neural tissues, neurodegenerative pathologies, psychiatric and behavioral dysfunctions, i.e. schizophrenia in its various guises, bipolar disorders, obesity, anorexia, drug addiction, and alcoholism. The possibility of using these new modalities to treat a number of cognitive dysfunctions is also under consideration. Because the DBS-CLDBS technology brings about a cross-fertilization between scientific investigation and surgical practice, it will also contribute to an enhanced understanding of brain function. Copyright © 2017. Published by Elsevier Inc.

The Basic Helix-Loop-Helix Transcription Factor MYC2 Directly Represses PLETHORA Expression during Jasmonate-Mediated Modulation of the Root Stem Cell Niche in Arabidopsis[W][OA

PubMed Central

Chen, Qian; Sun, Jiaqiang; Zhai, Qingzhe; Zhou, Wenkun; Qi, Linlin; Xu, Li; Wang, Bao; Chen, Rong; Jiang, Hongling; Qi, Jing; Li, Xugang; Palme, Klaus; Li, Chuanyou

2011-01-01

The root stem cell niche, which in the Arabidopsis thaliana root meristem is an area of four mitotically inactive quiescent cells (QCs) and the surrounding mitotically active stem cells, is critical for root development and growth. We report here that during jasmonate-induced inhibition of primary root growth, jasmonate reduces root meristem activity and leads to irregular QC division and columella stem cell differentiation. Consistently, jasmonate reduces the expression levels of the AP2-domain transcription factors PLETHORA1 (PLT1) and PLT2, which form a developmentally instructive protein gradient and mediate auxin-induced regulation of stem cell niche maintenance. Not surprisingly, the effects of jasmonate on root stem cell niche maintenance and PLT expression require the functioning of MYC2/JASMONATE INSENSITIVE1, a basic helix-loop-helix transcription factor that involves versatile aspects of jasmonate-regulated gene expression. Gel shift and chromatin immunoprecipitation experiments reveal that MYC2 directly binds the promoters of PLT1 and PLT2 and represses their expression. We propose that MYC2-mediated repression of PLT expression integrates jasmonate action into the auxin pathway in regulating root meristem activity and stem cell niche maintenance. This study illustrates a molecular framework for jasmonate-induced inhibition of root growth through interaction with the growth regulator auxin. PMID:21954460

miR-137 forms a regulatory loop with nuclear receptor TLX and LSD1 in neural stem cells.

PubMed

Sun, GuoQiang; Ye, Peng; Murai, Kiyohito; Lang, Ming-Fei; Li, Shengxiu; Zhang, Heying; Li, Wendong; Fu, Chelsea; Yin, Jason; Wang, Allen; Ma, Xiaoxiao; Shi, Yanhong

2011-11-08

miR-137 is a brain-enriched microRNA. Its role in neural development remains unknown. Here we show that miR-137 has an essential role in controlling embryonic neural stem cell fate determination. miR-137 negatively regulates cell proliferation and accelerates neural differentiation of embryonic neural stem cells. In addition, we show that the histone lysine-specific demethylase 1 (LSD1), a transcriptional co-repressor of nuclear receptor TLX, is a downstream target of miR-137. In utero electroporation of miR-137 in embryonic mouse brains led to premature differentiation and outward migration of the transfected cells. Introducing a LSD1 expression vector lacking the miR-137 recognition site rescued miR-137-induced precocious differentiation. Furthermore, we demonstrate that TLX, an essential regulator of neural stem cell self-renewal, represses the expression of miR-137 by recruiting LSD1 to the genomic regions of miR-137. Thus, miR-137 forms a feedback regulatory loop with TLX and LSD1 to control the dynamics between neural stem cell proliferation and differentiation during neural development.

Antigenic peptides containing large PEG loops designed to extend out of the HLA-A2 binding site form stable complexes with class I major histocompatibility complex molecules.

PubMed Central

Bouvier, M; Wiley, D C

1996-01-01

Recognition of peptides bound to class I major histocompatibility complex (MHC) molecules by specific receptors on T cells regulates the development and activity of the cellular immune system. We have designed and synthesized de novo cyclic peptides that incorporate PEG in the ring structure for binding to class I MHC molecules. The large PEG loops are positioned to extend out of the peptide binding site, thus creating steric effects aimed at preventing the recognition of class I MHC complexes by T-cell receptors. Peptides were synthesized and cyclized on polymer support using high molecular weight symmetrical PEG dicarboxylic acids to link the side chains of lysine residues substituted at positions 4 and 8 in the sequence of the HLA-A2-restricted human T-lymphotrophic virus type I Tax peptide. Cyclic peptides promoted the in vitro folding and assembly of HLA-A2 complexes. Thermal denaturation studies using circular dichroism spectroscopy showed that these complexes are as stable as complexes formed with antigenic peptides. Images Fig. 2 Fig. 4 PMID:8643447

A methodology for the synthesis of robust feedback systems. Ph.D. Thesis

NASA Technical Reports Server (NTRS)

Milich, David Albert

1988-01-01

A new methodology is developed for the synthesis of linear, time-variant (LTI) controllers for multivariable LTI systems. The resulting closed-loop system is nominally stable and exhibits a known level of performance. In addition, robustness of the feedback system is guaranteed, i.e., stability and performance are retained in the presence of multiple unstructured uncertainty blocks located at various points in the feedback loop. The design technique is referred to as the Causality Recovery Methodology (CRM). The CRM relies on the Youla parameterization of all stabilizing compensators to ensure nominal stability of the feedback system. A frequency-domain inequality in terms of the structured singular value mu defines the robustness specification. The optimal compensator, with respect to the mu condition, is shown to be noncausal in general. The aim of the CRM is to find a stable, causal transfer function matrix that approximates the robustness characteristics of the optimal solution. The CRM, via a series of infinite-dimensional convex programs, produces a closed-loop system whose performance robustness is at least as good as that of any initial design. The algorithm is approximated by a finite dimensional process for the purposes of implementation. Two numerical examples confirm the potential viability of the CRM concept; however, the robustness improvement comes at the expense of increased computational burden and compensator complexity.

The detailed 3D multi-loop aggregate/rosette chromatin architecture and functional dynamic organization of the human and mouse genomes.

PubMed

Knoch, Tobias A; Wachsmuth, Malte; Kepper, Nick; Lesnussa, Michael; Abuseiris, Anis; Ali Imam, A M; Kolovos, Petros; Zuin, Jessica; Kockx, Christel E M; Brouwer, Rutger W W; van de Werken, Harmen J G; van IJcken, Wilfred F J; Wendt, Kerstin S; Grosveld, Frank G

2016-01-01

The dynamic three-dimensional chromatin architecture of genomes and its co-evolutionary connection to its function-the storage, expression, and replication of genetic information-is still one of the central issues in biology. Here, we describe the much debated 3D architecture of the human and mouse genomes from the nucleosomal to the megabase pair level by a novel approach combining selective high-throughput high-resolution chromosomal interaction capture ( T2C ), polymer simulations, and scaling analysis of the 3D architecture and the DNA sequence. The genome is compacted into a chromatin quasi-fibre with ~5 ± 1 nucleosomes/11 nm, folded into stable ~30-100 kbp loops forming stable loop aggregates/rosettes connected by similar sized linkers. Minor but significant variations in the architecture are seen between cell types and functional states. The architecture and the DNA sequence show very similar fine-structured multi-scaling behaviour confirming their co-evolution and the above. This architecture, its dynamics, and accessibility, balance stability and flexibility ensuring genome integrity and variation enabling gene expression/regulation by self-organization of (in)active units already in proximity. Our results agree with the heuristics of the field and allow "architectural sequencing" at a genome mechanics level to understand the inseparable systems genomic properties.

The C-terminal domain of Tetrahymena thermophila telomerase holoenzyme protein p65 induces multiple structural changes in telomerase RNA

PubMed Central

Akiyama, Benjamin M.; Loper, John; Najarro, Kevin; Stone, Michael D.

2012-01-01

The unique cellular activity of the telomerase reverse transcriptase ribonucleoprotein (RNP) requires proper assembly of protein and RNA components into a functional complex. In the ciliate model organism Tetrahymena thermophila, the La-domain protein p65 is required for in vivo assembly of telomerase. Single-molecule and biochemical studies have shown that p65 promotes efficient RNA assembly with the telomerase reverse transcriptase (TERT) protein, in part by inducing a bend in the conserved stem IV region of telomerase RNA (TER). The domain architecture of p65 consists of an N-terminal domain, a La-RRM motif, and a C-terminal domain (CTD). Using single-molecule Förster resonance energy transfer (smFRET), we demonstrate the p65CTD is necessary for the RNA remodeling activity of the protein and is sufficient to induce a substantial conformational change in stem IV of TER. Moreover, nuclease protection assays directly map the site of p65CTD interaction to stem IV and reveal that, in addition to bending stem IV, p65 binding reorganizes nucleotides that comprise the low-affinity TERT binding site within stem–loop IV. PMID:22315458

Arabidopsis Myosins XI1, XI2, and XIK Are Crucial for Gravity-Induced Bending of Inflorescence Stems

PubMed Central

Talts, Kristiina; Ilau, Birger; Ojangu, Eve-Ly; Tanner, Krista; Peremyslov, Valera V.; Dolja, Valerian V.; Truve, Erkki; Paves, Heiti

2016-01-01

Myosins and actin filaments in the actomyosin system act in concert in regulating cell structure and dynamics and are also assumed to contribute to plant gravitropic response. To investigate the role of the actomyosin system in the inflorescence stem gravitropism, we used single and multiple mutants affecting each of the 17 Arabidopsis myosins of class VIII and XI. We show that class XI but not class VIII myosins are required for stem gravitropism. Simultaneous loss of function of myosins XI1, XI2, and XIK leads to impaired gravitropic bending that is correlated with altered growth, stiffness, and insufficient sedimentation of gravity sensing amyloplasts in stem endodermal cells. The gravitropic defect of the corresponding triple mutant xi1 xi2 xik could be rescued by stable expression of the functional XIK:YFP in the mutant background, indicating a role of class XI myosins in this process. Altogether, our results emphasize the critical contributions of myosins XI in stem gravitropism of Arabidopsis. PMID:28066484

Modeling and Control of Intelligent Flexible Structures

DTIC Science & Technology

1994-03-26

can be approximated as a simply supported beam in transverse vibration. Assuming that the Euler- Bernoulli beam assumptions hold, linear equations of...The assumptions made during the derivation are that the element can be modeled as an Euler- Bernoulli beam, that the cross-section is symmetric, and...parametes A,. and ,%. andc input maces 3,,. The closed loop system. ecuation (7), is stable when the 3.. 8 and output gain mantices H1., H., H. for

Structure-based discovery of NANOG variant with enhanced properties to promote self-renewal and reprogramming of pluripotent stem cells

DOE PAGES

Hayashi, Yohei; Caboni, Laura; Das, Debanu; ...

2015-03-30

NANOG (from Irish mythology Tír na nÓg) transcription factor plays a central role in maintaining pluripotency, cooperating with OCT4 (also known as POU5F1 or OCT3/4), SOX2, and other pluripotency factors. Although the physiological roles of the NANOG protein have been extensively explored, biochemical and biophysical properties in relation to its structural analysis are poorly understood. Here we determined the crystal structure of the human NANOG homeodomain (hNANOG HD) bound to an OCT4 promoter DNA, which revealed amino acid residues involved in DNA recognition that are likely to be functionally important. We generated a series of hNANOG HD alanine substitution mutantsmore » based on the protein–DNA interaction and evolutionary conservation and determined their biological activities. Some mutant proteins were less stable, resulting in loss or decreased affinity for DNA binding. Overexpression of the orthologous mouse NANOG (mNANOG) mutants failed to maintain self-renewal of mouse embryonic stem cells without leukemia inhibitory factor. These results suggest that these residues are critical for NANOG transcriptional activity. Interestingly, one mutant, hNANOG L122A, conversely enhanced protein stability and DNA-binding affinity. The mNANOG L122A, when overexpressed in mouse embryonic stem cells, maintained their expression of self-renewal markers even when retinoic acid was added to forcibly drive differentiation. When overexpressed in epiblast stem cells or human induced pluripotent stem cells, the L122A mutants enhanced reprogramming into ground-state pluripotency. These findings indicate that structural and biophysical information on key transcriptional factors provides insights into the manipulation of stem cell behaviors and a framework for rational protein engineering.« less

Structure-based discovery of NANOG variant with enhanced properties to promote self-renewal and reprogramming of pluripotent stem cells

PubMed Central

Hayashi, Yohei; Caboni, Laura; Das, Debanu; Yumoto, Fumiaki; Clayton, Thomas; Deller, Marc C.; Nguyen, Phuong; Farr, Carol L.; Chiu, Hsiu-Ju; Miller, Mitchell D.; Elsliger, Marc-André; Deacon, Ashley M.; Godzik, Adam; Lesley, Scott A.; Tomoda, Kiichiro; Conklin, Bruce R.; Wilson, Ian A.; Yamanaka, Shinya; Fletterick, Robert J.

2015-01-01

NANOG (from Irish mythology Tír na nÓg) transcription factor plays a central role in maintaining pluripotency, cooperating with OCT4 (also known as POU5F1 or OCT3/4), SOX2, and other pluripotency factors. Although the physiological roles of the NANOG protein have been extensively explored, biochemical and biophysical properties in relation to its structural analysis are poorly understood. Here we determined the crystal structure of the human NANOG homeodomain (hNANOG HD) bound to an OCT4 promoter DNA, which revealed amino acid residues involved in DNA recognition that are likely to be functionally important. We generated a series of hNANOG HD alanine substitution mutants based on the protein–DNA interaction and evolutionary conservation and determined their biological activities. Some mutant proteins were less stable, resulting in loss or decreased affinity for DNA binding. Overexpression of the orthologous mouse NANOG (mNANOG) mutants failed to maintain self-renewal of mouse embryonic stem cells without leukemia inhibitory factor. These results suggest that these residues are critical for NANOG transcriptional activity. Interestingly, one mutant, hNANOG L122A, conversely enhanced protein stability and DNA-binding affinity. The mNANOG L122A, when overexpressed in mouse embryonic stem cells, maintained their expression of self-renewal markers even when retinoic acid was added to forcibly drive differentiation. When overexpressed in epiblast stem cells or human induced pluripotent stem cells, the L122A mutants enhanced reprogramming into ground-state pluripotency. These findings demonstrate that structural and biophysical information on key transcriptional factors provides insights into the manipulation of stem cell behaviors and a framework for rational protein engineering. PMID:25825768

Structure-based discovery of NANOG variant with enhanced properties to promote self-renewal and reprogramming of pluripotent stem cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hayashi, Yohei; Caboni, Laura; Das, Debanu

NANOG (from Irish mythology Tír na nÓg) transcription factor plays a central role in maintaining pluripotency, cooperating with OCT4 (also known as POU5F1 or OCT3/4), SOX2, and other pluripotency factors. Although the physiological roles of the NANOG protein have been extensively explored, biochemical and biophysical properties in relation to its structural analysis are poorly understood. Here we determined the crystal structure of the human NANOG homeodomain (hNANOG HD) bound to an OCT4 promoter DNA, which revealed amino acid residues involved in DNA recognition that are likely to be functionally important. We generated a series of hNANOG HD alanine substitution mutantsmore » based on the protein–DNA interaction and evolutionary conservation and determined their biological activities. Some mutant proteins were less stable, resulting in loss or decreased affinity for DNA binding. Overexpression of the orthologous mouse NANOG (mNANOG) mutants failed to maintain self-renewal of mouse embryonic stem cells without leukemia inhibitory factor. These results suggest that these residues are critical for NANOG transcriptional activity. Interestingly, one mutant, hNANOG L122A, conversely enhanced protein stability and DNA-binding affinity. The mNANOG L122A, when overexpressed in mouse embryonic stem cells, maintained their expression of self-renewal markers even when retinoic acid was added to forcibly drive differentiation. When overexpressed in epiblast stem cells or human induced pluripotent stem cells, the L122A mutants enhanced reprogramming into ground-state pluripotency. These findings indicate that structural and biophysical information on key transcriptional factors provides insights into the manipulation of stem cell behaviors and a framework for rational protein engineering.« less

Optimal packaging of FIV genomic RNA depends upon a conserved long-range interaction and a palindromic sequence within gag.

PubMed

Rizvi, Tahir A; Kenyon, Julia C; Ali, Jahabar; Aktar, Suriya J; Phillip, Pretty S; Ghazawi, Akela; Mustafa, Farah; Lever, Andrew M L

2010-10-15

The feline immunodeficiency virus (FIV) is a lentivirus that is related to human immunodeficiency virus (HIV), causing a similar pathology in cats. It is a potential small animal model for AIDS and the FIV-based vectors are also being pursued for human gene therapy. Previous studies have mapped the FIV packaging signal (ψ) to two or more discontinuous regions within the 5' 511 nt of the genomic RNA and structural analyses have determined its secondary structure. The 5' and 3' sequences within ψ region interact through extensive long-range interactions (LRIs), including a conserved heptanucleotide interaction between R/U5 and gag. Other secondary structural elements identified include a conserved 150 nt stem-loop (SL2) and a small palindromic stem-loop within gag open reading frame that might act as a viral dimerization initiation site. We have performed extensive mutational analysis of these sequences and structures and ascertained their importance in FIV packaging using a trans-complementation assay. Disrupting the conserved heptanucleotide LRI to prevent base pairing between R/U5 and gag reduced packaging by 2.8-5.5 fold. Restoration of pairing using an alternative, non-wild type (wt) LRI sequence restored RNA packaging and propagation to wt levels, suggesting that it is the structure of the LRI, rather than its sequence, that is important for FIV packaging. Disrupting the palindrome within gag reduced packaging by 1.5-3-fold, but substitution with a different palindromic sequence did not restore packaging completely, suggesting that the sequence of this region as well as its palindromic nature is important. Mutation of individual regions of SL2 did not have a pronounced effect on FIV packaging, suggesting that either it is the structure of SL2 as a whole that is necessary for optimal packaging, or that there is redundancy within this structure. The mutational analysis presented here has further validated the previously predicted RNA secondary structure of FIV ψ. Copyright © 2010 Elsevier Ltd. All rights reserved.

Crystal structures of the archaeal RNase P protein Rpp38 in complex with RNA fragments containing a K-turn motif.

PubMed

Oshima, Kosuke; Gao, Xuzhu; Hayashi, Seiichiro; Ueda, Toshifumi; Nakashima, Takashi; Kimura, Makoto

2018-01-01

A characteristic feature of archaeal ribonuclease P (RNase P) RNAs is that they have extended helices P12.1 and P12.2 containing kink-turn (K-turn) motifs to which the archaeal RNase P protein Rpp38, a homologue of the human RNase P protein Rpp38, specifically binds. PhoRpp38 from the hyperthermophilic archaeon Pyrococcus horikoshii is involved in the elevation of the optimum temperature of the reconstituted RNase P by binding the K-turns in P12.1 and P12.2. Previously, the crystal structure of PhoRpp38 in complex with the K-turn in P12.2 was determined at 3.4 Å resolution. In this study, the crystal structure of PhoRpp38 in complex with the K-turn in P12.2 was improved to 2.1 Å resolution and the structure of PhoRpp38 in complex with the K-turn in P12.1 was also determined at a resolution of 3.1 Å. Both structures revealed that Lys35, Asn38 and Glu39 in PhoRpp38 interact with characteristic G·A and A·G pairs in the K-turn, while Thr37, Asp59, Lys84, Glu94, Ala96 and Ala98 in PhoRpp38 interact with the three-nucleotide bulge in the K-turn. Moreover, an extended stem-loop containing P10-P12.2 in complex with PhoRpp38, as well as PhoRpp21 and PhoRpp29, which are the archaeal homologues of the human proteins Rpp21 and Rpp29, respectively, was affinity-purified and crystallized. The crystals thus grown diffracted to a resolution of 6.35 Å. Structure determination of the crystals will demonstrate the previously proposed secondary structure of stem-loops including helices P12.1 and P12.2 and will also provide insight into the structural organization of the specificity domain in P. horikoshii RNase P RNA.

Crystal packing modifies ligand binding affinity: the case of aldose reductase.

PubMed

Cousido-Siah, Alexandra; Petrova, Tatiana; Hazemann, Isabelle; Mitschler, André; Ruiz, Francesc X; Howard, Eduardo; Ginell, Stephan; Atmanene, Cédric; Van Dorsselaer, Alain; Sanglier-Cianférani, Sarah; Joachimiak, Andrzej; Podjarny, Alberto

2012-11-01

The relationship between the structures of protein-ligand complexes existing in the crystal and in solution, essential in the case of fragment-based screening by X-ray crystallography (FBS-X), has been often an object of controversy. To address this question, simultaneous co-crystallization and soaking of two inhibitors with different ratios, Fidarestat (FID; K(d) = 6.5 nM) and IDD594 (594; K(d) = 61 nM), which bind to h-aldose reductase (AR), have been performed. The subatomic resolution of the crystal structures allows the differentiation of both inhibitors, even when the structures are almost superposed. We have determined the occupation ratio in solution by mass spectrometry (MS) Occ(FID)/Occ(594) = 2.7 and by X-ray crystallography Occ(FID)/Occ(594) = 0.6. The occupancies in the crystal and in solution differ 4.6 times, implying that ligand binding potency is influenced by crystal contacts. A structural analysis shows that the Loop A (residues 122-130), which is exposed to the solvent, is flexible in solution, and is involved in packing contacts within the crystal. Furthermore, inhibitor 594 contacts the base of Loop A, stabilizing it, while inhibitor FID does not. This is shown by the difference in B-factors of the Loop A between the AR-594 and AR-FID complexes. A stable loop diminishes the entropic energy barrier to binding, favoring 594 versus FID. Therefore, the effect of the crystal environment should be taken into consideration in the X-ray diffraction analysis of ligand binding to proteins. This conclusion highlights the need for additional methodologies in the case of FBS-X to validate this powerful screening technique, which is widely used. Copyright © 2012 Wiley Periodicals, Inc.

Energy Systems Integration News | Energy Systems Integration Facility |

Science.gov Websites

distribution feeder models for use in hardware-in-the-loop (HIL) experiments. Using this method, a full feeder ; proposes an additional control loop to improve frequency support while ensuring stable operation. The and Frequency Deviation," also proposes an additional control loop, this time to smooth the wind

CD4-binding site alterations in CCR5-using HIV-1 envelopes influencing gp120-CD4 interactions and fusogenicity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sterjovski, Jasminka; Churchill, Melissa J.; Roche, Michael

2011-02-20

CD4-binding site (CD4bs) alterations in gp120 contribute to different pathophysiological phenotypes of CCR5-using (R5) HIV-1 strains, but the potential structural basis is unknown. Here, we characterized functionally diverse R5 envelope (Env) clones (n = 16) to elucidate potential structural alterations within the gp120 CD4bs that influence Env function. Initially, we showed that the magnitude of gp120-CD4-binding correlates with increased fusogenicity and reduced CD4 dependence. Analysis of three-dimensional gp120 structural models revealed two CD4bs variants, D279 and N362, that were associated with reduced CD4 dependence. Further structural analysis showed that a wider aperture of the predicted CD4bs cavity, as constrained bymore » the inner-most atoms at the gp120 V1V2 stem and the V5 loop, was associated with amino acid alterations within V5 and correlated with increased gp120-CD4 binding and increased fusogenicity. Our results provide evidence that the gp120 V5 loop may alter CD4bs conformation and contribute to increased gp120-CD4 interactions and Env fusogenicity.« less

Closing the tau loop: the missing tau mutation

PubMed Central

McCarthy, Allan; Lonergan, Roisin; Olszewska, Diana A.; O’Dowd, Sean; Cummins, Gemma; Magennis, Brian; Fallon, Emer M.; Pender, Niall; Huey, Edward D.; Cosentino, Stephanie; O’Rourke, Killian; Kelly, Brendan D.; O’Connell, Martin; Delon, Isabelle; Farrell, Michael; Spillantini, Maria Grazia; Rowland, Lewis P.; Fahn, Stanley; Craig, Peter; Hutton, Michael

2015-01-01

Frontotemporal lobar degeneration comprises a group of disorders characterized by behavioural, executive, language impairment and sometimes features of parkinsonism and motor neuron disease. In 1994 we described an Irish-American family with frontotemporal dementia linked to chromosome 17 associated with extensive tau pathology. We named this disinhibition-dementia-parkinsonism-amyotrophy complex. We subsequently identified mutations in the MAPT gene. Eleven MAPT gene splice site stem loop mutations were identified over time except for 5’ splice site of exon 10. We recently identified another Irish family with autosomal dominant early amnesia and behavioural change or parkinsonism associated with the ‘missing’ +15 mutation at the intronic boundary of exon 10. We performed a clinical, neuropsychological and neuroimaging study on the proband and four siblings, including two affected siblings. We sequenced MAPT and performed segregation analysis. We looked for a biological effect of the tau variant by performing real-time polymerase chain reaction analysis of RNA extracted from human embryonic kidney cells transfected with exon trapping constructs. We found a c.915+15A>C exon 10/intron 10 stem loop mutation in all affected subjects but not in the unaffected. The c.915+15A>C variant caused a shift in tau splicing pattern to a predominantly exon 10+ pattern presumably resulting in predominant 4 repeat tau and little 3 repeat tau. This strongly suggests that the c.915+15A>C variant is a mutation and that it causes frontotemporal dementia linked to chromosome 17 in this pedigree by shifting tau transcription and translation to +4 repeat tau. Tau (MAPT) screening should be considered in families where amnesia or atypical parkinsonism coexists with behavioural disturbance early in the disease process. We describe the final missing stem loop tau mutation predicted 15 years ago. Mutations have now been identified at all predicted sites within the ‘stem’ when the stem-loop model was first proposed and no mutations have been found within the ‘loop’ region as expected. Therefore we ‘close the tau loop’ having ‘opened the loop’ 21 years ago. PMID:26297556

Disruption of a hydrogen bond network in human versus spider monkey cytochrome c affects heme crevice stability.

PubMed

Goldes, Matthew E; Jeakins-Cooley, Margaret E; McClelland, Levi J; Mou, Tung-Chung; Bowler, Bruce E

2016-05-01

The hypothesis that the recent rapid evolution of primate cytochromes c, which primarily involves residues in the least stable Ω-loop (Ω-loop C, residues 40-57), stabilizes the heme crevice of cytochrome c relative to other mammals, is tested. To accomplish this goal, we have compared the properties of human and spider monkey cytochrome c and a set of four variants produced in the process of converting human cytochrome c into spider monkey cytochrome c. The global stability of all variants has been measured by guanidine hydrochloride denaturation. The stability of the heme crevice has been assessed with the alkaline conformational transition. Structural insight into the effects of the five amino acid substitutions needed to convert human cytochrome c into spider monkey cytochrome c is provided by a 1.15Å resolution structure of spider monkey cytochrome c. The global stability for all variants is near 9.0kcal/mol at 25°C and pH7, which is higher than that observed for other mammalian cytochromes c. The heme crevice stability is more sensitive to the substitutions required to produce spider monkey cytochrome c with decreases of up to 0.5 units in the apparent pKa of the alkaline conformational transition relative to human cytochrome c. The structure of spider monkey cytochrome c indicates that the Y46F substitution destabilizes the heme crevice by disrupting an extensive hydrogen bond network that connects three surface loops including Ω-loop D (residues 70-85), which contains the Met80 heme ligand. Copyright © 2015 Elsevier Inc. All rights reserved.

Non-coding stem-bulge RNAs are required for cell proliferation and embryonic development in C. elegans

PubMed Central

Kowalski, Madzia P.; Baylis, Howard A.; Krude, Torsten

2015-01-01

ABSTRACT Stem bulge RNAs (sbRNAs) are a family of small non-coding stem-loop RNAs present in Caenorhabditis elegans and other nematodes, the function of which is unknown. Here, we report the first functional characterisation of nematode sbRNAs. We demonstrate that sbRNAs from a range of nematode species are able to reconstitute the initiation of chromosomal DNA replication in the presence of replication proteins in vitro, and that conserved nucleotide sequence motifs are essential for this function. By functionally inactivating sbRNAs with antisense morpholino oligonucleotides, we show that sbRNAs are required for S phase progression, early embryonic development and the viability of C. elegans in vivo. Thus, we demonstrate a new and essential role for sbRNAs during the early development of C. elegans. sbRNAs show limited nucleotide sequence similarity to vertebrate Y RNAs, which are also essential for the initiation of DNA replication. Our results therefore establish that the essential function of small non-coding stem-loop RNAs during DNA replication extends beyond vertebrates. PMID:25908866

Molecular determinants of Cytochrome C oxidase IV mRNA axonal trafficking

PubMed Central

Kar, Amar N.; Vargas, Jose Norberto S.; Chen, Cai-Yun; Kowalak, Jeffrey A; Gioio, Anthony E.; Kaplan, Barry B.

2017-01-01

In previous studies, we identified a putative 38-nucleotide stem-loop structure (zipcode) in the 3′ untranslated region of the cytochrome c oxidase subunit IV (COXIV) mRNA that was necessary and sufficient for the axonal localization of the message in primary superior cervical ganglion (SCG) neurons. However, little is known about the proteins that interact with the COXIV-zipcode and regulate the axonal trafficking and local translation of the COXIV message. To identify proteins involved in the axonal transport of the COXIV mRNA, we used the biotinylated 38-nucleotide COXIV RNA zipcode as bait in the affinity purification of COXIV zipcode binding proteins. Gel-shift assays of the biotinylated COXIV zipcode indicated that the putative stem-loop structure functions as a nucleation site for the formation of ribonucleoprotein complexes. Mass spectrometric analysis of the COXIV zipcode ribonucleoprotein complex led to the identification of a large number RNA binding proteins, including fused in sarcoma/translated in liposarcoma (FUS/TLS), and Y-box protein 1 (YB-1). Validation experiments, using western analyses, confirmed the presence of the candidate proteins in the COXIV zipcode affinity purified complexes obtained from SCG axons. Immunohistochemical studies show that FUS, and YB-1 are present in SCG axons. Importantly, RNA immunoprecipitation studies show that FUS, and YB-1 interact with endogenous axonal COXIV transcripts. siRNA-mediated downregulation of the candidate proteins FUS and YB-1 expression in the cell-bodies diminishes the levels of COXIV mRNA in the axon, suggesting functional roles for these proteins in the axonal trafficking of COXIV mRNA. PMID:28161363

A manifestation of the Ostwald step rule: molecular-dynamics simulations and free-energy landscape of the primary nucleation and melting of single-molecule polyethylene in dilute solution.

PubMed

Larini, L; Leporini, D

2005-10-08

The paper presents numerical results from extensive molecular-dynamics simulations of the crystallization process of a single polyethylene chain with N=500 monomers. The development of the ordered structure is seen to proceed along different routes involving either the global reorganization of the chain or, alternatively, well-separated connected nuclei. No dependence on the thermal history was observed at the late stages of the crystallization. The folding process involves several intermediate ordered metastable states, in strong analogy with the experiments, and ends up in a well-defined long-lived lamella with ten stems of approximately equal length, arranged into a regular, hexagonal pattern. This behavior may be seen as a microscopic manifestation of the Ostwald step rule. Both the metastable states and the long-lived one are evidenced as the local minima and the global one of the free-energy landscape, respectively. The study of the microscopic organization of the lamella evidenced that the two caps are rather flat, i.e., the loops connecting the stems are short. Interestingly, annealing the chain through the different metastable states leaves the average number of monomers per loop nearly unchanged. It is also seen that the chain ends, the so-called cilia, are localized on the surface of the lamella, in agreement with the experiments, and that structural fluctuations take place on the lamella surface, as noted by recent Monte Carlo simulations. The study of the melting process evidences that the degree of hysteresis is small.

Behaviour of fractional loop delay zero crossing digital phase locked loop (FR-ZCDPLL)

NASA Astrophysics Data System (ADS)

Nasir, Qassim

2018-01-01

This article analyses the performance of the first-order zero crossing digital phase locked loops (FR-ZCDPLL) when fractional loop delay is added to loop. The non-linear dynamics of the loop is presented, analysed and examined through bifurcation behaviour. Numerical simulation of the loop is conducted to proof the mathematical analysis of the loop operation. The results of the loop simulation show that the proposed FR-ZCDPLL has enhanced the performance compared to the conventional zero crossing DPLL in terms of wider lock range, captured range and stable operation region. In addition, extensive experimental simulation was conducted to find the optimum loop parameters for different loop environmental conditions. The addition of the fractional loop delay network in the conventional loop also reduces the phase jitter and its variance especially when the signal-to-noise ratio is low.

Translational control of ribosomal protein S15.

PubMed

Portier, C; Philippe, C; Dondon, L; Grunberg-Manago, M; Ebel, J P; Ehresmann, B; Ehresmann, C

1990-08-27

The expression of ribosomal protein S15 is shown to be translationally and negatively autocontrolled using a fusion within a reporter gene. Isolation and characterization of several deregulated mutants indicate that the regulatory site (the translational operator site) overlaps the ribosome loading site of the S15 messenger. In this region, three domains, each exhibiting a stem-loop structure, were determined using chemical and enzymatic probes. The most downstream hairpin carries the Shine-Dalgarno sequence and the initiation codon. Genetic and structural data derived from mutants constructed by site-directed mutagenesis show that the operator is a dynamic structure, two domains of which can form a pseudoknot. Binding of S15 to these two domains suggests that the pseudoknot could be stabilized by S15. A model is presented in which two alternative structures would explain the molecular basis of the S15 autocontrol.

Thermodynamic stability of RNA structures formed by CNG trinucleotide repeats. Implication for prediction of RNA structure.

PubMed

Broda, Magdalena; Kierzek, Elzbieta; Gdaniec, Zofia; Kulinski, Tadeusz; Kierzek, Ryszard

2005-08-16

Trinucleotide repeat expansion diseases (TREDs) are correlated with elongation of CNG DNA and RNA repeats to pathological level. This paper shows, for the first time, complete data concerning thermodynamic stabilities of RNA with CNG trinucleotide repeats. Our studies include the stability of oligoribonucleotides composed of two to seven of CAG, CCG, CGG, and CUG repeats. The thermodynamic parameters of helix propagation correlated with the presence of multiple N-N mismatches within CNG RNA duplexes were also determined. Moreover, the total stability of CNG RNA hairpins, as well as the contribution of trinucleotide repeats placed only in the stem or loop regions, was evaluated. The improved thermodynamic parameters allow to predict much more accurately the thermodynamic stabilities and structures of CNG RNAs.

A simple second-order digital phase-locked loop.

NASA Technical Reports Server (NTRS)

Tegnelia, C. R.

1972-01-01

A simple second-order digital phase-locked loop has been designed for the Viking Orbiter 1975 command system. Excluding analog-to-digital conversion, implementation of the loop requires only an adder/subtractor, two registers, and a correctable counter with control logic. The loop considers only the polarity of phase error and corrects system clocks according to a filtered sequence of this polarity. The loop is insensitive to input gain variation, and therefore offers the advantage of stable performance over long life. Predictable performance is guaranteed by extreme reliability of acquisition, yet in the steady state the loop produces only a slight degradation with respect to analog loop performance.

A palindrome-mediated mechanism distinguishes translocations involving LCR-B of chromosome 22q11.2.

PubMed

Gotter, Anthony L; Shaikh, Tamim H; Budarf, Marcia L; Rhodes, C Harker; Emanuel, Beverly S

2004-01-01

Two known recurrent constitutional translocations, t(11;22) and t(17;22), as well as a non-recurrent t(4;22), display derivative chromosomes that have joined to a common site within the low copy repeat B (LCR-B) region of 22q11.2. This breakpoint is located between two AT-rich inverted repeats that form a nearly perfect palindrome. Breakpoints within the 11q23, 17q11 and 4q35 partner chromosomes also fall near the center of palindromic sequences. In the present work the breakpoints of a fourth translocation involving LCR-B, a balanced ependymoma-associated t(1;22), were characterized not only to localize this junction relative to known genes, but also to further understand the mechanism underlying these rearrangements. FISH mapping was used to localize the 22q11.2 breakpoint to LCR-B and the 1p21 breakpoint to single BAC clones. STS mapping narrowed the 1p21.2 breakpoint to a 1990 bp AT-rich region, and junction fragments were amplified by nested PCR. Junction fragment-derived sequence indicates that the 1p21.2 breakpoint splits a 278 nt palindrome capable of forming stem-loop secondary structure. In contrast, the 1p21.2 reference genomic sequence from clones in the database does not exhibit this configuration, suggesting a predisposition for regional genomic instability perhaps etiologic for this rearrangement. Given its similarity to known chromosomal fragile site (FRA) sequences, this polymorphic 1p21.2 sequence may represent one of the FRA1 loci. Comparative analysis of the secondary structure of sequences surrounding translocation breakpoints that involve LCR-B with those not involving this region indicate a unique ability of the former to form stem-loop structures. The relative likelihood of forming these configurations appears to be related to the rate of translocation occurrence. Further analysis suggests that constitutional translocations in general occur between sequences of similar melting temperature and propensity for secondary structure.

Bovine leukemia virus nucleocapsid protein is an efficient nucleic acid chaperone

DOE Office of Scientific and Technical Information (OSTI.GOV)

Qualley, Dominic F., E-mail: dqualley@berry.edu; Sokolove, Victoria L.; Ross, James L.

2015-03-13

Nucleocapsid proteins (NCs) direct the rearrangement of nucleic acids to form the most thermodynamically stable structure, and facilitate many steps throughout the life cycle of retroviruses. NCs bind strongly to nucleic acids (NAs) and promote NA aggregation by virtue of their cationic nature; they also destabilize the NA duplex via highly structured zinc-binding motifs. Thus, they are considered to be NA chaperones. While most retroviral NCs are structurally similar, differences are observed both within and between retroviral genera. In this work, we compare the NA binding and chaperone activity of bovine leukemia virus (BLV) NC to that of two othermore » retroviral NCs: human immunodeficiency virus type 1 (HIV-1) NC, which is structurally similar to BLV NC but from a different retrovirus genus, and human T-cell leukemia virus type 1 (HTLV-1) NC, which possesses several key structural differences from BLV NC but is from the same genus. Our data show that BLV and HIV-1 NCs bind to NAs with stronger affinity in relation to HTLV-1 NC, and that they also accelerate the annealing of complementary stem-loop structures to a greater extent. Analysis of kinetic parameters derived from the annealing data suggests that while all three NCs stimulate annealing by a two-step mechanism as previously reported, the relative contributions of each step to the overall annealing equilibrium are conserved between BLV and HIV-1 NCs but are different for HTLV-1 NC. It is concluded that while BLV and HTLV-1 belong to the same genus of retroviruses, processes that rely on NC may not be directly comparable. - Highlights: • BLV NC binds strongly to DNA and RNA. • BLV NC promotes mini-TAR annealing as well as HIV-1 NC. • Annealing kinetics suggest a low degree of similarity between BLV NC and HTLV-1 NC.« less

Poly(ester-urethane) scaffolds: effect of structure on properties and osteogenic activity of stem cells.

PubMed

Kiziltay, Aysel; Marcos-Fernandez, Angel; San Roman, Julio; Sousa, Rui A; Reis, Rui L; Hasirci, Vasif; Hasirci, Nesrin

2015-08-01

The present study aimed to investigate the effect of structure (design and porosity) on the matrix stiffness and osteogenic activity of stem cells cultured on poly(ester-urethane) (PEU) scaffolds. Different three-dimensional (3D) forms of scaffold were prepared from lysine-based PEU using traditional salt-leaching and advanced bioplotting techniques. The resulting scaffolds were characterized by differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), scanning electron microscopy (SEM), mercury porosimetry and mechanical testing. The scaffolds had various pore sizes with different designs, and all were thermally stable up to 300 °C. In vitro tests, carried out using rat bone marrow stem cells (BMSCs) for bone tissue engineering, demonstrated better viability and higher cell proliferation on bioplotted scaffolds compared to salt-leached ones, most probably due to their larger and interconnected pores and stiffer nature, as shown by higher compressive moduli, which were measured by compression testing. Similarly, SEM, von Kossa staining and EDX analyses indicated higher amounts of calcium deposition on bioplotted scaffolds during cell culture. It was concluded that the design with larger interconnected porosity and stiffness has an effect on the osteogenic activity of the stem cells. Copyright © 2013 John Wiley & Sons, Ltd.

Output Feedback Pole-Placement in the Design of Compensators for Suboptimal Linear Quadratic Regulators.

DTIC Science & Technology

1979-06-01

also extended to the class of stabilizable systems and the required compensator shown to possess a separation property. Finally the design methodology...Page 1.1. Block diagram of transfer function given in (1.28) ........... 15 3.3.1. Compensator structure for controllable and stabilizable systems ...response will be stable. The implemented output feedback control law will stabilize the total closed loop system . n nn Let [uin and iJi= 1 be the

RNApdbee 2.0: multifunctional tool for RNA structure annotation.

PubMed

Zok, Tomasz; Antczak, Maciej; Zurkowski, Michal; Popenda, Mariusz; Blazewicz, Jacek; Adamiak, Ryszard W; Szachniuk, Marta

2018-04-30

In the field of RNA structural biology and bioinformatics, an access to correctly annotated RNA structure is of crucial importance, especially in the secondary and 3D structure predictions. RNApdbee webserver, introduced in 2014, primarily aimed to address the problem of RNA secondary structure extraction from the PDB files. Its new version, RNApdbee 2.0, is a highly advanced multifunctional tool for RNA structure annotation, revealing the relationship between RNA secondary and 3D structure given in the PDB or PDBx/mmCIF format. The upgraded version incorporates new algorithms for recognition and classification of high-ordered pseudoknots in large RNA structures. It allows analysis of isolated base pairs impact on RNA structure. It can visualize RNA secondary structures-including that of quadruplexes-with depiction of non-canonical interactions. It also annotates motifs to ease identification of stems, loops and single-stranded fragments in the input RNA structure. RNApdbee 2.0 is implemented as a publicly available webserver with an intuitive interface and can be freely accessed at http://rnapdbee.cs.put.poznan.pl/.

Optimization of the structural characteristics of CaO and its effective stabilization yield high-capacity CO2 sorbents.

PubMed

Naeem, Muhammad Awais; Armutlulu, Andac; Imtiaz, Qasim; Donat, Felix; Schäublin, Robin; Kierzkowska, Agnieszka; Müller, Christoph R

2018-06-19

Calcium looping, a CO 2 capture technique, may offer a mid-term if not near-term solution to mitigate climate change, triggered by the yet increasing anthropogenic CO 2 emissions. A key requirement for the economic operation of calcium looping is the availability of highly effective CaO-based CO 2 sorbents. Here we report a facile synthesis route that yields hollow, MgO-stabilized, CaO microspheres featuring highly porous multishelled morphologies. As a thermal stabilizer, MgO minimized the sintering-induced decay of the sorbents' CO 2 capacity and ensured a stable CO 2 uptake over multiple operation cycles. Detailed electron microscopy-based analyses confirm a compositional homogeneity which is identified, together with the characteristics of its porous structure, as an essential feature to yield a high-performance sorbent. After 30 cycles of repeated CO 2 capture and sorbent regeneration, the best performing material requires as little as 11 wt.% MgO for structural stabilization and exceeds the CO 2 uptake of the limestone-derived reference material by ~500%.

Molecular characterization and application of lipase from Bacillus sp. PU1 and investigation of structural changes based on pH and temperature using MD simulation.

PubMed

Esakkiraj, Palanichamy; Antonyraj, Christian Bharathi; Meleppat, Balraj; Ankaiah, Dasari; Ayyanna, Repally; Ahamed, Syed Ibrahim Basheer; Arul, Venkatesan

2017-10-01

A gene coding lipase from Bacillus sp. PU1 was cloned and expressed in E. coli BL21(DE3) pLysS. The purified lipase has a molecular weight of 23kDa, is highly alkaline (pH range 8-10) and mesophilic (20-50°C). Three dimensional structure of the lipase was modeled by comparative homology and identified as a typical serine lipase by the presence of conserved Ser77, Asp133, His156. The molecular stability and behavior of the lipase was carried out using MD simulation studies at different pH and temperature was performed in comparison with biochemical analysis. Structural modifications of the lipase under these conditions were trapped by dihedral based FEL analysis and the functional loops (loop-H5/B4 and loop-H6/B5 of lipase) are identified which would cause the catalytic behavior of the lipase by high flexibility. Further characteristic feature of lipase are observed as follows; SDS completely inhibits the lipase activity and enzyme activity is enhanced with non-ionic surfactants. The lipase was highly stable in different organic solvents and also it could tolerate NaCl (0.4-0.8M). This enzyme was found to disrupt the biofilm of tested pathogenic bacterial strains. Copyright © 2017 Elsevier B.V. All rights reserved.

Structural basis for the appearance of a molten globule state in chimeric molecules derived from lysozyme and alpha-lactalbumin.

PubMed

Joniau, M; Haezebrouck, P; Noyelle, K; Van Dael, H

2001-07-01

The problem as to why alpha-lactalbumin, in the absence of Ca(2+), forms a molten globule intermediate, in contrast to its structural homologue lysozyme, has been addressed by the construction of chimeras of human lysozyme in which either the Ca(2+)-binding loop or a part of helix C of bovine alpha-lactalbumin were transplanted. Previously, we have shown that the introduction of both structural elements together in the lysozyme matrix causes the apo form of the resulting chimera to display molten globule behavior during the course of thermal denaturation. In this article, we demonstrate that this molten globule character is not correlated with the Ca(2+)-binding loop. Also, the Del 101 mutant in which Arg101 was deleted to simulate the alpha-lactalbumin conformation of the connecting loop between helix C and helix D, does not show a stable equilibrium intermediate. Rather, the molten globule character of the chimeras has to be related with a specific part of helix C. More particularly, attention is drawn to the four hydrophobic side-chains I93, V96, I99, and L100, the lysozyme counterparts of which are constituted of less bulky valines and alanine. Our observations are discussed in terms of decreased stability of the native form and increased stability of the intermediate molten globule. Copyright 2001 Wiley-Liss, Inc.

An Elegant Biosensor Molecular Beacon Probe: Challenges and Recent Solutions

PubMed Central

Kolpashchikov, Dmitry M.

2012-01-01

Molecular beacon (MB) probes are fluorophore- and quencher-labeled short synthetic DNAs folded in a stem-loop shape. Since the first report by Tyagi and Kramer, it has become a widely accepted tool for nucleic acid analysis and triggered a cascade of related developments in the field of molecular sensing. The unprecedented success of MB probes stems from their ability to detect specific DNA or RNA sequences immediately after hybridization with no need to wash out the unbound probe (instantaneous format). Importantly, the hairpin structure of the probe is responsible for both the low fluorescent background and improved selectivity. Furthermore, the signal is generated in a reversible manner; thus, if the analyte is removed, the signal is reduced to the background. This paper highlights the advantages of MB probes and discusses the approaches that address the challenges in MB probe design. Variations of MB-based assays tackle the problem of stem invasion, improve SNP genotyping and signal-to-noise ratio, as well as address the challenges of detecting folded RNA and DNA. PMID:24278758

The p53–Mdm2 feedback loop protects against DNA damage by inhibiting p53 activity but is dispensable for p53 stability, development, and longevity

PubMed Central

Pant, Vinod; Xiong, Shunbin; Jackson, James G.; Post, Sean M.; Abbas, Hussein A.; Quintás-Cardama, Alfonso; Hamir, Amirali N.; Lozano, Guillermina

2013-01-01

The p53–Mdm2 feedback loop is perceived to be critical for regulating stress-induced p53 activity and levels. However, this has never been tested in vivo. Using a genetically engineered mouse with mutated p53 response elements in the Mdm2 P2 promoter, we show that feedback loop-deficient Mdm2P2/P2 mice are viable and aphenotypic and age normally. p53 degradation kinetics after DNA damage in radiosensitive tissues remains similar to wild-type controls. Nonetheless, DNA damage response is elevated in Mdm2P2/P2 mice. Enhanced p53-dependent apoptosis sensitizes hematopoietic stem cells (HSCs), causing drastic myeloablation and lethality. These results suggest that while basal Mdm2 levels are sufficient to regulate p53 in most tissues under homeostatic conditions, the p53–Mdm2 feedback loop is critical for regulating p53 activity and sustaining HSC function after DNA damage. Therefore, transient disruption of p53–Mdm2 interaction could be explored as a potential adjuvant/therapeutic strategy for targeting stem cells in hematological malignancies. PMID:23973961

Synthesis of aspartyl-tRNA(Asp) in Escherichia coli--a snapshot of the second step.

PubMed Central

Eiler, S; Dock-Bregeon, A; Moulinier, L; Thierry, J C; Moras, D

1999-01-01

The 2.4 A crystal structure of the Escherichia coli aspartyl-tRNA synthetase (AspRS)-tRNA(Asp)-aspartyl-adenylate complex shows the two substrates poised for the transfer of the aspartic acid moiety from the adenylate to the 3'-hydroxyl of the terminal adenosine of the tRNA. A general molecular mechanism is proposed for the second step of the aspartylation reaction that accounts for the observed conformational changes, notably in the active site pocket. The stabilization of the transition state is mediated essentially by two amino acids: the class II invariant arginine of motif 2 and the eubacterial-specific Gln231, which in eukaryotes and archaea is replaced by a structurally non-homologous serine. Two archetypal RNA-protein modes of interactions are observed: the anticodon stem-loop, including the wobble base Q, binds to the N-terminal beta-barrel domain through direct protein-RNA interactions, while the binding of the acceptor stem involves both direct and water-mediated hydrogen bonds in an original recognition scheme. PMID:10562565

Ribosomal frameshifting and transcriptional slippage: From genetic steganography and cryptography to adventitious use.

PubMed

Atkins, John F; Loughran, Gary; Bhatt, Pramod R; Firth, Andrew E; Baranov, Pavel V

2016-09-06

Genetic decoding is not 'frozen' as was earlier thought, but dynamic. One facet of this is frameshifting that often results in synthesis of a C-terminal region encoded by a new frame. Ribosomal frameshifting is utilized for the synthesis of additional products, for regulatory purposes and for translational 'correction' of problem or 'savior' indels. Utilization for synthesis of additional products occurs prominently in the decoding of mobile chromosomal element and viral genomes. One class of regulatory frameshifting of stable chromosomal genes governs cellular polyamine levels from yeasts to humans. In many cases of productively utilized frameshifting, the proportion of ribosomes that frameshift at a shift-prone site is enhanced by specific nascent peptide or mRNA context features. Such mRNA signals, which can be 5' or 3' of the shift site or both, can act by pairing with ribosomal RNA or as stem loops or pseudoknots even with one component being 4 kb 3' from the shift site. Transcriptional realignment at slippage-prone sequences also generates productively utilized products encoded trans-frame with respect to the genomic sequence. This too can be enhanced by nucleic acid structure. Together with dynamic codon redefinition, frameshifting is one of the forms of recoding that enriches gene expression. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Characterization of regulatory elements within the coat protein (CP) coding region of Tobacco mosaic virus affecting subgenomic transcription and green fluorescent protein expression from the CP subgenomic RNA promoter.

PubMed

Man, Michal; Epel, Bernard L

2004-06-01

A replicon based on Tobacco mosaic virus that was engineered to express the open reading frame (ORF) of the green fluorescent protein (GFP) gene in place of the native coat protein (CP) gene from a minimal CP subgenomic (sg) RNA promoter was found to accumulate very low levels of GFP. Regulatory regions within the CP ORF were identified that, when presented as untranslated regions flanking the GFP ORF, enhanced or inhibited sg transcription and GFP expression. Full GFP expression from the CP sgRNA promoter required more than the first 20 nt of the CP ORF but not beyond the first 56 nt. Further analysis indicated the presence of an enhancer element between nt +25 and +55 with respect to the CP translation start site. The inclusion of this enhancer sequence upstream of the GFP ORF led to elevated sg transcription and to a 50-fold increase in GFP accumulation in comparison with a minimal CP promoter in which the entire CP ORF was displaced by the GFP ORF. Inclusion of the 3'-terminal 22 nt had a minor positive effect on GFP accumulation, but the addition of extended untranslated sequences from the 3' terminus of the CP ORF downstream of the GFP ORF was basically found to inhibit sg transcription. Secondary structure analysis programs predicted the CP sgRNA promoter to reside within two stable stem-loop structures, which are followed by an enhancer region.

A Stem-Loop Structure in Potato Leafroll Virus Open Reading Frame 5 (ORF5) Is Essential for Readthrough Translation of the Coat Protein ORF Stop Codon 700 Bases Upstream.

PubMed

Xu, Yi; Ju, Ho-Jong; DeBlasio, Stacy; Carino, Elizabeth J; Johnson, Richard; MacCoss, Michael J; Heck, Michelle; Miller, W Allen; Gray, Stewart M

2018-06-01

Translational readthrough of the stop codon of the capsid protein (CP) open reading frame (ORF) is used by members of the Luteoviridae to produce their minor capsid protein as a readthrough protein (RTP). The elements regulating RTP expression are not well understood, but they involve long-distance interactions between RNA domains. Using high-resolution mass spectrometry, glutamine and tyrosine were identified as the primary amino acids inserted at the stop codon of Potato leafroll virus (PLRV) CP ORF. We characterized the contributions of a cytidine-rich domain immediately downstream and a branched stem-loop structure 600 to 700 nucleotides downstream of the CP stop codon. Mutations predicted to disrupt and restore the base of the distal stem-loop structure prevented and restored stop codon readthrough. Motifs in the downstream readthrough element (DRTE) are predicted to base pair to a site within 27 nucleotides (nt) of the CP ORF stop codon. Consistent with a requirement for this base pairing, the DRTE of Cereal yellow dwarf virus was not compatible with the stop codon-proximal element of PLRV in facilitating readthrough. Moreover, deletion of the complementary tract of bases from the stop codon-proximal region or the DRTE of PLRV prevented readthrough. In contrast, the distance and sequence composition between the two domains was flexible. Mutants deficient in RTP translation moved long distances in plants, but fewer infection foci developed in systemically infected leaves. Selective 2'-hydroxyl acylation and primer extension (SHAPE) probing to determine the secondary structure of the mutant DRTEs revealed that the functional mutants were more likely to have bases accessible for long-distance base pairing than the nonfunctional mutants. This study reveals a heretofore unknown combination of RNA structure and sequence that reduces stop codon efficiency, allowing translation of a key viral protein. IMPORTANCE Programmed stop codon readthrough is used by many animal and plant viruses to produce key viral proteins. Moreover, such "leaky" stop codons are used in host mRNAs or can arise from mutations that cause genetic disease. Thus, it is important to understand the mechanism(s) of stop codon readthrough. Here, we shed light on the mechanism of readthrough of the stop codon of the coat protein ORFs of viruses in the Luteoviridae by identifying the amino acids inserted at the stop codon and RNA structures that facilitate this "leakiness" of the stop codon. Members of the Luteoviridae encode a C-terminal extension to the capsid protein known as the readthrough protein (RTP). We characterized two RNA domains in Potato leafroll virus (PLRV), located 600 to 700 nucleotides apart, that are essential for efficient RTP translation. We further determined that the PLRV readthrough process involves both local structures and long-range RNA-RNA interactions. Genetic manipulation of the RNA structure altered the ability of PLRV to translate RTP and systemically infect the plant. This demonstrates that plant virus RNA contains multiple layers of information beyond the primary sequence and extends our understanding of stop codon readthrough. Strategic targets that can be exploited to disrupt the virus life cycle and reduce its ability to move within and between plant hosts were revealed. Copyright © 2018 American Society for Microbiology.

Tornado-like Evolution of a Kink-unstable Solar Prominence

NASA Astrophysics Data System (ADS)

Wang, Wensi; Liu, Rui; Wang, Yuming

2017-01-01

We report on the tornado-like evolution of a quiescent prominence on 2014 November 1. The eastern section of the prominence first rose slowly, transforming into an arch-shaped structure as high as ˜150 Mm above the limb; the arch then writhed moderately in a left-handed sense, while the original dark prominence material emitted in the Fe ix 171 Å passband, and a braided structure appeared at the eastern edge of the warped arch. The unraveling of the braided structure was associated with a transient brightening in the EUV and apparently contributed to the formation of a curtain-like structure (CLS). The CLS consisted of myriad thread-like loops rotating counterclockwise about the vertical if viewed from above. Heated prominence material was observed to slide along these loops and land outside the filament channel. The tornado eventually disintegrated and the remaining material flew along a left-handed helical path constituting approximately a full turn, as corroborated through stereoscopic reconstruction, into the cavity of the stable, western section of the prominence. We suggest that the tornado-like evolution of the prominence was governed by the helical kink instability, and that the CLS formed through magnetic reconnections between the prominence field and the overlying coronal field.

Loop L5 Assumes Three Distinct Orientations during the ATPase Cycle of the Mitotic Kinesin Eg5

PubMed Central

Muretta, Joseph M.; Behnke-Parks, William M.; Major, Jennifer; Petersen, Karl J.; Goulet, Adeline; Moores, Carolyn A.; Thomas, David D.; Rosenfeld, Steven S.

2013-01-01

Members of the kinesin superfamily of molecular motors differ in several key structural domains, which probably allows these molecular motors to serve the different physiologies required of them. One of the most variable of these is a stem-loop motif referred to as L5. This loop is longest in the mitotic kinesin Eg5, and previous structural studies have shown that it can assume different conformations in different nucleotide states. However, enzymatic domains often consist of a mixture of conformations whose distribution shifts in response to substrate binding or product release, and this information is not available from the “static” images that structural studies provide. We have addressed this issue in the case of Eg5 by attaching a fluorescent probe to L5 and examining its fluorescence, using both steady state and time-resolved methods. This reveals that L5 assumes an equilibrium mixture of three orientations that differ in their local environment and segmental mobility. Combining these studies with transient state kinetics demonstrates that there is a major shift in this distribution during transitions that interconvert weak and strong microtubule binding states. Finally, in conjunction with previous cryo-EM reconstructions of Eg5·microtubule complexes, these fluorescence studies suggest a model in which L5 regulates both nucleotide and microtubule binding through a set of reversible interactions with helix α3. We propose that these features facilitate the production of sustained opposing force by Eg5, which underlies its role in supporting formation of a bipolar spindle in mitosis. PMID:24145034

Characterization of the complete mitochondrial genome of Chilo auricilius and comparison with three other rice stem borers.

PubMed

Cao, Shuang-Shuang; Du, Yu-Zhou

2014-09-15

The mitogenome of Chilo auricilius (Lepidoptera: Pyraloidea: Crambidae) was a circular molecule made up of 15,367 bp. Sesamia inferens, Chilo suppressalis, Tryporyza incertulas, and C. auricilius, are closely related, well known rice stem borers that are widely distributed in the main rice-growing regions of China. The gene order and orientation of all four stem borers were similar to that of other insect mitogenomes. Among the four stem borers, all AT contents were below 83%, while all AT contents of tRNA genes were above 80%. The genomes were compact, with only 121-257 bp of non-coding intergenic spacer. There are 56 or 62-bp overlapping nucleotides in Crambidae moths, but were only 25-bp overlapping nucleotides in the noctuid moth S. inferens. There was a conserved motif 'ATACTAAA' between trnS2 (UCN) and nad1 in Crambidae moths, but this same region was 'ATCATA' in the noctuid S. inferens. And there was a 6-bp motif 'ATGATAA' of overlapping nucleotides, which was conserved in Lepidoptera, and a 14-bp motif 'TAAGCTATTTAAAT' conserved in the three Crambidae moths (C. suppressalis, C. auricilius and T. incertulas), but not in the noctuid. Finally, there were no stem-and-loop structures in the two Chilo moths. Copyright © 2014 Elsevier B.V. All rights reserved.

Encapsulation of Mesenchymal Stem Cells Improves Vascularization of Alginate-Based Scaffolds.

PubMed

Steiner, Dominik; Lingens, Lara; Fischer, Laura; Köhn, Katrin; Detsch, Rainer; Boccaccini, Aldo R; Fey, Tobias; Greil, Peter; Weis, Christian; Beier, Justus P; Horch, Raymund E; Arkudas, Andreas

2018-05-09

Vascularization of bioartificial tissues can be significantly enhanced by the generation of an arteriovenous (AV) loop. Besides the surgical vascularization, the choice of the scaffold and the applied cells are indispensable cofactors. The combination of alginate dialdehyde and gelatin (ADA-GEL) and mesenchymal stem cells (MSCs) is a promising approach with regard to biocompatibility, biodegradation, as well as de novo tissue formation. In this study, we targeted the investigation of the vascularization of ADA-GEL with and in the absence of encapsulated MSCs in the AV loop model. A Teflon chamber filled with ADA-GEL microcapsules was placed in the groin of Lewis rats and an AV loop was placed into the chamber. Group A encompassed the ADA-GEL without MSCs, whereas group B contained 2 × 10 6 DiI-labeled MSCs/mL ADA-GEL. Four weeks postoperatively, tissue formation and vascularization were investigated by histology and microcomputed tomography. We were able to prove vascularization originating from the AV loop in both groups with statistically significant more vessels in group B containing MSCs. Moreover, encapsulated MSCs promoted biodegradation of the ADA-GEL microcapsules. In the present study, we were able to demonstrate for the first time, the successful vascularization of ADA-GEL microcapsules by means of the AV loop. Furthermore, ADA-GEL displayed a good biocompatibility and encapsulation of MSCs into ADA-GEL microcapsule-enhanced vascularization as well as biodegradation.

Structural and Dynamical Characterization of DNA and RNA Quadruplexes Obtained from the GGGGCC and GGGCCT Hexanucleotide Repeats Associated with C9FTD/ALS and SCA36 Diseases.

PubMed

Zhang, Yuan; Roland, Christopher; Sagui, Celeste

2018-05-16

A (GGGGCC) hexanucleotide repeat (HR) expansion in the C9ORF72 gene has been considered the major cause behind both frontotemporal dementia and amyotrophic lateral sclerosis, while a (GGGCCT) is associated with spinocerebellar ataxia 36. Recent experiments involving NMR, CD, optical melting and 1D 1 H NMR spectroscopy, suggest that the r(GGGGCC) HR can adopt a hairpin structure with G-G mismatches in equilibrium with a G-quadruplex structure. G-Quadruplexes have also been identified for d(GGGGCC). As these experiments lack molecular resolution, we have used molecular dynamics microsecond simulations to obtain a structural characterization of the G-quadruplexes associated with both HRs. All DNA G-quadruplexes, parallel or antiparallel, with or without loops are stable, while only parallel and one antiparallel (stabilized by diagonal loops) RNA G-quadruplexes are stable. It is known that antiparallel G-quadruplexes require alternating guanines to be in a syn conformation that is hindered by the C3'-endo pucker preferred by RNA. Initial RNA antiparallel quadruplexes built with C2'-endo sugars evolve such that the transition (C2'-endo)-to-(C3'-endo) triggers unwinding and buckling of the flat G-tetrads, resulting in the unfolding of the RNA antiparallel quadruplex. Finally, a parallel G-quadruplex stabilizes an adjacent C-tetrad in both DNA and RNA (thus effectively becoming a mixed quadruplex of 5 layers). The C-tetrad is stabilized by the stacking interactions with the preceding G-tetrad, by cyclical hydrogen bonds C(N4)-(O2), and by an ion between the G-tetrad and the C-tetrad. In addition, antiparallel DNA G-quadruplexes also stabilize flat C-layers at the ends of the quadruplexes.

Characterization of electrical noise limits in ultra-stable laser systems.

PubMed

Zhang, J; Shi, X H; Zeng, X Y; Lü, X L; Deng, K; Lu, Z H

2016-12-01

We demonstrate thermal noise limited and shot noise limited performance of ultra-stable diode laser systems. The measured heterodyne beat linewidth between such two independent diode lasers reaches 0.74 Hz. The frequency instability of one single laser approaches 1.0 × 10 -15 for averaging time between 0.3 s and 10 s, which is close to the thermal noise limit of the reference cavity. Taking advantage of these two ultra-stable laser systems, we systematically investigate the ultimate electrical noise contributions, and derive expressions for the closed-loop spectral density of laser frequency noise. The measured power spectral density of the beat frequency is compared with the theoretically calculated closed-loop spectral density of the laser frequency noise, and they agree very well. It illustrates the power and generality of the derived closed-loop spectral density formula of the laser frequency noise. Our result demonstrates that a 10 -17 level locking in a wide frequency range is feasible with careful design.

The multidrug ABC transporter BmrC/BmrD of Bacillus subtilis is regulated via a ribosome-mediated transcriptional attenuation mechanism

PubMed Central

Reilman, Ewoud; Mars, Ruben A. T.; van Dijl, Jan Maarten; Denham, Emma L.

2014-01-01

Expression of particular drug transporters in response to antibiotic pressure is a critical element in the development of bacterial multidrug resistance, and represents a serious concern for human health. To obtain a better understanding of underlying regulatory mechanisms, we have dissected the transcriptional activation of the ATP-binding cassette (ABC) transporter BmrC/BmrD of the Gram-positive model bacterium Bacillus subtilis. By using promoter-GFP fusions and live cell array technology, we demonstrate a temporally controlled transcriptional activation of the bmrCD genes in response to antibiotics that target protein synthesis. Intriguingly, bmrCD expression only occurs during the late-exponential and stationary growth stages, irrespective of the timing of the antibiotic challenge. We show that this is due to tight transcriptional control by the transition state regulator AbrB. Moreover, our results show that the bmrCD genes are co-transcribed with bmrB (yheJ), a small open reading frame immediately upstream of bmrC that harbors three alternative stem-loop structures. These stem-loops are apparently crucial for antibiotic-induced bmrCD transcription. Importantly, the antibiotic-induced bmrCD expression requires translation of bmrB, which implies that BmrB serves as a regulatory leader peptide. Altogether, we demonstrate for the first time that a ribosome-mediated transcriptional attenuation mechanism can control the expression of a multidrug ABC transporter. PMID:25217586

Sequence-structure relationships in RNA loops: establishing the basis for loop homology modeling.

PubMed

Schudoma, Christian; May, Patrick; Nikiforova, Viktoria; Walther, Dirk

2010-01-01

The specific function of RNA molecules frequently resides in their seemingly unstructured loop regions. We performed a systematic analysis of RNA loops extracted from experimentally determined three-dimensional structures of RNA molecules. A comprehensive loop-structure data set was created and organized into distinct clusters based on structural and sequence similarity. We detected clear evidence of the hallmark of homology present in the sequence-structure relationships in loops. Loops differing by <25% in sequence identity fold into very similar structures. Thus, our results support the application of homology modeling for RNA loop model building. We established a threshold that may guide the sequence divergence-based selection of template structures for RNA loop homology modeling. Of all possible sequences that are, under the assumption of isosteric relationships, theoretically compatible with actual sequences observed in RNA structures, only a small fraction is contained in the Rfam database of RNA sequences and classes implying that the actual RNA loop space may consist of a limited number of unique loop structures and conserved sequences. The loop-structure data sets are made available via an online database, RLooM. RLooM also offers functionalities for the modeling of RNA loop structures in support of RNA engineering and design efforts.

Control technology development

NASA Astrophysics Data System (ADS)

Schaechter, D. B.

1982-03-01

The main objectives of the control technology development task are given in the slide below. The first is to develop control design techniques based on flexible structural models, rather than simple rigid-body models. Since large space structures are distributed parameter systems, a new degree of freedom, that of sensor/actuator placement, may be exercised for improving control system performance. Another characteristic of large space structures is numerous oscillatory modes within the control bandwidth. Reduced-order controller design models must be developed which produce stable closed-loop systems when combined with the full-order system. Since the date of an actual large-space-structure flight is rapidly approaching, it is vitally important that theoretical developments are tested in actual hardware. Experimental verification is a vital counterpart of all current theoretical developments.

Stimulating Fracture Healing in Ischemic Environments: Does Oxygen Direct Stem Cell Fate during Fracture Healing?

PubMed Central

Miclau, Katherine R.; Brazina, Sloane A.; Bahney, Chelsea S.; Hankenson, Kurt D.; Hunt, Thomas K.; Marcucio, Ralph S.; Miclau, Theodore

2017-01-01

Bone fractures represent an enormous societal and economic burden as one of the most prevalent causes of disability worldwide. Each year, nearly 15 million people are affected by fractures in the United States alone. Data indicate that the blood supply is critical for fracture healing; as data indicate that concomitant bone and vascular injury are major risk factors for non-union. However, the various role(s) that the vasculature plays remains speculative. Fracture stabilization dictates stem cell fate choices during repair. In stabilized fractures stem cells differentiate directly into osteoblasts and heal the injury by intramembranous ossification. In contrast, in non-stable fractures stem cells differentiate into chondrocytes and the bone heals through endochondral ossification, where a cartilage template transforms into bone as the chondrocytes transform into osteoblasts. One suggested role of the vasculature has been to participate in the stem cell fate decisions due to delivery of oxygen. In stable fractures, the blood vessels are thought to remain intact and promote osteogenesis, while in non-stable fractures, continual disruption of the vasculature creates hypoxia that favors formation of cartilage, which is avascular. However, recent data suggests that non-stable fractures are more vascularized than stable fractures, that oxygen does not appear associated with differentiation of stem cells into chondrocytes and osteoblasts, that cartilage is not hypoxic, and that oxygen, not sustained hypoxia, is required for angiogenesis. These unexpected results, which contrast other published studies, are indicative of the need to better understand the complex, spatio-temporal regulation of vascularization and oxygenation in fracture healing. This work has also revealed that oxygen, along with the promotion of angiogenesis, may be novel adjuvants that can stimulate healing in select patient populations. PMID:28523266

Three Dimensional Structure and Time Development of Radio Emission from Solar Active Regions.

DTIC Science & Technology

1983-01-15

8217 surrounded by a weaker unpolarized halo whose angular extent ranges between 5’ and 9’. The bright (106K) sunspot-associated cores, which were intepreted in...shorter intervals. Examination of the He film indicates that the dominant He emission was stable for periods of at least six hours. Figure 8 and 9 also...the cool loops and may occupy a substantial fraction of the region above sunspots. This intepretation has, in fact, been supported by the model of

[Circular RNA in human disease and their potential clinic significance].

PubMed

Chen, Yonghua; Li, Cheng; Tan, Chunlu; Mai, Gang; Liu, Xubao

2017-02-10

Circular RNAs (circ RNAs) are a novel type of RNA that, unlike linear RNAs, form a covalently closed continuous loop and are highly represented in the eukaryotic transcriptome. They share a stable structure, high expression and often exhibit tissue/developmental-stage-specific expression. Emerging evidence indicates that circRNAs might play important roles in human disease, such as cancer, neurological disorders and atherosclerotic vascular disease risk. The huge potentials of circRNAs are recently being discovered from the laboratory to the clinic. CircRNAs might be developed as a potential novel and stable biomarker and potential drugs used in disease diagnosis and treatment. Here, we review the current understanding of the roles of circRNAs in human disease and their potential clinic significance in disease.

Solution nuclear magnetic resonance analyses of the anticodon arms of proteinogenic and nonproteinogenic tRNA(Gly).

PubMed

Chang, Andrew T; Nikonowicz, Edward P

2012-05-01

Although the fate of most tRNA molecules in the cell is aminoacylation and delivery to the ribosome, some tRNAs are destined to fulfill other functional roles. In addition to their central role in translation, tRNA molecules participate in processes such as regulation of gene expression, bacterial cell wall biosynthesis, viral replication, antibiotic biosynthesis, and suppression of alternative splicing. In bacteria, glycyl-tRNA molecules with anticodon sequences GCC and UCC exhibit multiple extratranslational functions, including transcriptional regulation and cell wall biosynthesis. We have determined the high-resolution structures of three glycyl-tRNA anticodon arms with anticodon sequences GCC and UCC. Two of the tRNA molecules are proteinogenic (tRNA(Gly,GCC) and tRNA(Gly,UCC)), and the third is nonproteinogenic (np-tRNA(Gly,UCC)) and participates in cell wall biosynthesis. The UV-monitored thermal melting curves show that the anticodon arm of tRNA(Gly,UCC) with a loop-closing C-A(+) base pair melts at a temperature 10 °C lower than those of tRNA(Gly,GCC) and np-tRNA(Gly,UCC). U-A and C-G pairs close the loops of the latter two molecules and enhance stem stability. Mg(2+) stabilizes the tRNA(Gly,UCC) anticodon arm and reduces the T(m) differential. The structures of the three tRNA(Gly) anticodon arms exhibit small differences among one another, but none of them form the classical U-turn motif. The anticodon loop of tRNA(Gly,GCC) becomes more dynamic and disordered in the presence of multivalent cations, whereas metal ion coordination in the anticodon loops of tRNA(Gly,UCC) and np-tRNA(Gly,UCC) establishes conformational homogeneity. The conformational similarity of the molecules is greater than their functional differences might suggest. Because aminoacylation of full-length tRNA molecules is accomplished by one tRNA synthetase, the similar structural context of the loop may facilitate efficient recognition of each of the anticodon sequences.

Reconstruction of structural evolution in the trnL intron P6b loop of symbiotic Nostoc (Cyanobacteria).

PubMed

Olsson, Sanna; Kaasalainen, Ulla; Rikkinen, Jouko

2012-02-01

In this study we reconstruct the structural evolution of the hyper-variable P6b region of the group I trnLeu intron in a monophyletic group of lichen-symbiotic Nostoc strains and establish it as a useful marker in the phylogenetic analysis of these organisms. The studied cyanobacteria occur as photosynthetic and/or nitrogen-fixing symbionts in lichen species of the diverse Nephroma guild. Phylogenetic analyses and secondary structure reconstructions are used to improve the understanding of the replication mechanisms in the P6b stem-loop and to explain the observed distribution patterns of indels. The variants of the P6b region in the Nostoc clade studied consist of different combinations of five sequence modules. The distribution of indels together with the ancestral character reconstruction performed enables the interpretation of the evolution of each sequence module. Our results indicate that the indel events are usually associated with single nucleotide changes in the P6b region and have occurred several times independently. In spite of their homoplasy, they provide phylogenetic information for closely related taxa. Thus we recognize that features of the P6b region can be used as molecular markers for species identification and phylogenetic studies involving symbiotic Nostoc cyanobacteria.

Flexibility and mutagenic resiliency of glycosyltransferases.

PubMed

Bay, Marie Lund; Cuesta-Seijo, Jose A; Weadge, Joel T; Persson, Mattias; Palcic, Monica M

2014-10-01

The human blood group A and B antigens are synthesized by two highly homologous enzymes, glycosyltransferase A (GTA) and glycosyltransferase B (GTB), respectively. These enzymes catalyze the transfer of either GalNAc or Gal from their corresponding UDP-donors to αFuc1-2βGal-R terminating acceptors. GTA and GTB differ at only four of 354 amino acids (R176G, G235S, L266M, G268A), which alter the donor specificity from UDP-GalNAc to UDP-Gal. Blood type O individuals synthesize truncated or non-functional enzymes. The cloning, crystallization and X-ray structure elucidations for GTA and GTB have revealed key residues responsible for donor discrimination and acceptor binding. Structural studies suggest that numerous conformational changes occur during the catalytic cycle. Over 300 ABO alleles are tabulated in the blood group antigen mutation database (BGMUT) that provides a framework for structure-function studies. Natural mutations are found in all regions of GTA and GTB from the active site, flexible loops, stem region and surfaces remote from the active site. Our characterizations of natural mutants near a flexible loop (V175M), on a remote surface site (P156L), in the metal binding motif (M212V) and near the acceptor binding site (L232P) demonstrate the resiliency of GTA and GTB to mutagenesis.

A Positive Regulatory Loop between a Wnt-Regulated Non-coding RNA and ASCL2 Controls Intestinal Stem Cell Fate.

PubMed

Giakountis, Antonis; Moulos, Panagiotis; Zarkou, Vasiliki; Oikonomou, Christina; Harokopos, Vaggelis; Hatzigeorgiou, Artemis G; Reczko, Martin; Hatzis, Pantelis

2016-06-21

The canonical Wnt pathway plays a central role in stem cell maintenance, differentiation, and proliferation in the intestinal epithelium. Constitutive, aberrant activity of the TCF4/β-catenin transcriptional complex is the primary transforming factor in colorectal cancer. We identify a nuclear long non-coding RNA, termed WiNTRLINC1, as a direct target of TCF4/β-catenin in colorectal cancer cells. WiNTRLINC1 positively regulates the expression of its genomic neighbor ASCL2, a transcription factor that controls intestinal stem cell fate. WiNTRLINC1 interacts with TCF4/β-catenin to mediate the juxtaposition of its promoter with the regulatory regions of ASCL2. ASCL2, in turn, regulates WiNTRLINC1 transcriptionally, closing a feedforward regulatory loop that controls stem cell-related gene expression. This regulatory circuitry is highly amplified in colorectal cancer and correlates with increased metastatic potential and decreased patient survival. Our results uncover the interplay between non-coding RNA-mediated regulation and Wnt signaling and point to the diagnostic and therapeutic potential of WiNTRLINC1. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Functional formation of domain V of the poliovirus noncoding region: significance of unpaired bases.

PubMed

Rowe, A; Burlison, J; Macadam, A J; Minor, P D

2001-10-10

Previously we have shown that polioviruses with mutations that disrupt the predicted secondary structure of the 5' noncoding region of domain V are temperature sensitive for growth. Non-temperature-sensitive revertant viruses had mutations that re-formed secondary structure by a direct back mutation of changes in the opposite strand. We mutated unpaired regions and selected revertants of viruses with single base deletions, where no obvious back mutation was available in order to gain information on secondary structure. Results indicated that conservation of length of a three base loop between two double-stranded stems was essential for a functional domain V to form. The requirement for the unpaired "hinge" base at 484 which is implicated in the attenuation of Sabin 2 was also confirmed. Results also underline the necessity for functional folding over local secondary structure stability. Copyright 2001 Academic Press.

Effects of magnesium ions on the stabilization of RNA oligomers of defined structures.

PubMed Central

Serra, Martin J; Baird, John D; Dale, Taraka; Fey, Bridget L; Retatagos, Kimberly; Westhof, Eric

2002-01-01

Optical melting was used to determine the stabilities of 11 small RNA oligomers of defined secondary structure as a function of magnesium ion concentration. The oligomers included helices composed of Watson-Crick base pairs, GA tandem base pairs, GU tandem base pairs, and loop E motifs (both eubacterial and eukaryotic). The effect of magnesium ion concentration on stability was interpreted in terms of two simple models. The first assumes an uptake of metal ion upon duplex formation. The second assumes nonspecific electrostatic attraction of metal ions to the RNA oligomer. For all oligomers, except the eubacterial loop E, the data could best be interpreted as nonspecific binding of metal ions to the RNAs. The effect of magnesium ions on the stability of the eubacterial loop E was distinct from that seen with the other oligomers in two ways. First, the extent of stabilization by magnesium ions (as measured by either change in melting temperature or free energy) was three times greater than that observed for the other helical oligomers. Second, the presence of magnesium ions produces a doubling of the enthalpy for the melting transition. These results indicate that magnesium ion stabilizes the eubacterial loop E sequence by chelating the RNA specifically. Further, these results on a rather small system shed light on the large enthalpy changes observed upon thermal unfolding of large RNAs like group I introns. It is suggested that parts of those large enthalpy changes observed in the folding of RNAs may be assigned to variations in the hydration states and types of coordinating atoms in some specifically bound magnesium ions and to an increase in the observed cooperativity of the folding transition due to the binding of those magnesium ions coupling the two stems together. Brownian dynamic simulations, carried out to visualize the metal ion binding sites, reveal rather delocalized ionic densities in all oligomers, except for the eubacterial loop E, in which precisely located ion densities were previously calculated. PMID:12003491

Efficient HIV-1 inhibition by a 16 nt-long RNA aptamer designed by combining in vitro selection and in silico optimisation strategies

PubMed Central

Sánchez-Luque, Francisco J.; Stich, Michael; Manrubia, Susanna; Briones, Carlos; Berzal-Herranz, Alfredo

2014-01-01

The human immunodeficiency virus type-1 (HIV-1) genome contains multiple, highly conserved structural RNA domains that play key roles in essential viral processes. Interference with the function of these RNA domains either by disrupting their structures or by blocking their interaction with viral or cellular factors may seriously compromise HIV-1 viability. RNA aptamers are amongst the most promising synthetic molecules able to interact with structural domains of viral genomes. However, aptamer shortening up to their minimal active domain is usually necessary for scaling up production, what requires very time-consuming, trial-and-error approaches. Here we report on the in vitro selection of 64 nt-long specific aptamers against the complete 5′-untranslated region of HIV-1 genome, which inhibit more than 75% of HIV-1 production in a human cell line. The analysis of the selected sequences and structures allowed for the identification of a highly conserved 16 nt-long stem-loop motif containing a common 8 nt-long apical loop. Based on this result, an in silico designed 16 nt-long RNA aptamer, termed RNApt16, was synthesized, with sequence 5′-CCCCGGCAAGGAGGGG-3′. The HIV-1 inhibition efficiency of such an aptamer was close to 85%, thus constituting the shortest RNA molecule so far described that efficiently interferes with HIV-1 replication. PMID:25175101

First Mitochondrial Genome from Nemouridae (Plecoptera) Reveals Novel Features of the Elongated Control Region and Phylogenetic Implications

PubMed Central

Chen, Zhi-Teng; Du, Yu-Zhou

2017-01-01

The complete mitochondrial genome (mitogenome) of Nemoura nankinensis (Plecoptera: Nemouridae) was sequenced as the first reported mitogenome from the family Nemouridae. The N. nankinensis mitogenome was the longest (16,602 bp) among reported plecopteran mitogenomes, and it contains 37 genes including 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes and two ribosomal RNA (rRNA) genes. Most PCGs used standard ATN as start codons, and TAN as termination codons. All tRNA genes of N. nankinensis could fold into the cloverleaf secondary structures except for trnSer (AGN), whose dihydrouridine (DHU) arm was reduced to a small loop. There was also a large non-coding region (control region, CR) in the N. nankinensis mitogenome. The 1751 bp CR was the longest and had the highest A+T content (81.8%) among stoneflies. A large tandem repeat region, five potential stem-loop (SL) structures, four tRNA-like structures and four conserved sequence blocks (CSBs) were detected in the elongated CR. The presence of these tRNA-like structures in the CR has never been reported in other plecopteran mitogenomes. These novel features of the elongated CR in N. nankinensis may have functions associated with the process of replication and transcription. Finally, phylogenetic reconstruction suggested that Nemouridae was the sister-group of Capniidae. PMID:28475163

First Mitochondrial Genome from Nemouridae (Plecoptera) Reveals Novel Features of the Elongated Control Region and Phylogenetic Implications.

PubMed

Chen, Zhi-Teng; Du, Yu-Zhou

2017-05-05

The complete mitochondrial genome (mitogenome) of Nemoura nankinensis (Plecoptera: Nemouridae) was sequenced as the first reported mitogenome from the family Nemouridae. The N. nankinensis mitogenome was the longest (16,602 bp) among reported plecopteran mitogenomes, and it contains 37 genes including 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes and two ribosomal RNA (rRNA) genes. Most PCGs used standard ATN as start codons, and TAN as termination codons. All tRNA genes of N. nankinensis could fold into the cloverleaf secondary structures except for trnSer ( AGN ), whose dihydrouridine (DHU) arm was reduced to a small loop. There was also a large non-coding region (control region, CR) in the N. nankinensis mitogenome. The 1751 bp CR was the longest and had the highest A+T content (81.8%) among stoneflies. A large tandem repeat region, five potential stem-loop (SL) structures, four tRNA-like structures and four conserved sequence blocks (CSBs) were detected in the elongated CR. The presence of these tRNA-like structures in the CR has never been reported in other plecopteran mitogenomes. These novel features of the elongated CR in N. nankinensis may have functions associated with the process of replication and transcription. Finally, phylogenetic reconstruction suggested that Nemouridae was the sister-group of Capniidae.

Porphyrinoids as a platform of stable radicals

PubMed Central

Shimizu, Daiki

2018-01-01

The non-innocent ligand nature of porphyrins was observed for compound I in enzymatic cycles of cytochrome P450. Such porphyrin radicals were first regarded as reactive intermediates in catabolism, but recent studies have revealed that porphyrinoids, including porphyrins, ring-contracted porphyrins, and ring-expanded porphyrins, display excellent radical-stabilizing abilities to the extent that radicals can be handled like usual closed-shell organic molecules. This review surveys four types of stable porphyrinoid radical and covers their synthetic methods and properties such as excellent redox properties, NIR absorption, and magnetic properties. The radical-stabilizing abilities of porphyrinoids stem from their unique macrocyclic conjugated systems with high electronic and structural flexibilities. PMID:29675188

Upregulation of the PatAB Transporter Confers Fluoroquinolone Resistance to Streptococcus pseudopneumoniae

PubMed Central

Alvarado, María; Martín-Galiano, Antonio J.; Ferrándiz, María J.; Zaballos, Ángel; de la Campa, Adela G.

2017-01-01

We characterized the mechanism of fluoroquinolone-resistance in two isolates of Streptococcus pseudopneumoniae having fluoroquinolone-efflux as unique mechanism of resistance. Whole genome sequencing and genetic transformation experiments were performed together with phenotypic determinations of the efflux mechanism. The PatAB pump was identified as responsible for efflux of ciprofloxacin (MIC of 4 μg/ml), ethidium bromide (MICs of 8–16 μg/ml) and acriflavine (MICs of 4–8 μg/ml) in both isolates. These MICs were at least 8-fold lower in the presence of the efflux inhibitor reserpine. Complete genome sequencing indicated that the sequence located between the promoter of the patAB operon and the initiation codon of patA, which putatively forms an RNA stem-loop structure, may be responsible for the efflux phenotype. RT-qPCR determinations performed on RNAs of cultures treated or not treated with subinhibitory ciprofloxacin concentrations were performed. While no significant changes were observed in wild-type Streptococcus pneumoniae R6 strain, increases in transcription were detected in the ciprofloxacin-efflux transformants obtained with DNA from efflux-positive isolates, in the ranges of 1.4 to 3.4-fold (patA) and 2.1 to 2.9-fold (patB). Ciprofloxacin-induction was related with a lower predicted free energy for the stem-loop structure in the RNA of S. pseudopneumoniae isolates (−13.81 and −8.58) than for R6 (−15.32 kcal/mol), which may ease transcription. The presence of these regulatory variations in commensal S. pseudopneumoniae isolates, and the possibility of its transfer to Streptococcus pneumoniae by genetic transformation, could increase fluoroquinolone resistance in this important pathogen. PMID:29123510

Structural variant of the intergenic internal ribosome entry site elements in dicistroviruses and computational search for their counterparts

PubMed Central

HATAKEYAMA, YOSHINORI; SHIBUYA, NORIHIRO; NISHIYAMA, TAKASHI; NAKASHIMA, NOBUHIKO

2004-01-01

The intergenic region (IGR) located upstream of the capsid protein gene in dicistroviruses contains an internal ribosome entry site (IRES). Translation initiation mediated by the IRES does not require initiator methionine tRNA. Comparison of the IGRs among dicistroviruses suggested that Taura syndrome virus (TSV) and acute bee paralysis virus have an extra side stem loop in the predicted IRES. We examined whether the side stem is responsible for translation activity mediated by the IGR using constructs with compensatory mutations. In vitro translation analysis showed that TSV has an IGR-IRES that is structurally distinct from those previously described. Because IGR-IRES elements determine the translation initiation site by virtue of their own tertiary structure formation, the discovery of this initiation mechanism suggests the possibility that eukaryotic mRNAs might have more extensive coding regions than previously predicted. To test this hypothesis, we searched full-length cDNA databases and whole genome sequences of eukaryotes using the pattern matching program, Scan For Matches, with parameters that can extract sequences containing secondary structure elements resembling those of IGR-IRES. Our search yielded several sequences, but their predicted secondary structures were suggested to be unstable in comparison to those of dicistroviruses. These results suggest that RNAs structurally similar to dicistroviruses are not common. If some eukaryotic mRNAs are translated independently of an initiator methionine tRNA, their structures are likely to be significantly distinct from those of dicistroviruses. PMID:15100433

Protein loop modeling using a new hybrid energy function and its application to modeling in inaccurate structural environments.

PubMed

Park, Hahnbeom; Lee, Gyu Rie; Heo, Lim; Seok, Chaok

2014-01-01

Protein loop modeling is a tool for predicting protein local structures of particular interest, providing opportunities for applications involving protein structure prediction and de novo protein design. Until recently, the majority of loop modeling methods have been developed and tested by reconstructing loops in frameworks of experimentally resolved structures. In many practical applications, however, the protein loops to be modeled are located in inaccurate structural environments. These include loops in model structures, low-resolution experimental structures, or experimental structures of different functional forms. Accordingly, discrepancies in the accuracy of the structural environment assumed in development of the method and that in practical applications present additional challenges to modern loop modeling methods. This study demonstrates a new strategy for employing a hybrid energy function combining physics-based and knowledge-based components to help tackle this challenge. The hybrid energy function is designed to combine the strengths of each energy component, simultaneously maintaining accurate loop structure prediction in a high-resolution framework structure and tolerating minor environmental errors in low-resolution structures. A loop modeling method based on global optimization of this new energy function is tested on loop targets situated in different levels of environmental errors, ranging from experimental structures to structures perturbed in backbone as well as side chains and template-based model structures. The new method performs comparably to force field-based approaches in loop reconstruction in crystal structures and better in loop prediction in inaccurate framework structures. This result suggests that higher-accuracy predictions would be possible for a broader range of applications. The web server for this method is available at http://galaxy.seoklab.org/loop with the PS2 option for the scoring function.

Stable multi-wavelength fiber lasers for temperature measurements using an optical loop mirror.

PubMed

Diaz, Silvia; Socorro, Abian Bentor; Martínez Manuel, Rodolfo; Fernandez, Ruben; Monasterio, Ioseba

2016-10-10

In this work, two novel stable multi-wavelength fiber laser configurations are proposed and demonstrated by using a spool of a single-mode fiber as an optical loop mirror and one or two fiber ring cavities, respectively. The lasers are comprised of fiber Bragg grating reflectors as the oscillation wavelength selecting filters. The influence of the length of the spool of fiber on the laser stability both in terms of wavelength and laser output power was investigated. An application for temperature measurement is also shown.

Multiwavelength erbium-doped fiber laser employing a nonlinear optical loop mirror

NASA Astrophysics Data System (ADS)

Feng, Xinhuan; Tam, Hwa-yaw; Liu, Heliang; Wai, P. K. A.

2006-12-01

A stable and broad bandwidth multiwavelength erbium-doped fiber laser is proposed and demonstrated successfully. A nonlinear optical loop mirror which induces wavelength-dependent cavity loss and behaves as an amplitude equalizer is employed to ensure stable room-temperature multiwavelength operation. Up to 50 wavelengths lasing oscillations with wavelength spacing of 0.8 nm within a 3-dB spectral range of 1562-1605 nm has been achieved. The measured power fluctuation of each wavelength is about 0.1 dB within a 2-h period.

Value of routine investigations to predict loop diuretic down-titration success in stable heart failure.

PubMed

Martens, Pieter; Verbrugge, Frederik H; Boonen, Levinia; Nijst, Petra; Dupont, Matthias; Mullens, Wilfried

2018-01-01

Guidelines advocate down-titration of loop diuretics in chronic heart failure (CHF) when patients have no signs of volume overload. Limited data are available on the expected success rate of this practice or how routine diagnostic tests might help steering this process. Fifty ambulatory CHF-patients on stable neurohumoral blocker/diuretic therapy for at least 3months without any clinical sign of volume overload were prospectively included to undergo loop diuretic down-titration. All patients underwent a similar pre-down-titration evaluation consisting of a dyspnea scoring, physical examination, transthoracic echocardiography (diastolic function, right ventricular function, cardiac filling pressures and valvular disease), blood sample (serum creatinine, plasma NT-pro-BNP and neurohormones). Loop diuretic maintenance dose was subsequently reduced by 50% or stopped if dose was ≤40mg furosemide equivalents. Successful down-titration was defined as a persistent dose reduction after 30days without weight increase >1.5kg or new-onset symptoms of worsening heart failure. At 30-day follow-up, down-titration was successful in 62% (n=31). In 12/19 patients exhibiting down-titration failure, this occurred within the first week. Physical examination, transthoracic echocardiography and laboratory analysis had limited predictive capability to detect patients with down-titration success/failure (positive likelihood-ratios below 1.5, or area under the curve [AUC] non-statically different from AUC=0.5). Loop diuretic down-titration is feasible in a majority of stable CHF patients in which the treating clinician felt continuation of loops was unnecessary to sustain euvolemia. Importantly, routine diagnostics which suggest euvolemia, have limited diagnostic impact on the post-test probability. Copyright © 2017 Elsevier B.V. All rights reserved.

Modeling and stability of segmented reflector telescopes - A decentralized approach

NASA Technical Reports Server (NTRS)

Ryaciotaki-Boussalis, Helen A.; Ih, Che-Hang Charles

1990-01-01

The decentralization of a segmented reflector telescope based on a finite-element model of its structure is considered. The decentralization of the system at the panel level is considered. Each panel is originally treated as an isolated subsystem so that the controller design is performed independently at the local level, and then applied to the composite system for stability analysis. The panel-level control laws were designed by means of pole placement using local output feedback. Simulation results show a better 1000:1 vibration attenuation in panel position when compared to the open-loop system. It is shown that the overall closed-loop system is exponentially stable provided that certain conditions are met. The advantage to the decentralized approach is that the design is performed in terms of the low-dimensionality subsystems, thus drastically reducing the design computational complexities.

The analysis of novel microRNA mimic sequences in cancer cells reveals lack of specificity in stem-loop RT-qPCR-based microRNA detection.

PubMed

Winata, Patrick; Williams, Marissa; McGowan, Eileen; Nassif, Najah; van Zandwijk, Nico; Reid, Glen

2017-11-17

MicroRNAs are frequently downregulated in cancer, and restoring expression has tumour suppressive activity in tumour cells. Our recent phase I clinical trial investigated microRNA-based therapy in patients with malignant pleural mesothelioma. Treatment with TargomiRs, microRNA mimics with novel sequence packaged in EGFR antibody-targeted bacterial minicells, revealed clear signs of clinical activity. In order to detect delivery of microRNA mimics to tumour cells in future clinical trials, we tested hydrolysis probe-based assays specific for the sequence of the novel mimics in transfected mesothelioma cell lines using RT-qPCR. The custom assays efficiently and specifically amplified the consensus mimics. However, we found that these assays gave a signal when total RNA from untransfected and control mimic-transfected cells were used as templates. Further investigation revealed that the reverse transcription step using stem-loop primers appeared to introduce substantial non-specific amplification with either total RNA or synthetic RNA templates. This suggests that reverse transcription using stem-loop primers suffers from an intrinsic lack of specificity for the detection of highly similar microRNAs in the same family, especially when analysing total RNA. These results suggest that RT-qPCR is unlikely to be an effective means to detect delivery of microRNA mimic-based drugs to tumour cells in patients.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chatman, Shawn ME; Zarzycki, Piotr P.; Preocanin, Tajana

Time dependent potentiometric pH titrations were used to study the effect of atomic scale surface structure on the protonation behavior of the structurally well defined hematite/aqueous electrolyte interfaces. Our recently proposed thermodynamic model [1,23] was applied to measured acidimetric and alkalimetric titration hysteresis loops, collected from highly organized (001), (012), and (113) crystal face terminations using pH equilibration times ranging from 15 to 30 mins. Hysteresis loop areas indicate that (001) faces equilibrate faster than the (012) and (113) faces, consistent with the different expected ensembles of singly, doubly, and triply coordinated surface sites on each face. Strongly non-linear hystereticmore » pH-potential relationships were found, with slopes exceeding Nernstian, collectively indicating that protonation and deprotonation is much more complex than embodied in present day surface complexation models. The asymmetrical shape of the acidimetric and alkalimetric titration branches were used to illustrate a proposed steric "leaky screen" repulsion/trapping interaction mechanism that stems from high affinity singly-coordinated sites electrostatically and sterically screening lower affinity doubly and triply coordinated sites. Our data indicate that site interaction is the dominant phenomenon defining surface potential accumulation behavior on single crystal faces of metal oxide minerals.« less

Functional organization of the Sm core in the crystal structure of human U1 snRNP.

PubMed

Weber, Gert; Trowitzsch, Simon; Kastner, Berthold; Lührmann, Reinhard; Wahl, Markus C

2010-12-15

U1 small nuclear ribonucleoprotein (snRNP) recognizes the 5'-splice site early during spliceosome assembly. It represents a prototype spliceosomal subunit containing a paradigmatic Sm core RNP. The crystal structure of human U1 snRNP obtained from natively purified material by in situ limited proteolysis at 4.4 Å resolution reveals how the seven Sm proteins, each recognize one nucleotide of the Sm site RNA using their Sm1 and Sm2 motifs. Proteins D1 and D2 guide the snRNA into and out of the Sm ring, and proteins F and E mediate a direct interaction between the Sm site termini. Terminal extensions of proteins D1, D2 and B/B', and extended internal loops in D2 and B/B' support a four-way RNA junction and a 3'-terminal stem-loop on opposite sides of the Sm core RNP, respectively. On a higher organizational level, the core RNP presents multiple attachment sites for the U1-specific 70K protein. The intricate, multi-layered interplay of proteins and RNA rationalizes the hierarchical assembly of U snRNPs in vitro and in vivo.

Comparison of stem morphology and anatomy of two alfalfa clonal lines exhibiting divergent cell wall composition.

PubMed

Gronwald, John W; Bucciarelli, Bruna

2013-08-30

In previous research, two alfalfa clonal lines (252 and 1283) were identified that exhibited environmentally stable differences in stem cell walls. Compared with stems of 1283, stems of 252 have a higher cell wall concentration and greater amounts of lignin and cellulose but reduced levels of pectic sugar residues. These results suggest greater deposition of secondary xylem and a reduction in pith in stems of 252 compared with 1283. The stem morphology and anatomy of first-cut and second-cut harvests of field-grown 1283 and 252 were examined. For both harvests, stems of 1283 were thicker and had a higher leaf/stem ratio compared with stems of 252. Stem cross-sections of both genotypes were stained for lignin, and the proportions of stem area that were pith and secondary xylem were measured using ImageJ. Stems of 252 exhibited greater deposition of secondary xylem and a reduction in pith proportion compared with stems of 1283 for the first-cut harvest, but this difference was not statistically significant for the second-cut harvest. The results indicate that the proportions of secondary xylem and pith are not environmentally stable in these two genotypes and hence cannot be the sole basis for the differences in cell wall concentration/composition. © 2012 Society of Chemical Industry.

Simulations of Living Cell Origins Using a Cellular Automata Model

NASA Astrophysics Data System (ADS)

Ishida, Takeshi

2014-04-01

Understanding the generalized mechanisms of cell self-assembly is fundamental for applications in various fields, such as mass producing molecular machines in nanotechnology. Thus, the details of real cellular reaction networks and the necessary conditions for self-organized cells must be elucidated. We constructed a 2-dimensional cellular automata model to investigate the emergence of biological cell formation, which incorporated a looped membrane and a membrane-bound information system (akin to a genetic code and gene expression system). In particular, with an artificial reaction system coupled with a thermal system, the simultaneous formation of a looped membrane and an inner reaction process resulted in a more stable structure. These double structures inspired the primitive biological cell formation process from chemical evolution stage. With a model to simulate cellular self-organization in a 2-dimensional cellular automata model, 3 phenomena could be realized: (1) an inner reaction system developed as an information carrier precursor (akin to DNA); (2) a cell border emerged (akin to a cell membrane); and (3) these cell structures could divide into 2. This double-structured cell was considered to be a primary biological cell. The outer loop evolved toward a lipid bilayer membrane, and inner polymeric particles evolved toward precursor information carriers (evolved toward DNA). This model did not completely clarify all the necessary and sufficient conditions for biological cell self-organization. Further, our virtual cells remained unstable and fragile. However, the "garbage bag model" of Dyson proposed that the first living cells were deficient; thus, it would be reasonable that the earliest cells were more unstable and fragile than the simplest current unicellular organisms.

Simulations of living cell origins using a cellular automata model.

PubMed

Ishida, Takeshi

2014-04-01

Understanding the generalized mechanisms of cell self-assembly is fundamental for applications in various fields, such as mass producing molecular machines in nanotechnology. Thus, the details of real cellular reaction networks and the necessary conditions for self-organized cells must be elucidated. We constructed a 2-dimensional cellular automata model to investigate the emergence of biological cell formation, which incorporated a looped membrane and a membrane-bound information system (akin to a genetic code and gene expression system). In particular, with an artificial reaction system coupled with a thermal system, the simultaneous formation of a looped membrane and an inner reaction process resulted in a more stable structure. These double structures inspired the primitive biological cell formation process from chemical evolution stage. With a model to simulate cellular self-organization in a 2-dimensional cellular automata model, 3 phenomena could be realized: (1) an inner reaction system developed as an information carrier precursor (akin to DNA); (2) a cell border emerged (akin to a cell membrane); and (3) these cell structures could divide into 2. This double-structured cell was considered to be a primary biological cell. The outer loop evolved toward a lipid bilayer membrane, and inner polymeric particles evolved toward precursor information carriers (evolved toward DNA). This model did not completely clarify all the necessary and sufficient conditions for biological cell self-organization. Further, our virtual cells remained unstable and fragile. However, the "garbage bag model" of Dyson proposed that the first living cells were deficient; thus, it would be reasonable that the earliest cells were more unstable and fragile than the simplest current unicellular organisms.

Mining protein loops using a structural alphabet and statistical exceptionality

PubMed Central

2010-01-01

Background Protein loops encompass 50% of protein residues in available three-dimensional structures. These regions are often involved in protein functions, e.g. binding site, catalytic pocket... However, the description of protein loops with conventional tools is an uneasy task. Regular secondary structures, helices and strands, have been widely studied whereas loops, because they are highly variable in terms of sequence and structure, are difficult to analyze. Due to data sparsity, long loops have rarely been systematically studied. Results We developed a simple and accurate method that allows the description and analysis of the structures of short and long loops using structural motifs without restriction on loop length. This method is based on the structural alphabet HMM-SA. HMM-SA allows the simplification of a three-dimensional protein structure into a one-dimensional string of states, where each state is a four-residue prototype fragment, called structural letter. The difficult task of the structural grouping of huge data sets is thus easily accomplished by handling structural letter strings as in conventional protein sequence analysis. We systematically extracted all seven-residue fragments in a bank of 93000 protein loops and grouped them according to the structural-letter sequence, named structural word. This approach permits a systematic analysis of loops of all sizes since we consider the structural motifs of seven residues rather than complete loops. We focused the analysis on highly recurrent words of loops (observed more than 30 times). Our study reveals that 73% of loop-lengths are covered by only 3310 highly recurrent structural words out of 28274 observed words). These structural words have low structural variability (mean RMSd of 0.85 Å). As expected, half of these motifs display a flanking-region preference but interestingly, two thirds are shared by short (less than 12 residues) and long loops. Moreover, half of recurrent motifs exhibit a significant level of amino-acid conservation with at least four significant positions and 87% of long loops contain at least one such word. We complement our analysis with the detection of statistically over-represented patterns of structural letters as in conventional DNA sequence analysis. About 30% (930) of structural words are over-represented, and cover about 40% of loop lengths. Interestingly, these words exhibit lower structural variability and higher sequential specificity, suggesting structural or functional constraints. Conclusions We developed a method to systematically decompose and study protein loops using recurrent structural motifs. This method is based on the structural alphabet HMM-SA and not on structural alignment and geometrical parameters. We extracted meaningful structural motifs that are found in both short and long loops. To our knowledge, it is the first time that pattern mining helps to increase the signal-to-noise ratio in protein loops. This finding helps to better describe protein loops and might permit to decrease the complexity of long-loop analysis. Detailed results are available at http://www.mti.univ-paris-diderot.fr/publication/supplementary/2009/ACCLoop/. PMID:20132552

Mining protein loops using a structural alphabet and statistical exceptionality.

PubMed

Regad, Leslie; Martin, Juliette; Nuel, Gregory; Camproux, Anne-Claude

2010-02-04

Protein loops encompass 50% of protein residues in available three-dimensional structures. These regions are often involved in protein functions, e.g. binding site, catalytic pocket... However, the description of protein loops with conventional tools is an uneasy task. Regular secondary structures, helices and strands, have been widely studied whereas loops, because they are highly variable in terms of sequence and structure, are difficult to analyze. Due to data sparsity, long loops have rarely been systematically studied. We developed a simple and accurate method that allows the description and analysis of the structures of short and long loops using structural motifs without restriction on loop length. This method is based on the structural alphabet HMM-SA. HMM-SA allows the simplification of a three-dimensional protein structure into a one-dimensional string of states, where each state is a four-residue prototype fragment, called structural letter. The difficult task of the structural grouping of huge data sets is thus easily accomplished by handling structural letter strings as in conventional protein sequence analysis. We systematically extracted all seven-residue fragments in a bank of 93000 protein loops and grouped them according to the structural-letter sequence, named structural word. This approach permits a systematic analysis of loops of all sizes since we consider the structural motifs of seven residues rather than complete loops. We focused the analysis on highly recurrent words of loops (observed more than 30 times). Our study reveals that 73% of loop-lengths are covered by only 3310 highly recurrent structural words out of 28274 observed words). These structural words have low structural variability (mean RMSd of 0.85 A). As expected, half of these motifs display a flanking-region preference but interestingly, two thirds are shared by short (less than 12 residues) and long loops. Moreover, half of recurrent motifs exhibit a significant level of amino-acid conservation with at least four significant positions and 87% of long loops contain at least one such word. We complement our analysis with the detection of statistically over-represented patterns of structural letters as in conventional DNA sequence analysis. About 30% (930) of structural words are over-represented, and cover about 40% of loop lengths. Interestingly, these words exhibit lower structural variability and higher sequential specificity, suggesting structural or functional constraints. We developed a method to systematically decompose and study protein loops using recurrent structural motifs. This method is based on the structural alphabet HMM-SA and not on structural alignment and geometrical parameters. We extracted meaningful structural motifs that are found in both short and long loops. To our knowledge, it is the first time that pattern mining helps to increase the signal-to-noise ratio in protein loops. This finding helps to better describe protein loops and might permit to decrease the complexity of long-loop analysis. Detailed results are available at http://www.mti.univ-paris-diderot.fr/publication/supplementary/2009/ACCLoop/.

Structural and thermodynamic analysis of modified nucleosides in self-assembled DNA cross-tiles.

PubMed

Hakker, Lauren; Marchi, Alexandria N; Harris, Kimberly A; LaBean, Thomas H; Agris, Paul F

2014-01-01

DNA Holliday junctions are important natural strand-exchange structures that form during homologous recombination. Immobile four-arm junctions, analogs to Holliday junctions, have been designed to self-assemble into cross-tile structures by maximizing Watson-Crick base pairing and fixed crossover points. The cross-tiles, self-assembled from base pair recognition between designed single-stranded DNAs, form higher order lattice structures through cohesion of self-associating sticky ends. These cross-tiles have 16 unpaired nucleosides in the central loop at the junction of the four duplex stems. The importance of the centralized unpaired nucleosides to the structure's thermodynamic stability and self-assembly is unknown. Cross-tile DNA nanostructures were designed and constructed from nine single-stranded DNAs with four shell strands, four arms, and a central loop containing 16 unpaired bases. The 16 unpaired bases were either 2'-deoxyribothymidines, 2'-O-methylribouridines, or abasic 1',2'-dideoxyribonucleosides. Thermodynamic profiles and structural base-stacking contributions were assessed using UV absorption spectroscopy during thermal denaturation and circular dichroism spectroscopy, respectively, and the resulting structures were observed by atomic force microscopy. There were surprisingly significant changes in the thermodynamic and structural properties of lattice formation as a result of altering only the 16 unpaired, centralized nucleosides. The 16 unpaired 2'-O-methyluridines were stabilizing and produced uniform tubular structures. In contrast, the abasic nucleosides were destabilizing producing a mixture of structures. These results strongly indicate the importance of a small number of centrally located unpaired nucleosides within the structures. Since minor modifications lead to palpable changes in lattice formation, DNA cross-tiles present an easily manipulated structure convenient for applications in biomedical and biosensing devices.

Modeling Aggressive Medulloblastoma Using Human Induced Pluripotent Stem Cells

DTIC Science & Technology

2017-09-01

and Myc in turn induces expression of AT1R creating a positive feedback loop and development of aggression tumor phenotype. The therapeutic...strengths are the relevant expertise of the applicant and his collaborating team, the novel paracrine positive feedback loop in EC-tumor cell...to as MYC-driven MB. The molecular mechanisms that drive MYC hyper -activation in MB remain incompletely understood. MB cells in actual tumors interact

A designed glycoprotein analogue of Gc-MAF exhibits native-like phagocytic activity.

PubMed

Bogani, Federica; McConnell, Elizabeth; Joshi, Lokesh; Chang, Yung; Ghirlanda, Giovanna

2006-06-07

Rational protein design has been successfully used to create mimics of natural proteins that retain native activity. In the present work, de novo protein engineering is explored to develop a mini-protein analogue of Gc-MAF, a glycoprotein involved in the immune system activation that has shown anticancer activity in mice. Gc-MAF is derived in vivo from vitamin D binding protein (VDBP) via enzymatic processing of its glycosaccharide to leave a single GalNAc residue located on an exposed loop. We used molecular modeling tools in conjunction with structural analysis to splice the glycosylated loop onto a stable three-helix bundle (alpha3W, PDB entry 1LQ7). The resulting 69-residue model peptide, MM1, has been successfully synthesized by solid-phase synthesis both in the aglycosylated and the glycosylated (GalNAc-MM1) form. Circular dichroism spectroscopy confirmed the expected alpha-helical secondary structure. The thermodynamic stability as evaluated from chemical and thermal denaturation is comparable with that of the scaffold protein, alpha3W, indicating that the insertion of the exogenous loop of Gc-MAF did not significantly perturb the overall structure. GalNAc-MM1 retains the macrophage stimulation activity of natural Gc-MAF; in vitro tests show an identical enhancement of Fc-receptor-mediated phagocytosis in primary macrophages. GalNAc-MM1 provides a framework for the development of mutants with increased activity that could be used in place of Gc-MAF as an immunomodulatory agent in therapy.

Structural dynamics of possible late-stage intermediates in folding of quadruplex DNA studied by molecular simulations

PubMed Central

Stadlbauer, Petr; Krepl, Miroslav; Cheatham, Thomas E.; Koča, Jaroslav; Šponer, Jiří

2013-01-01

Explicit solvent molecular dynamics simulations have been used to complement preceding experimental and computational studies of folding of guanine quadruplexes (G-DNA). We initiate early stages of unfolding of several G-DNAs by simulating them under no-salt conditions and then try to fold them back using standard excess salt simulations. There is a significant difference between G-DNAs with all-anti parallel stranded stems and those with stems containing mixtures of syn and anti guanosines. The most natural rearrangement for all-anti stems is a vertical mutual slippage of the strands. This leads to stems with reduced numbers of tetrads during unfolding and a reduction of strand slippage during refolding. The presence of syn nucleotides prevents mutual strand slippage; therefore, the antiparallel and hybrid quadruplexes initiate unfolding via separation of the individual strands. The simulations confirm the capability of G-DNA molecules to adopt numerous stable locally and globally misfolded structures. The key point for a proper individual folding attempt appears to be correct prior distribution of syn and anti nucleotides in all four G-strands. The results suggest that at the level of individual molecules, G-DNA folding is an extremely multi-pathway process that is slowed by numerous misfolding arrangements stabilized on highly variable timescales. PMID:23700306

A loop 2 cytidine-stem 1 minor groove interaction as a positive determinant for pseudoknot-stimulated -1 ribosomal frameshifting.

PubMed

Cornish, Peter V; Hennig, Mirko; Giedroc, David P

2005-09-06

The molecular determinants of stimulation of -1 programmed ribosomal frameshifting (-1 PRF) by RNA pseudoknots are poorly understood. Sugarcane yellow leaf virus (ScYLV) encodes a 28-nt mRNA pseudoknot that promotes -1 PRF between the P1 (protease) and P2 (polymerase) genes in plant luteoviruses. The solution structure of the ScYLV pseudoknot reveals a well ordered loop 2 (L2) that exhibits continuous stacking of A20 through C27 in the minor groove of the upper stem 1 (S1), with C25 flipped out of the triple-stranded stack. Five consecutive triple base pairs flank the helical junction where the 3' nucleotide of L2, C27, adopts a cytidine 27 N3-cytidine 14 2'-OH hydrogen bonding interaction with the C14-G7 base pair. This interaction is isosteric with the adenosine N1-2'-OH interaction in the related mRNA from beet western yellows virus (BWYV); however, the ScYLV and BWYV mRNA structures differ in their detailed L2-S1 hydrogen bonding and L2 stacking interactions. Functional analyses of ScYLV/BWYV chimeric pseudoknots reveal that the ScYLV RNA stimulates a higher level of -1 PRF (15 +/- 2%) relative to the BWYV pseudoknot (6 +/- 1%), a difference traced largely to the identity of the 3' nucleotide of L2 (C27 vs. A25 in BWYV). Strikingly, C27A ScYLV RNA is a poor frameshift stimulator (2.0%) and is destabilized by approximately 1.5 kcal x mol(-1) (pH 7.0, 37 degrees C) with respect to the wild-type pseudoknot. These studies establish that the precise network of weak interactions nearest the helical junction in structurally similar pseudoknots make an important contribution to setting the frameshift efficiency in mRNAs.

A loop 2 cytidine-stem 1 minor groove interaction as a positive determinant for pseudoknot-stimulated –1 ribosomal frameshifting

PubMed Central

Cornish, Peter V.; Hennig, Mirko; Giedroc, David P.

2005-01-01

The molecular determinants of stimulation of –1 programmed ribosomal frameshifting (–1 PRF) by RNA pseudoknots are poorly understood. Sugarcane yellow leaf virus (ScYLV) encodes a 28-nt mRNA pseudoknot that promotes –1 PRF between the P1 (protease) and P2 (polymerase) genes in plant luteoviruses. The solution structure of the ScYLV pseudoknot reveals a well ordered loop 2 (L2) that exhibits continuous stacking of A20 through C27 in the minor groove of the upper stem 1 (S1), with C25 flipped out of the triple-stranded stack. Five consecutive triple base pairs flank the helical junction where the 3′ nucleotide of L2, C27, adopts a cytidine 27 N3-cytidine 14 2′-OH hydrogen bonding interaction with the C14-G7 base pair. This interaction is isosteric with the adenosine N1–2′-OH interaction in the related mRNA from beet western yellows virus (BWYV); however, the ScYLV and BWYV mRNA structures differ in their detailed L2–S1 hydrogen bonding and L2 stacking interactions. Functional analyses of ScYLV/BWYV chimeric pseudoknots reveal that the ScYLV RNA stimulates a higher level of –1 PRF (15 ± 2%) relative to the BWYV pseudoknot (6 ± 1%), a difference traced largely to the identity of the 3′ nucleotide of L2 (C27 vs. A25 in BWYV). Strikingly, C27A ScYLV RNA is a poor frameshift stimulator (2.0%) and is destabilized by ≈1.5 kcal·mol–1 (pH 7.0, 37°C) with respect to the wild-type pseudoknot. These studies establish that the precise network of weak interactions nearest the helical junction in structurally similar pseudoknots make an important contribution to setting the frameshift efficiency in mRNAs. PMID:16123125

Multiple roles of mobile active center loops in the E1 component of the Escherichia coli pyruvate dehydrogenase complex - Linkage of protein dynamics to catalysis

PubMed Central

Jordan, Frank; Arjunan, Palaniappa; Kale, Sachin; Nemeria, Natalia S.; Furey, William

2009-01-01

The region encompassing residues 401–413 on the E1 component of the pyruvate dehydrogenase multienzyme complex from Escherichia coli comprises a loop (the inner loop) which was not seen in the X-ray structure in the presence of thiamin diphosphate, the required cofactor for the enzyme. This loop is seen in the presence of a stable analogue of the pre-decarboxylation intermediate, the covalent adduct between the substrate analogue methyl acetylphosphonate and thiamin diphosphate, C2α-phosphonolactylthiamin diphosphate. It has been shown that the residue H407 and several other residues on this loop are required to reduce the mobility of the loop so electron density corresponding to it can be seen once the pre-decarboxylation intermediate is formed. Concomitantly, the loop encompassing residues 541–557 (the outer loop) appears to work in tandem with the inner loop and there is a hydrogen bond between the two loops ensuring their correlated motion. The inner loop was shown to: a) sequester the active center from carboligase side reactions; b) assist the interaction between the E1 and the E2 components, thereby affecting the overall reaction rate of the entire multienzyme complex; c) control substrate access to the active center. Using viscosity effects on kinetics it was shown that formation of the pre-decarboxylation intermediate is specifically affected by loop movement. A cysteine-less variant was created for the E1 component, onto which cysteines were substituted at selected loop positions. Introducing an electron spin resonance spin label and an 19F NMR label onto these engineered cysteines, the loop mobility was examined: a) both methods suggested that in the absence of ligand, the loop exists in two conformations; b) line-shape analysis of the NMR signal at different temperatures, enabled estimation of the rate constant for loop movement, and this rate constant was found to be of the same order of magnitude as the turnover number for the enzyme under the same conditions. Furthermore, this analysis gave important insights into rate-limiting thermal loop dynamics. Overall, the results suggest that the dynamic properties correlate with catalytic events on the E1 component of the pyruvate dehydrogenase complex. PMID:20160956

Conformational Sampling in Template-Free Protein Loop Structure Modeling: An Overview

PubMed Central

Li, Yaohang

2013-01-01

Accurately modeling protein loops is an important step to predict three-dimensional structures as well as to understand functions of many proteins. Because of their high flexibility, modeling the three-dimensional structures of loops is difficult and is usually treated as a “mini protein folding problem” under geometric constraints. In the past decade, there has been remarkable progress in template-free loop structure modeling due to advances of computational methods as well as stably increasing number of known structures available in PDB. This mini review provides an overview on the recent computational approaches for loop structure modeling. In particular, we focus on the approaches of sampling loop conformation space, which is a critical step to obtain high resolution models in template-free methods. We review the potential energy functions for loop modeling, loop buildup mechanisms to satisfy geometric constraints, and loop conformation sampling algorithms. The recent loop modeling results are also summarized. PMID:24688696

Conformational sampling in template-free protein loop structure modeling: an overview.

PubMed

Li, Yaohang

2013-01-01

Accurately modeling protein loops is an important step to predict three-dimensional structures as well as to understand functions of many proteins. Because of their high flexibility, modeling the three-dimensional structures of loops is difficult and is usually treated as a "mini protein folding problem" under geometric constraints. In the past decade, there has been remarkable progress in template-free loop structure modeling due to advances of computational methods as well as stably increasing number of known structures available in PDB. This mini review provides an overview on the recent computational approaches for loop structure modeling. In particular, we focus on the approaches of sampling loop conformation space, which is a critical step to obtain high resolution models in template-free methods. We review the potential energy functions for loop modeling, loop buildup mechanisms to satisfy geometric constraints, and loop conformation sampling algorithms. The recent loop modeling results are also summarized.

Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

PubMed Central

Liu, Yuanjie; Mao, Richeng; Mitra, Bidisha; Cai, Dawei; Yan, Ran; Guo, Ju-Tao; Block, Timothy M.; Mechti, Nadir

2017-01-01

Hepatitis B virus (HBV) replicates its DNA genome through reverse transcription of a viral RNA pregenome. We report herein that the interferon (IFN) stimulated exoribonuclease gene of 20 KD (ISG20) inhibits HBV replication through degradation of HBV RNA. ISG20 expression was observed at basal level and was highly upregulated upon IFN treatment in hepatocytes, and knock down of ISG20 resulted in elevation of HBV replication and attenuation of IFN-mediated antiviral effect. The sequence element conferring the susceptibility of HBV RNA to ISG20-mediated RNA degradation was mapped at the HBV RNA terminal redundant region containing epsilon (ε) stem-loop. Furthermore, ISG20-induced HBV RNA degradation relies on its ribonuclease activity, as the enzymatic inactive form ISG20D94G was unable to promote HBV RNA decay. Interestingly, ISG20D94G retained antiviral activity against HBV DNA replication by preventing pgRNA encapsidation, resulting from a consequence of ISG20-ε interaction. This interaction was further characterized by in vitro electrophoretic mobility shift assay (EMSA) and ISG20 was able to bind HBV ε directly in absence of any other cellular proteins, indicating a direct ε RNA binding capability of ISG20; however, cofactor(s) may be required for ISG20 to efficiently degrade ε. In addition, the lower stem portion of ε is the major ISG20 binding site, and the removal of 4 base pairs from the bottom portion of ε abrogated the sensitivity of HBV RNA to ISG20, suggesting that the specificity of ISG20-ε interaction relies on both RNA structure and sequence. Furthermore, the C-terminal Exonuclease III (ExoIII) domain of ISG20 was determined to be responsible for interacting with ε, as the deletion of ExoIII abolished in vitro ISG20-ε binding and intracellular HBV RNA degradation. Taken together, our study sheds light on the underlying mechanisms of IFN-mediated HBV inhibition and the antiviral mechanism of ISG20 in general. PMID:28399146

Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA.

PubMed

Liu, Yuanjie; Nie, Hui; Mao, Richeng; Mitra, Bidisha; Cai, Dawei; Yan, Ran; Guo, Ju-Tao; Block, Timothy M; Mechti, Nadir; Guo, Haitao

2017-04-01

Hepatitis B virus (HBV) replicates its DNA genome through reverse transcription of a viral RNA pregenome. We report herein that the interferon (IFN) stimulated exoribonuclease gene of 20 KD (ISG20) inhibits HBV replication through degradation of HBV RNA. ISG20 expression was observed at basal level and was highly upregulated upon IFN treatment in hepatocytes, and knock down of ISG20 resulted in elevation of HBV replication and attenuation of IFN-mediated antiviral effect. The sequence element conferring the susceptibility of HBV RNA to ISG20-mediated RNA degradation was mapped at the HBV RNA terminal redundant region containing epsilon (ε) stem-loop. Furthermore, ISG20-induced HBV RNA degradation relies on its ribonuclease activity, as the enzymatic inactive form ISG20D94G was unable to promote HBV RNA decay. Interestingly, ISG20D94G retained antiviral activity against HBV DNA replication by preventing pgRNA encapsidation, resulting from a consequence of ISG20-ε interaction. This interaction was further characterized by in vitro electrophoretic mobility shift assay (EMSA) and ISG20 was able to bind HBV ε directly in absence of any other cellular proteins, indicating a direct ε RNA binding capability of ISG20; however, cofactor(s) may be required for ISG20 to efficiently degrade ε. In addition, the lower stem portion of ε is the major ISG20 binding site, and the removal of 4 base pairs from the bottom portion of ε abrogated the sensitivity of HBV RNA to ISG20, suggesting that the specificity of ISG20-ε interaction relies on both RNA structure and sequence. Furthermore, the C-terminal Exonuclease III (ExoIII) domain of ISG20 was determined to be responsible for interacting with ε, as the deletion of ExoIII abolished in vitro ISG20-ε binding and intracellular HBV RNA degradation. Taken together, our study sheds light on the underlying mechanisms of IFN-mediated HBV inhibition and the antiviral mechanism of ISG20 in general.

Secondary Structure Predictions for Long RNA Sequences Based on Inversion Excursions and MapReduce.

PubMed

Yehdego, Daniel T; Zhang, Boyu; Kodimala, Vikram K R; Johnson, Kyle L; Taufer, Michela; Leung, Ming-Ying

2013-05-01

Secondary structures of ribonucleic acid (RNA) molecules play important roles in many biological processes including gene expression and regulation. Experimental observations and computing limitations suggest that we can approach the secondary structure prediction problem for long RNA sequences by segmenting them into shorter chunks, predicting the secondary structures of each chunk individually using existing prediction programs, and then assembling the results to give the structure of the original sequence. The selection of cutting points is a crucial component of the segmenting step. Noting that stem-loops and pseudoknots always contain an inversion, i.e., a stretch of nucleotides followed closely by its inverse complementary sequence, we developed two cutting methods for segmenting long RNA sequences based on inversion excursions: the centered and optimized method. Each step of searching for inversions, chunking, and predictions can be performed in parallel. In this paper we use a MapReduce framework, i.e., Hadoop, to extensively explore meaningful inversion stem lengths and gap sizes for the segmentation and identify correlations between chunking methods and prediction accuracy. We show that for a set of long RNA sequences in the RFAM database, whose secondary structures are known to contain pseudoknots, our approach predicts secondary structures more accurately than methods that do not segment the sequence, when the latter predictions are possible computationally. We also show that, as sequences exceed certain lengths, some programs cannot computationally predict pseudoknots while our chunking methods can. Overall, our predicted structures still retain the accuracy level of the original prediction programs when compared with known experimental secondary structure.

New Approaches to Hepatitis A Vaccine Development

DTIC Science & Technology

1991-04-22

and-mouth virus ( FMDV ) (Bittle et al., 1982). Peptide immunogens are highly stable reagents; they are potentially very inexpensive and extremely safe...of particular interest as it occurs within the "foot-and-mouth disease virus ( FMDV ) loop" of mengovirus. In FMDV , this loop contains a linear

Tornado-Like Evolution of A Kink-Unstable Solar Prominence

NASA Astrophysics Data System (ADS)

Wang, W.; Liu, R.; Wang, Y.

2016-12-01

We report on the tornado-like evolution of a quiescent prominence on 2014 November 1. The evolution started with a slow rise of the eastern section of the prominence into an arch-shaped structure as high as 150 Mm above the limb, and then the arch experienced a moderate, left-handed writhing. Following the writhing, the originally dark prominence material became in emission in the Fe IX 171 °A passband, and a braiding structure appeared at the eastern edge of the writhing prominence body, whose unraveling process was associated with a transient brightening in EUV and apparently contributed to the formation of a curtain-like structure (CLS), which consisted of myriads of thread-like loops rotating counterclockwise about the vertical if viewed from above. Material sliding along these loops landed outside of the prominence channel. The tornado was eventually disintegrated and the remaining material flew along a lefthanded helical path of approximately a full turn, as confirmed through stereoscopic reconstruction, into the cavity of the stable, western section of the prominence. We suggest that the tornado-like evolution of the prominence was regulated by the helical kink instability, and that the CLS forms through magnetic reconnections between the flux-rope flux and the overlying flux.

Search for and Study of Nearly Periodic Orbits in the Plane Problem of Three Equal-Mass Bodies

NASA Astrophysics Data System (ADS)

Martynova, A. I.; Orlov, V. V.

2005-09-01

We analyze nearly periodic solutions in the plane problem of three equal-mass bodies by numerically simulating the dynamics of triple systems. We identify families of orbits in which all three points are on one straight line (syzygy) at the initial time. In this case, at fixed total energy of a triple system, the set of initial conditions is a bounded region in four-dimensional parameter space. We scan this region and identify sets of trajectories in which the coordinates and velocities of all bodies are close to their initial values at certain times (which are approximately multiples of the period). We classify the nearly periodic orbits by the structure of trajectory loops over one period. We have found the families of orbits generated by von Schubart’s stable periodic orbit revealed in the rectilinear three-body problem. We have also found families of hierarchical, nearly periodic trajectories with prograde and retrograde motions. In the orbits with prograde motions, the trajectory loops of two close bodies form looplike structures. The trajectories with retrograde motions are characterized by leafed structures. Orbits with central and axial symmetries are identified among the families found.

Comparison of stem morphology and anatomy of two alfalfa clonal lines exhibiting divergent cell wall composition

USDA-ARS?s Scientific Manuscript database

In previous research, two alfalfa clonal lines (252, 1283) were identified that exhibited environmentally stable differences in stem cell walls. Compared to stems of 1283, stems of 252 have a higher cell wall concentration and greater amounts of lignin and cellulose but reduced levels of pectic suga...

Using emergent order to shape a space society

NASA Technical Reports Server (NTRS)

Graps, Amara L.

1993-01-01

A fast-growing movement in the scientific community is reshaping the way that we view the world around us. The short-hand name for this movement is 'chaos'. Chaos is a science of the global, nonlinear nature of systems. The center of this set of ideas is that simple, deterministic systems can breed complexity. Systems as complex as the human body, ecology, the mind or a human society. While it is true that simple laws can breed complexity, the other side is that complex systems can breed order. It is the latter that I will focus on in this paper. In the past, nonlinear was nearly synonymous with unsolvable because no general analytic solutions exist. Mathematically, an essential difference exists between linear and nonlinear systems. For linear systems, you just break up the complicated system into many simple pieces and patch together the separated solutions for each piece to form a solution to the full problem. In contrast, solutions to a nonlinear system cannot be added to form a new solution. The system must be treated in its full complexity. While it is true that no general analytical approach exists for reducing a complex system such as a society, it can be modeled. The technical involves a mathematical construct called phase space. In this space stable structures can appear which I use as analogies for the stable structures that appear in a complex system such as an ecology, the mind or a society. The common denominator in all of these systems is that they rely on a process called feedback loops. Feedback loops link the microscopic (individual) parts to the macroscopic (global) parts. The key, then, in shaping a space society, is in effectively using feedback loops. This paper will illustrate how one can model a space society by using methods that chaoticists have developed over the last hundred years. And I will show that common threads exist in the modeling of biological, economical, philosophical, and sociological systems.

Digital control algorithms for microgravity isolation systems

NASA Technical Reports Server (NTRS)

Sinha, Alok; Wang, Yung-Peng

1992-01-01

New digital control algorithms were developed to achieve the desired acceleration transmissibility function. The attractive electromagnets have been taken as actuators. The relative displacement and the acceleration of the mass were used as feedback signals. Two approaches were developed to find that controller transfer function in Z-domain, which yields the desired transmissibility at each frequency. In the first approach, the controller transfer function is obtained by assuming that the desired transmissibility is known in Z-domain. Since the desired transmissibility H sub d(S) = 1/(tauS+1)(exp 2) is given in S-domain, the first task is to obtain the desired transmissibility in Z-domain. There are three methods to perform this task: bilinear transformation, and backward and forward rectangular rules. The bilinear transformation and backward rectangular rule lead to improper controller transfer functions, which are physically not realizable. The forward rectangular rule does lead to a physically realizable controller. However, this controller is found to be marginally stable because of a pole at Z=1. In order to eliminate this pole, a hybrid control structure is proposed. Here the control input is composed of two parts: analog and digital. The analog input simply represents the velocity (or the integral of acceleration) feedback; and the digital controller which uses only relative displacement signal, is then obtained to achieve the desired closed-loop transfer function. The stability analysis indicates that the controller transfer function is stable for typical values of sampling period. In the second approach, the aforementioned hybrid control structure is again used. First, an analog controller transfer function corresponding to relative displacement feedback is obtained to achieve the transmissibility as 1/(tauS+1)(exp 2). Then the transfer function for the digital control input is obtained by discretizing this analog controller transfer function via bilinear transformation. The stability of the resulting Z-domain closed loop system is analyzed. Also, the frequency response of the Z-domain closed-loop transfer function is determined to evaluate the performance of the control system.

Entropic stabilization of a deubiquitinase provides conformational plasticity and slow unfolding kinetics beneficial for functioning on the proteasome

PubMed Central

Lee, Yun-Tzai Cloud; Chang, Chia-Yun; Chen, Szu-Yu; Pan, Yun-Ru; Ho, Meng-Ru; Hsu, Shang-Te Danny

2017-01-01

Human ubiquitin C-terminal hydrolyase UCH-L5 is a topologically knotted deubiquitinase that is activated upon binding to the proteasome subunit Rpn13. The length of its intrinsically disordered cross-over loop is essential for substrate recognition. Here, we showed that the catalytic domain of UCH-L5 exhibits higher equilibrium folding stability with an unfolding rate on the scale of 10−8 s−1, over four orders of magnitudes slower than its paralogs, namely UCH-L1 and -L3, which have shorter cross-over loops. NMR relaxation dynamics analysis confirmed the intrinsic disorder of the cross-over loop. Hydrogen deuterium exchange analysis further revealed a positive correlation between the length of the cross-over loop and the degree of local fluctuations, despite UCH-L5 being thermodynamically and kinetically more stable than the shorter UCHs. Considering the role of UCH-L5 in removing K48-linked ubiquitin to prevent proteasomal degradation of ubiquitinated substrates, our findings offered mechanistic insights into the evolution of UCH-L5. Compared to its paralogs, it is entropically stabilized to withstand mechanical unfolding by the proteasome while maintaining structural plasticity. It can therefore accommodate a broad range of substrate geometries at the cost of unfavourable entropic loss. PMID:28338014

Tunable erbium-doped fiber laser based on optical fiber Sagnac interference loop with angle shift spliced polarization maintaining fibers

NASA Astrophysics Data System (ADS)

Ding, Zhenming; Wang, Zhaokun; Zhao, Chunliu; Wang, Dongning

2018-05-01

In this paper, we propose and experimentally demonstrate a tunable erbium-doped fiber laser (EDFL) with Sagnac interference loop with 45° angle shift spliced polarization maintaining fibers (PMFs). In the Sagnac loop, two PMFs with similar lengths. The Sagnac loop outputs a relatively complex interference spectrum since two beams transmitted in clockwise and counterclockwise encounter at the 3 dB coupler, interfere, and form two interference combs when the light transmitted in the Sagnac loop. The laser will excite and be stable when two interference lines in these two interference combs overlap together. Then by adjusting the polarization controller, the wide wavelength tuning is realized. Experimental results show that stable single wavelength laser can be realized in the wavelength range of 1585 nm-1604 nm under the pump power 157.1 mW. The side-mode suppression ratio is not less than 53.9 dB. The peak power fluctuation is less than 0.29 dB within 30 min monitor time and the side-mode suppression ratio is great than 57.49 dB when the pump power is to 222.7 mW.

Robust controller design for flexible structures using normalized coprime factor plant descriptions

NASA Technical Reports Server (NTRS)

Armstrong, Ernest S.

1993-01-01

Stabilization is a fundamental requirement in the design of feedback compensators for flexible structures. The search for the largest neighborhood around a given design plant for which a single controller produces closed-loop stability can be formulated as an H(sub infinity) control problem. The use of normalized coprime factor plant descriptions, in which the plant perturbations are defined as additive modifications to the coprime factors, leads to a closed-form expression for the maximum neighborhood boundary allowing optimal and suboptimal H(sub infinity) compensators to be computed directly without the usual gamma iteration. A summary of the theory on robust stabilization using normalized coprime factor plant descriptions is presented, and the application of the theory to the computation of robustly stable compensators for the phase version of the Control-Structures Interaction (CSI) Evolutionary Model is described. Results from the application indicate that the suboptimal version of the theory has the potential of providing the bases for the computation of low-authority compensators that are robustly stable to expected variations in design model parameters and additive unmodeled dynamics.

RNA2D3D: a program for generating, viewing, and comparing 3-dimensional models of RNA.

PubMed

Martinez, Hugo M; Maizel, Jacob V; Shapiro, Bruce A

2008-06-01

Using primary and secondary structure information of an RNA molecule, the program RNA2D3D automatically and rapidly produces a first-order approximation of a 3-dimensional conformation consistent with this information. Applicable to structures of arbitrary branching complexity and pseudoknot content, it features efficient interactive graphical editing for the removal of any overlaps introduced by the initial generating procedure and for making conformational changes favorable to targeted features and subsequent refinement. With emphasis on fast exploration of alternative 3D conformations, one may interactively add or delete base-pairs, adjacent stems can be coaxially stacked or unstacked, single strands can be shaped to accommodate special constraints, and arbitrary subsets can be defined and manipulated as rigid bodies. Compaction, whereby base stacking within stems is optimally extended into connecting single strands, is also available as a means of strategically making the structures more compact and revealing folding motifs. Subsequent refinement of the first-order approximation, of modifications, and for the imposing of tertiary constraints is assisted with standard energy refinement techniques. Previously determined coordinates for any part of the molecule are readily incorporated, and any part of the modeled structure can be output as a PDB or XYZ file. Illustrative applications in the areas of ribozymes, viral kissing loops, viral internal ribosome entry sites, and nanobiology are presented.

Structure and dynamics of an upland old- growth forest at Redwood National Park, California

USGS Publications Warehouse

van Mantgem, Philip J.; Stuart, John D.

2011-01-01

Many current redwood forest management targets are based on old-growth conditions, so it is critical that we understand the variability and range of conditions that constitute these forests. Here we present information on the structure and dynamics from six one-hectare forest monitoring plots in an upland old-growth forest at Redwood National Park, California. We surveyed all stems =20 cm DBH in 1995 and 2010, allowing us to estimate any systematic changes in these stands. Stem size distributions for all species and for redwood (Sequoia sempervirens (D. Don) Endl.) alone did not appreciably change over the 15 year observation interval. Recruitment and mortality rates were roughly balanced, as were basal area dynamics (gains from recruitment and growth versus losses from mortality). Similar patterns were found for Sequoia alone. The spatial structure of stems at the plots suggested a random distribution of trees, though the pattern for Sequoia alone was found to be significantly clumped at small scales (< 5 m) at three of the six plots. These results suggest that these forests, including populations of Sequoia, have been generally stable over the past 15 years at this site, though it is possible that fire exclusion may be affecting recruitment of smaller Sequoia (< 20 cm DBH). The non-uniform spatial arrangement of stems also suggests that restoration prescriptions for second-growth redwood forests that encourage uniform spatial arrangements do not appear to mimic current upland old-growth conditions.

HCV IRES domain IIb affects the configuration of coding RNA in the 40S subunit's decoding groove

PubMed Central

Filbin, Megan E.; Kieft, Jeffrey S.

2011-01-01

Hepatitis C virus (HCV) uses a structured internal ribosome entry site (IRES) RNA to recruit the translation machinery to the viral RNA and begin protein synthesis without the ribosomal scanning process required for canonical translation initiation. Different IRES structural domains are used in this process, which begins with direct binding of the 40S ribosomal subunit to the IRES RNA and involves specific manipulation of the translational machinery. We have found that upon initial 40S subunit binding, the stem–loop domain of the IRES that contains the start codon unwinds and adopts a stable configuration within the subunit's decoding groove. This configuration depends on the sequence and structure of a different stem–loop domain (domain IIb) located far from the start codon in sequence, but spatially proximal in the IRES•40S complex. Mutation of domain IIb results in misconfiguration of the HCV RNA in the decoding groove that includes changes in the placement of the AUG start codon, and a substantial decrease in the ability of the IRES to initiate translation. Our results show that two distal regions of the IRES are structurally communicating at the initial step of 40S subunit binding and suggest that this is an important step in driving protein synthesis. PMID:21606179

TORNADO-LIKE EVOLUTION OF A KINK-UNSTABLE SOLAR PROMINENCE

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Wensi; Liu, Rui; Wang, Yuming, E-mail: rliu@ustc.edu.cn

We report on the tornado-like evolution of a quiescent prominence on 2014 November 1. The eastern section of the prominence first rose slowly, transforming into an arch-shaped structure as high as ∼150 Mm above the limb; the arch then writhed moderately in a left-handed sense, while the original dark prominence material emitted in the Fe ix 171 Å passband, and a braided structure appeared at the eastern edge of the warped arch. The unraveling of the braided structure was associated with a transient brightening in the EUV and apparently contributed to the formation of a curtain-like structure (CLS). The CLSmore » consisted of myriad thread-like loops rotating counterclockwise about the vertical if viewed from above. Heated prominence material was observed to slide along these loops and land outside the filament channel. The tornado eventually disintegrated and the remaining material flew along a left-handed helical path constituting approximately a full turn, as corroborated through stereoscopic reconstruction, into the cavity of the stable, western section of the prominence. We suggest that the tornado-like evolution of the prominence was governed by the helical kink instability, and that the CLS formed through magnetic reconnections between the prominence field and the overlying coronal field.« less

LPV control for the full region operation of a wind turbine integrated with synchronous generator.

PubMed

Cao, Guoyan; Grigoriadis, Karolos M; Nyanteh, Yaw D

2015-01-01

Wind turbine conversion systems require feedback control to achieve reliable wind turbine operation and stable current supply. A robust linear parameter varying (LPV) controller is proposed to reduce the structural loads and improve the power extraction of a horizontal axis wind turbine operating in both the partial load and the full load regions. The LPV model is derived from the wind turbine state space models extracted by FAST (fatigue, aerodynamics, structural, and turbulence) code linearization at different operating points. In order to assure a smooth transition between the two regions, appropriate frequency-dependent varying scaling parametric weighting functions are designed in the LPV control structure. The solution of a set of linear matrix inequalities (LMIs) leads to the LPV controller. A synchronous generator model is connected with the closed LPV control loop for examining the electrical subsystem performance obtained by an inner speed control loop. Simulation results of a 1.5 MW horizontal axis wind turbine model on the FAST platform illustrates the benefit of the LPV control and demonstrates the advantages of this proposed LPV controller, when compared with a traditional gain scheduling PI control and prior LPV control configurations. Enhanced structural load mitigation, improved power extraction, and good current performance were obtained from the proposed LPV control.

LPV Control for the Full Region Operation of a Wind Turbine Integrated with Synchronous Generator

PubMed Central

Grigoriadis, Karolos M.; Nyanteh, Yaw D.

2015-01-01

Wind turbine conversion systems require feedback control to achieve reliable wind turbine operation and stable current supply. A robust linear parameter varying (LPV) controller is proposed to reduce the structural loads and improve the power extraction of a horizontal axis wind turbine operating in both the partial load and the full load regions. The LPV model is derived from the wind turbine state space models extracted by FAST (fatigue, aerodynamics, structural, and turbulence) code linearization at different operating points. In order to assure a smooth transition between the two regions, appropriate frequency-dependent varying scaling parametric weighting functions are designed in the LPV control structure. The solution of a set of linear matrix inequalities (LMIs) leads to the LPV controller. A synchronous generator model is connected with the closed LPV control loop for examining the electrical subsystem performance obtained by an inner speed control loop. Simulation results of a 1.5 MW horizontal axis wind turbine model on the FAST platform illustrates the benefit of the LPV control and demonstrates the advantages of this proposed LPV controller, when compared with a traditional gain scheduling PI control and prior LPV control configurations. Enhanced structural load mitigation, improved power extraction, and good current performance were obtained from the proposed LPV control. PMID:25884036

Constrained Combinatorial Libraries of Gp2 Proteins Enhance Discovery of PD-L1 Binders.

PubMed

Kruziki, Max A; Sarma, Vidur; Hackel, Benjamin J

2018-06-05

Engineered protein ligands are used for molecular therapy, diagnostics, and industrial biotechnology. The Gp2 domain is a 45-amino acid scaffold that has been evolved for specific, high-affinity binding to multiple targets by diversification of two solvent-exposed loops. Inspired by sitewise enrichment of select amino acids, including cysteine pairs, in earlier Gp2 discovery campaigns, we hypothesized that the breadth and efficiency of de novo Gp2 discovery will be aided by sitewise amino acid constraint within combinatorial library design. We systematically constructed eight libraries and comparatively evaluated their efficacy for binder discovery via yeast display against a panel of targets. Conservation of a cysteine pair at the termini of the first diversified paratope loop increased binder discovery 16-fold ( p < 0.001). Yet two other libraries with conserved cysteine pairs, within the second loop or an interloop pair, did not aid discovery thereby indicating site-specific impact. Via a yeast display protease resistance assay, Gp2 variants from the loop one cysteine pair library were 3.3 ± 2.1-fold ( p = 0.005) more stable than nonconstrained variants. Sitewise constraint of noncysteine residues-guided by previously evolved binders, natural Gp2 homology, computed stability, and structural analysis-did not aid discovery. A panel of binders to programmed death ligand 1 (PD-L1), a key target in cancer immunotherapy, were discovered from the loop 1 cysteine constraint library. Affinity maturation via loop walking resulted in strong, specific cellular PD-L1 affinity ( K d = 6-9 nM).

EXPLAINING INVERTED-TEMPERATURE LOOPS IN THE QUIET SOLAR CORONA WITH MAGNETOHYDRODYNAMIC WAVE-MODE CONVERSION

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schiff, Avery J.; Cranmer, Steven R.

Coronal loops trace out bipolar, arch-like magnetic fields above the Sun’s surface. Recent measurements that combine rotational tomography, extreme-ultraviolet imaging, and potential-field extrapolation have shown the existence of large loops with inverted-temperature profiles, i.e., loops for which the apex temperature is a local minimum, not a maximum. These “down loops” appear to exist primarily in equatorial quiet regions near solar minimum. We simulate both these and the more prevalent large-scale “up loops” by modeling coronal heating as a time-steady superposition of (1) dissipation of incompressible Alfvén wave turbulence and (2) dissipation of compressive waves formed by mode conversion from themore » initial population of Alfvén waves. We found that when a large percentage (>99%) of the Alfvén waves undergo this conversion, heating is greatly concentrated at the footpoints and stable “down loops” are created. In some cases we found loops with three maxima that are also gravitationally stable. Models that agree with the tomographic temperature data exhibit higher gas pressures for “down loops” than for “up loops,” which is consistent with observations. These models also show a narrow range of Alfvén wave amplitudes: 3 to 6 km s{sup -1} at the coronal base. This is low in comparison to typical observed amplitudes of 20–30 km s{sup -1} in bright X-ray loops. However, the large-scale loops we model are believed to compose a weaker diffuse background that fills much of the volume of the corona. By constraining the physics of loops that underlie quiescent streamers, we hope to better understand the formation of the slow solar wind.« less

Stem loop-mediated isothermal amplification test: comparative analysis with classical LAMP and PCR in detection of Entamoeba histolytica in Kenya.

PubMed

Mwendwa, Fridah; Mbae, Cecilia K; Kinyua, Johnson; Mulinge, Erastus; Mburugu, Gitonga Nkanata; Njiru, Zablon K

2017-03-31

Entamoeba histolytica, the causative agent for amoebiasis is a considerable burden to population in the developing countries where it accounts for over 50 million infections. The tools for detection of amoebiasis are inadequate and diagnosis relies on microscopy which means a significant percent of cases remain undiagnosed. Moreover, tests formats that can be rapidly applied in rural endemic areas are not available. In this study, a loop-mediated isothermal test (LAMP) based on 18S small subunit ribosomal RNA gene was designed with extra reaction accelerating primers (stem primers) and compared with the published LAMP and PCR tests in detection of E. histolytica DNA in clinical samples. The stem LAMP test indicated shorter time to results by an average 11 min and analytical sensitivity of 10 -7 (~30 pg/ml) compared to the standard LAMP and PCR which showed sensitivities levels of 10 -5 (~3 ng/ml) and 10 -4 (~30 ng/ml) respectively using tenfold serial dilution of DNA. In the analysis of clinical specimens positive for Entamoeba spp. trophozoites and cysts using microscopy, the stem LAMP test detected E. histolytica DNA in 36/126, standard LAMP test 20/126 and PCR 17/126 cases respectively. There was 100% agreement in detection of the stem LAMP test product using fluorescence of SYTO-9 dye in real time machine, through addition of 1/10 dilution of SYBR ® Green I and electrophoresis in 2% agarose gel stained with ethidium bromide. The stem LAMP test developed in this study indicates potential towards detection of E. histolytica.

Unfolding and melting of DNA (RNA) hairpins: the concept of structure-specific 2D dynamic landscapes.

PubMed

Lin, Milo M; Meinhold, Lars; Shorokhov, Dmitry; Zewail, Ahmed H

2008-08-07

A 2D free-energy landscape model is presented to describe the (un)folding transition of DNA/RNA hairpins, together with molecular dynamics simulations and experimental findings. The dependence of the (un)folding transition on the stem sequence and the loop length is shown in the enthalpic and entropic contributions to the free energy. Intermediate structures are well defined by the two coordinates of the landscape during (un)zipping. Both the free-energy landscape model and the extensive molecular dynamics simulations totaling over 10 mus predict the existence of temperature-dependent kinetic intermediate states during hairpin (un)zipping and provide the theoretical description of recent ultrafast temperature-jump studies which indicate that hairpin (un)zipping is, in general, not a two-state process. The model allows for lucid prediction of the collapsed state(s) in simple 2D space and we term it the kinetic intermediate structure (KIS) model.

Systematically frameshifting by deletion of every 4th or 4th and 5th nucleotides during mitochondrial transcription: RNA self-hybridization regulates delRNA expression.

PubMed

Seligmann, Hervé

2016-01-01

In mitochondria, secondary structures punctuate post-transcriptional RNA processing. Recently described transcripts match the human mitogenome after systematic deletions of every 4th, respectively every 4th and 5th nucleotides, called delRNAs. Here I explore predicted stem-loop hairpin formation by delRNAs, and their associations with delRNA transcription and detected peptides matching their translation. Despite missing 25, respectively 40% of the nucleotides in the original sequence, del-transformed sequences form significantly more secondary structures than corresponding randomly shuffled sequences, indicating biological function, independently of, and in combination with, previously detected delRNA and thereof translated peptides. Self-hybridization decreases delRNA abundances, indicating downregulation. Systematic deletions of the human mitogenome reveal new, unsuspected coding and structural informations. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Application of STEM characterization for investigating radiation effects in BCC Fe-based alloys

DOE Office of Scientific and Technical Information (OSTI.GOV)

Parish, Chad M.; Field, Kevin G.; Certain, Alicia G.

2015-04-20

This paper provides a general overview of advanced scanning transmission electron microscopy (STEM) techniques used for characterization of irradiated BCC Fe-based alloys. Advanced STEM methods provide the high-resolution imaging and chemical analysis necessary to understand the irradiation response of BCC Fe-based alloys. The use of STEM with energy dispersive x-ray spectroscopy (EDX) for measurement of radiation-induced segregation (RIS) is described, with an illustrated example of RIS in proton- and self-ion irradiated T91. Aberration-corrected STEM-EDX for nanocluster/nanoparticle imaging and chemical analysis is also discussed, and examples are provided from ion-irradiated oxide dispersion strengthened (ODS) alloys. In conclusion, STEM techniques for void,more » cavity, and dislocation loop imaging are described, with examples from various BCC Fe-based alloys.« less

The Neural Cell Adhesion Molecule-Derived (NCAM)-Peptide FG Loop (FGL) Mobilizes Endogenous Neural Stem Cells and Promotes Endogenous Regenerative Capacity after Stroke.

PubMed

Klein, Rebecca; Mahlberg, Nicolas; Ohren, Maurice; Ladwig, Anne; Neumaier, Bernd; Graf, Rudolf; Hoehn, Mathias; Albrechtsen, Morten; Rees, Stephen; Fink, Gereon Rudolf; Rueger, Maria Adele; Schroeter, Michael

2016-12-01

The neural cell adhesion molecule (NCAM)-derived peptide FG loop (FGL) modulates synaptogenesis, neurogenesis, and stem cell proliferation, enhances cognitive capacities, and conveys neuroprotection after stroke. Here we investigated the effect of subcutaneously injected FGL on cellular compartments affected by degeneration and regeneration after stroke due to middle cerebral artery occlusion (MCAO), namely endogenous neural stem cells (NSC), oligodendrocytes, and microglia. In addition to immunohistochemistry, we used non-invasive positron emission tomography (PET) imaging with the tracer [ 18 F]-fluoro-L-thymidine ([ 18 F]FLT) to visualize endogenous NSC in vivo. FGL significantly increased endogenous NSC mobilization in the neurogenic niches as evidenced by in vivo and ex vivo methods, and it induced remyelination. Moreover, FGL affected neuroinflammation. Extending previous in vitro results, our data show that the NCAM mimetic peptide FGL mobilizes endogenous NSC after focal ischemia and enhances regeneration by amplifying remyelination and modulating neuroinflammation via affecting microglia. Results suggest FGL as a promising candidate to promote recovery after stroke.

The multidrug ABC transporter BmrC/BmrD of Bacillus subtilis is regulated via a ribosome-mediated transcriptional attenuation mechanism.

PubMed

Reilman, Ewoud; Mars, Ruben A T; van Dijl, Jan Maarten; Denham, Emma L

2014-10-01

Expression of particular drug transporters in response to antibiotic pressure is a critical element in the development of bacterial multidrug resistance, and represents a serious concern for human health. To obtain a better understanding of underlying regulatory mechanisms, we have dissected the transcriptional activation of the ATP-binding cassette (ABC) transporter BmrC/BmrD of the Gram-positive model bacterium Bacillus subtilis. By using promoter-GFP fusions and live cell array technology, we demonstrate a temporally controlled transcriptional activation of the bmrCD genes in response to antibiotics that target protein synthesis. Intriguingly, bmrCD expression only occurs during the late-exponential and stationary growth stages, irrespective of the timing of the antibiotic challenge. We show that this is due to tight transcriptional control by the transition state regulator AbrB. Moreover, our results show that the bmrCD genes are co-transcribed with bmrB (yheJ), a small open reading frame immediately upstream of bmrC that harbors three alternative stem-loop structures. These stem-loops are apparently crucial for antibiotic-induced bmrCD transcription. Importantly, the antibiotic-induced bmrCD expression requires translation of bmrB, which implies that BmrB serves as a regulatory leader peptide. Altogether, we demonstrate for the first time that a ribosome-mediated transcriptional attenuation mechanism can control the expression of a multidrug ABC transporter. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

On the combinatorics of sparsification.

PubMed

Huang, Fenix Wd; Reidys, Christian M

2012-10-22

We study the sparsification of dynamic programming based on folding algorithms of RNA structures. Sparsification is a method that improves significantly the computation of minimum free energy (mfe) RNA structures. We provide a quantitative analysis of the sparsification of a particular decomposition rule, Λ∗. This rule splits an interval of RNA secondary and pseudoknot structures of fixed topological genus. Key for quantifying sparsifications is the size of the so called candidate sets. Here we assume mfe-structures to be specifically distributed (see Assumption 1) within arbitrary and irreducible RNA secondary and pseudoknot structures of fixed topological genus. We then present a combinatorial framework which allows by means of probabilities of irreducible sub-structures to obtain the expectation of the Λ∗-candidate set w.r.t. a uniformly random input sequence. We compute these expectations for arc-based energy models via energy-filtered generating functions (GF) in case of RNA secondary structures as well as RNA pseudoknot structures. Furthermore, for RNA secondary structures we also analyze a simplified loop-based energy model. Our combinatorial analysis is then compared to the expected number of Λ∗-candidates obtained from the folding mfe-structures. In case of the mfe-folding of RNA secondary structures with a simplified loop-based energy model our results imply that sparsification provides a significant, constant improvement of 91% (theory) to be compared to an 96% (experimental, simplified arc-based model) reduction. However, we do not observe a linear factor improvement. Finally, in case of the "full" loop-energy model we can report a reduction of 98% (experiment). Sparsification was initially attributed a linear factor improvement. This conclusion was based on the so called polymer-zeta property, which stems from interpreting polymer chains as self-avoiding walks. Subsequent findings however reveal that the O(n) improvement is not correct. The combinatorial analysis presented here shows that, assuming a specific distribution (see Assumption 1), of mfe-structures within irreducible and arbitrary structures, the expected number of Λ∗-candidates is Θ(n2). However, the constant reduction is quite significant, being in the range of 96%. We furthermore show an analogous result for the sparsification of the Λ∗-decomposition rule for RNA pseudoknotted structures of genus one. Finally we observe that the effect of sparsification is sensitive to the employed energy model.

Hyperbolic chaos in the klystron-type microwave vacuum tube oscillator

NASA Astrophysics Data System (ADS)

Emel'yanov, V. V.; Kuznetsov, S. P.; Ryskin, N. M.

2010-12-01

The ring-loop oscillator consisting of two coupled klystrons which is capable of generating hyperbolic chaotic signal in the microwave band is considered. The system of delayed-differential equations describing the dynamics of the oscillator is derived. This system is further reduced to the two-dimensional return map under the assumption of the instantaneous build-up of oscillations in the cavities. The results of detailed numerical simulation for both models are presented showing that there exists large enough range of control parameters where the sustained regime corresponds to the structurally stable hyperbolic chaos.

A molecular beacon microarray based on a quantum dot label for detecting single nucleotide polymorphisms.

PubMed

Guo, Qingsheng; Bai, Zhixiong; Liu, Yuqian; Sun, Qingjiang

2016-03-15

In this work, we report the application of streptavidin-coated quantum dot (strAV-QD) in molecular beacon (MB) microarray assays by using the strAV-QD to label the immobilized MB, avoiding target labeling and meanwhile obviating the use of amplification. The MBs are stem-loop structured oligodeoxynucleotides, modified with a thiol and a biotin at two terminals of the stem. With the strAV-QD labeling an "opened" MB rather than a "closed" MB via streptavidin-biotin reaction, a sensitive and specific detection of label-free target DNA sequence is demonstrated by the MB microarray, with a signal-to-background ratio of 8. The immobilized MBs can be perfectly regenerated, allowing the reuse of the microarray. The MB microarray also is able to detect single nucleotide polymorphisms, exhibiting genotype-dependent fluorescence signals. It is demonstrated that the MB microarray can perform as a 4-to-2 encoder, compressing the genotype information into two outputs. Copyright © 2015 Elsevier B.V. All rights reserved.

Magnetic suspension system for an Annular Momentum Control Device (AMCD)

NASA Technical Reports Server (NTRS)

1979-01-01

A technique to control a rim suspended in a magnetic field was developed. A complete system was developed, incorporating a support structure, magnetic actuators, a rim drive mechanism, an emergency fail-safe system, servo control system, and control electronics. Open loop and closed loop response of the system at zero speed and at 500 revolutions per minute (r/min) of the rim was obtained and analyzed. The rim was then dynamically balanced and a rim speed of 725 r/min was achieved. An analog simulation of the hardware was developed and tested with the actual control electronics connected to the analog computer. The system under development is stable at rim speeds below 700 r/min. Test results indicate that the rim under test is not rigid. The rim has a warp and a number of binding modes which prevented achievement of higher speeds. Further development efforts are required to achieve higher rim speeds.

Label-free logic modules and two-layer cascade based on stem-loop probes containing a G-quadruplex domain.

PubMed

Guo, Yahui; Cheng, Junjie; Wang, Jine; Zhou, Xiaodong; Hu, Jiming; Pei, Renjun

2014-09-01

A simple, versatile, and label-free DNA computing strategy was designed by using toehold-mediated strand displacement and stem-loop probes. A full set of logic gates (YES, NOT, OR, NAND, AND, INHIBIT, NOR, XOR, XNOR) and a two-layer logic cascade were constructed. The probes contain a G-quadruplex domain, which was blocked or unfolded through inputs initiating strand displacement and the obviously distinguishable light-up fluorescent signal of G-quadruplex/NMM complex was used as the output readout. The inputs are the disease-specific nucleotide sequences with potential for clinic diagnosis. The developed versatile computing system based on our label-free and modular strategy might be adapted in multi-target diagnosis through DNA hybridization and aptamer-target interaction. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Loop vertex expansion for higher-order interactions

NASA Astrophysics Data System (ADS)

Rivasseau, Vincent

2018-05-01

This note provides an extension of the constructive loop vertex expansion to stable interactions of arbitrarily high order, opening the way to many applications. We treat in detail the example of the (\\bar{φ } φ )^p field theory in zero dimension. We find that the important feature to extend the loop vertex expansion is not to use an intermediate field representation, but rather to force integration of exactly one particular field per vertex of the initial action.

Tone based command system for reception of very weak signals

NASA Technical Reports Server (NTRS)

Bokulic, Robert Steven (Inventor); Jensen, James Robert (Inventor)

2006-01-01

This disclosure presents a communication receiver system for spacecraft that includes an open loop receiver adapted to receive a communication signal. An ultrastable oscillator (USO) and a tone detector are connected to the open loop receiver. The open loop receiver translates the communication signal to an intermediate frequency signal using a highly stable reference frequency from the USO. The tone detector extracts commands from the communication signal by evaluating the difference between tones of the communication signal.

A 5' UTR-Overlapping LncRNA Activates the Male-Determining Gene doublesex1 in the Crustacean Daphnia magna.

PubMed

Kato, Yasuhiko; Perez, Christelle Alexa G; Mohamad Ishak, Nur Syafiqah; Nong, Quang D; Sudo, Yuumi; Matsuura, Tomoaki; Wada, Tadashi; Watanabe, Hajime

2018-06-04

Long noncoding RNAs (lncRNAs) are pervasively transcribed in the eukaryotic genome [1] and are important for the control of master regulatory genes that are involved in cell differentiation and development [2, 3]. Here, we show that a 5' UTR-overlapping lncRNA regulates the male-specific expression of the DM-domain gene doublesex1 (dsx1) in the crustacean Daphnia magna, which produces males in response to environmental stimuli. This lncRNA, named doublesex1 alpha promoter-associated long RNA (DAPALR), is transcribed upstream the transcription start site (TSS) in a sense orientation and subjected to 5' end capping and 3' end processing at a stem-loop structure before the dsx1 coding exon. Similar to dsx1, its expression is only activated in males by the juvenile hormone (JH) and basic-leucine zipper (bZIP) transcription factor Vrille (Vri) and is maintained during embryogenesis. Knockdown of DAPALR in males silenced dsx1 and led to feminization, including egg production, whereas ectopic expression of DAPALR in dsx1-silenced females resulted in the de-repression of dsx1. We further demonstrate that the DAPALR transcript overlaps the dsx1 5'-UTR, and this overlapping region is required for dsx1 activation. Our results suggest that DAPALR can transactivate and possibly maintain dsx1 expression. This might be important for converting transient environmental signals into stable male development, controlled by the continuous expression of dsx1. Copyright © 2018 Elsevier Ltd. All rights reserved.

Cryopreservation of Human Stem Cells for Clinical Application: A Review

PubMed Central

Hunt, Charles J.

2011-01-01

Summary Stem cells have been used in a clinical setting for many years. Haematopoietic stem cells have been used for the treatment of both haematological and non-haematological disease; while more recently mesenchymal stem cells derived from bone marrow have been the subject of both laboratory and early clinical studies. Whilst these cells show both multipotency and expansion potential, they nonetheless do not form stable cell lines in culture which is likely to limit the breadth of their application in the field of regenerative medicine. Human embryonic stem cells are pluripotent cells, capable of forming stable cell lines which retain the capacity to differentiate into cells from all three germ layers. This makes them of special significance in both regenerative medicine and toxicology. Induced pluripotent stem (iPS) cells may also provide a similar breadth of utility without some of the confounding ethical issues surrounding embryonic stem cells. An essential pre-requisite to the commercial and clinical application of stem cells are suitable cryopreservation protocols for long-term storage. Whilst effective methods for cryopreservation and storage have been developed for haematopoietic and mesenchymal stem cells, embryonic cells and iPS cells have proved more refractory. This paper reviews the current state of cryopreservation as it pertains to stem cells and in particular the embryonic and iPS cell. PMID:21566712

Cryopreservation of Human Stem Cells for Clinical Application: A Review.

PubMed

Hunt, Charles J

2011-01-01

SUMMARY: Stem cells have been used in a clinical setting for many years. Haematopoietic stem cells have been used for the treatment of both haematological and non-haematological disease; while more recently mesenchymal stem cells derived from bone marrow have been the subject of both laboratory and early clinical studies. Whilst these cells show both multipotency and expansion potential, they nonetheless do not form stable cell lines in culture which is likely to limit the breadth of their application in the field of regenerative medicine. Human embryonic stem cells are pluripotent cells, capable of forming stable cell lines which retain the capacity to differentiate into cells from all three germ layers. This makes them of special significance in both regenerative medicine and toxicology. Induced pluripotent stem (iPS) cells may also provide a similar breadth of utility without some of the confounding ethical issues surrounding embryonic stem cells. An essential pre-requisite to the commercial and clinical application of stem cells are suitable cryopreservation protocols for long-term storage. Whilst effective methods for cryopreservation and storage have been developed for haematopoietic and mesenchymal stem cells, embryonic cells and iPS cells have proved more refractory. This paper reviews the current state of cryopreservation as it pertains to stem cells and in particular the embryonic and iPS cell.

A single EBV-based vector for stable episomal maintenance and expression of GFP in human embryonic stem cells.

PubMed

Thyagarajan, Bhaskar; Scheyhing, Kelly; Xue, Haipeng; Fontes, Andrew; Chesnut, Jon; Rao, Mahendra; Lakshmipathy, Uma

2009-03-01

Stable expression of transgenes in stem cells has been a challenge due to the nonavailability of efficient transfection methods and the inability of transgenes to support sustained gene expression. Several methods have been reported to stably modify both embryonic and adult stem cells. These methods rely on integration of the transgene into the genome of the host cell, which could result in an expression pattern dependent on the number of integrations and the genomic locus of integration. To overcome this issue, site-specific integration methods mediated by integrase, adeno-associated virus or via homologous recombination have been used to generate stable human embryonic stem cell (hESC) lines. In this study, we describe a vector that is maintained episomally in hESCs. The vector used in this study is based on components derived from the Epstein-Barr virus, containing the Epstein-Barr virus nuclear antigen 1 expression cassette and the OriP origin of replication. The vector also expresses the drug-resistance marker gene hygromycin, which allows for selection and long-term maintenance of cells harboring the plasmid. Using this vector system, we show sustained expression of green fluorescent protein in undifferentiated hESCs and their differentiating embryoid bodies. In addition, the stable hESC clones show comparable expression with and without drug selection. Consistent with this observation, bulk-transfected adipose tissue-derived mesenchymal stem cells showed persistent marker gene expression as they differentiate into adipocytes, osteoblasts and chondroblasts. Episomal vectors offer a fast and efficient method to create hESC reporter lines, which in turn allows one to test the effect of overexpression of various genes on stem cell growth, proliferation and differentiation.

Versatile retraction mechanics: Implant assisted en-masse retraction with a boot loop.

PubMed

Philip, Pramod; Jose, Nidhin Philip

2015-03-01

The purpose of this paper is to explain the versatility offered by the use of arch wires with boot loops in retraction mechanics while taking direct anchorage from mini-screws. The materials include the mini screws placed at the appropriate location and retraction arches made of 0.019 X 0.025 SS with boot loops placed distal to the lateral incisors. Mini screw provides a stable anchorage for enmasse retraction of the anterior teeth with the help of a boot loop using sliding and/or loop mechanics. The arch wires with boot loops have a definite advantage over the soldered/crimpable hooks because of the versatility it offers during the process of retraction. An innovative approach combining the advantages of absolute anchorage using mini implants and a retraction arch with boot loop is presented here.

Investigation of Stress-Strain History Modeling at Stress Risers

DTIC Science & Technology

1977-06-01

GJ6(A ’/2 = 0.0149) 21 7 Hysteresis Loops for Test GJ9(A f/2 = 0.0211) 22 8 Hysteresis Loops for Test GJI (6 ir/2 = 0.0294) 23 9 Cyclic and Monotonic...61 Hysteresis Loops for Test GJ9(A c/2=0.021 1) 105 62 Hysteresis Loops for Test GJI (At/2=0.0294) 106 63 Cyclic Locus Curve 107 64 Stable Hysteresis...tests GJI through GJ37 show that 7075-T651 aluminum both hardens and softens. The response ]or four of these tests is plotted in Figures 59 through 62

Following isotopes in pulse-chase enriched aspen seedlings

NASA Astrophysics Data System (ADS)

Norris, C. E.; Wasylishen, R. E.; Landhäusser, S.; Quideau, S. A.

2011-12-01

One method to quantitatively trace biogeochemical fluxes through ecosystems, such as organic matter decomposition, is to use plant material enriched with stable isotopes. However, as plant macromolecules are known to vary in their rate of formation and decomposition, both the enrichment levels and the location of enrichment within the plant material should be characterized prior to decomposition and tracing studies. Aspen (Populus tremuloides Michx.) is a common tree species with a diverse organic matter chemical structure found in the western Canadian boreal forest. This study used a multi pulse and multi chase enrichment of stable isotopes (15N and 13C) on aspen seedlings to determine the seedling enrichment, isotope movement among plant tissues and translocation of isotopes within plant macromolecules e.g., carbohydrates and lignin. As expected, all tissues experienced increased enrichment with multiple pulses. An initial enrichment with 13C was observed in the leaves followed by translocation to the stems and roots while the 15N moved upward from the roots to leaves. The macromolecular chemistry of the organic carbon was further characterized using 13C solid state nuclear magnetic resonance spectroscopy. After the initial two hour chase period enrichment of the O-alkyl type (carbohydrate) carbon within the leaves was identified, followed by redistribution to more complex carbon compounds after the one week chase period. Root and stem tissues did not show the same pattern. Rather, changes in 13C enrichment were observed in shifting ethyl and methyl alkyl (lipid) carbon peak intensities for the stem samples while roots did not preferentially allocate 13C to a specific macromolecule. These results confirm that stable isotope enrichment of plants was non-uniform across macromolecules and tissue types. Enrichment of aspen seedlings was therefore dependant on the pulse-chase sequence used.

Generation of mechanical oscillation applicable to vibratory rate gyroscopes

NASA Technical Reports Server (NTRS)

Lemkin, Mark A. (Inventor); Juneau, Thor N. (Inventor); Clark, William A. (Inventor); Roessig, Allen W. (Inventor)

2001-01-01

To achieve a drive-axis oscillation with improved frequency and amplitude stability, additional feedback loops are used to adjust force-feedback loop parameters. An amplitude-control loop measures oscillation amplitude, compares this value to the desired level, and adjusts damping of the mechanical sense-element to grow or shrink oscillation amplitude as appropriate. A frequency-tuning loop measures the oscillation frequency, compares this value with a highly stable reference, and adjusts the gain in the force-feedback loop to keep the drive-axis oscillation frequency at the reference value. The combined topology simultaneously controls both amplitude and frequency. Advantages of the combined topology include improved stability, fast oscillation start-up, low power consumption, and excellent shock rejection.

The three-dimensional feto-maternal vascular interrelationship during early bovine placental development: a scanning electron microscopical study

PubMed Central

PFARRER, CHRISTIANE; EBERT, BRIGITTE; MIGLINO, MARIA ANGELICA; KLISCH, KARL; LEISER, RUDOLF

2001-01-01

Both the fetal and maternal microvasculature of bovine placentomes was examined by scanning electron microscopy of vascular casts. So far the development of the vascular architecture of the bovine placentome in early gestation has only been studied 2-dimensionally due to technical difficulties arising from the fragility of the early placental blood vessels. Repeated experiments led to the selection of the microvascular corrosion casts presented here. The vasculature of the maternal compartment is supplied by large caruncular stalk or spiral arteries, which release short maternal stem arteries. In the 3rd month of gestation, these arteries branch into several arterioles at their base, thus providing the vascular framework for the lower part of the septal walls of the primary crypts. In the 4th month, due to progressive longitudinal growth of the stem arteries, branching into arterioles occurs not only at the base, but over the whole length of the stem arteries. These arterioles supply the capillary complexes of the septa which resemble the major part of the septal vasculature and face the secondary crypts. Further indentation results in the formation of tertiary crypt capillary complexes, encircling the earlier secondary unit. From the 6th month of gestation the architecture resembles the fully developed maternal placenta with stem arteries running directly to the fetal side to branch into 4 to 6 arterioles, which turn back to enter secondary and tertiary septa. Maternal venules, collecting the blood from the capillary bed of secondary and tertiary septa, converge onto stem veins leaving the caruncle via branches of the uterine vein. The fetal part of the placentome is supplied by the cotyledonary arteries, which branch into fetal stem arteries that are the tributary to single villous trees. Over their whole course towards the maternal side, these give off arterioles entering secondary villi. The tertiary or terminal villous vasculature consists of capillaries, which are organised in serial capillary loops. This system is progressively elaborated in the course of gestation. In the 4th month there are only finger-like loops, whereas from the 6th month large fan-like structures can be observed. In early gestation the maternal and fetal blood vessels meet predominantly in a countercurrent fashion, changing to the less efficient crosscurrent exchange when the tertiary unit develops. These results indicate the development of a highly elaborated fetomaternal villous-crypt exchange system, already established in the 1st half of gestation, thus meeting the increasing needs of the fetus. PMID:11430698

[Comparative analysis of clustered regularly interspaced short palindromic repeats (CRISPRs) loci in the genomes of halophilic archaea].

PubMed

Zhang, Fan; Zhang, Bing; Xiang, Hua; Hu, Songnian

2009-11-01

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) is a widespread system that provides acquired resistance against phages in bacteria and archaea. Here we aim to genome-widely analyze the CRISPR in extreme halophilic archaea, of which the whole genome sequences are available at present time. We used bioinformatics methods including alignment, conservation analysis, GC content and RNA structure prediction to analyze the CRISPR structures of 7 haloarchaeal genomes. We identified the CRISPR structures in 5 halophilic archaea and revealed a conserved palindromic motif in the flanking regions of these CRISPR structures. In addition, we found that the repeat sequences of large CRISPR structures in halophilic archaea were greatly conserved, and two types of predicted RNA secondary structures derived from the repeat sequences were likely determined by the fourth base of the repeat sequence. Our results support the proposal that the leader sequence may function as recognition site by having palindromic structures in flanking regions, and the stem-loop secondary structure formed by repeat sequences may function in mediating the interaction between foreign genetic elements and CAS-encoded proteins.

Role of Modulators in Controlling the Colloidal Stability and Polydispersity of the UiO-66 Metal–Organic Framework

DOE Office of Scientific and Technical Information (OSTI.GOV)

Morris, William; Wang, Shunzhi; Cho, David

2017-04-07

Nanoscale UiO-66 Zr6(OH)4O4(C8O4H4)6 has been synthesized with a series of carboxylic acid modulators, R-COOH (where R = H, CH3, CF3, and CHCl2). The phase purity and size of each MOF was confirmed by powder X-ray diffraction, BET surface area analysis, and scanning transmission electron microscopy (STEM). Size control of UiO-66 crystals from 20 nm to over 1 μm was achieved, and confirmed by STEM. The colloidal stability of each MOF was evaluated by dynamic light scattering and was found to be highly dependent on the modulator conditions utilized in the synthesis, with both lower pKa and higher acid concentration resultingmore » in more stable structures. Furthermore, STEM was carried out on both colloidally stable samples and those that exhibited a large degree of aggregation, which allowed for visualization of the different degrees of dispersion of the samples. The use of modulators at higher concentrations and with lower pKas leads to the formation of more defects, as a consequence of terephthalic acid ligands being replaced by modulator molecules, thereby enhancing the colloidal stability of the UiO-66 nanoparticles. These findings could have a significant impact on nanoscale MOF material syntheses and applications, especially in the areas of catalysis and drug delivery.« less

Production and characterization of genetically modified human IL-11 variants.

PubMed

Sano, Emiko; Takei, Toshiaki; Ueda, Takuya; Tsumoto, Kouhei

2017-02-01

Interleukin-11 (IL-11) has been expected as a drug on severe thrombocytopenia caused by myelo-suppressive chemotherapy. Whereas, development of IL-11 inhibitor is also expected for a treatment against IL-11 related cancer progression. Here, we will demonstrate the creation of various kinds of genetically modified hIL-11s. Modified vectors were constructed by introducing N- or O-glycosylation site on the region of hIL-11 that does not belong to the core α-helical motif based on the predicted secondary structure. N-terminal (N: between 22 to 23 aa), the first loop (M1:70 to 71 aa), the second loop (M2:114-115 aa), the third loop (M3:160-161 aa) and C-terminal (C: 200- aa) were selected for modification. A large scale production system was established and the characteristics of modified hIL-11s were evaluated. The structure was analyzed by amino acid sequence and composition analysis and CD-spectra. Glycan was assessed by monosaccharide composition analysis. Growth promoting activity and biological stability were analyzed by proliferation of T1165 cells. N-terminal modified proteins were well glycosylated and produced. Growth activity of 3NN with NASNASNAS sequence on N-terminal was about tenfold higher than wild type (WT). Structural and biological stabilities of 3NN were also better than WT and residence time in mouse blood was longer than WT. M1 variants lacked growth activity though they are well glycosylated and secondary structure is very stable. Both of 3NN and OM1 with AAATPAPG on M1 associated with hIL-11R strongly. These results indicate N-terminal and M1 variants will be expected for practical use as potent agonists or antagonists of hIL-11. Copyright © 2016 Elsevier B.V. All rights reserved.

Upward and downward heat and mass transfer with miniature periodically operating loop thermosyphons

NASA Astrophysics Data System (ADS)

Fantozzi, Fabio; Filippeschi, Sauro; Latrofa, Enrico Maria

2004-03-01

Upward and downward two-phase heat and mass transfer has been considered in the present paper. The heat and mass transfer with the condenser located below the evaporator has been obtained by inserting an accumulator tank in the liquid line of a loop thermosyphon and enforcing a pressure pulsation. In previous papers these heat transfer devices have been called pulsated two phase thermosyphons (PTPT). A mini PTPT has been experimentally investigated. It has shown a stable periodic heat transfer regime weakly influenced by the position of the condenser with respect to the evaporator. In contrast a classical loop mini thermosyphon (diameter of connecting pipes 4 mm) did not achieve a stable functioning for the investigated level differences between evaporator and condenser lower than 0.37 m. The present study shows that the functioning of a PTPT device does not directly depend on the level difference or the presence of noncondensable gas. In order to obtain a natural circulation in mini or micro loops, a periodically operating heat transfer regime should therefore be considered.

ERECTA-family receptor kinases regulate stem cell homeostasis via buffering its cytokinin responsiveness in the shoot apical meristem.

PubMed

Uchida, Naoyuki; Shimada, Masanori; Tasaka, Masao

2013-03-01

Shoot apical meristems (SAMs), which are maintained at the tips of stems, are indeterminate structures and sources of stem cells from which all aerial organs are ultimately derived. Although mechanisms that regulate the homeostasis of the stem cells have been extensively investigated, identification of further unknown regulators should provide better understanding of the regulation. Here, we report that members of the Arabidopsis ERECTA (ER) receptor kinase family redundantly play a significant role in the regulation of stem cell homeostasis. In wild-type seedlings, the expression of WUSCHEL (WUS), a central regulator of the stem cell population, is stimulated by cytokinin. Interestingly, however, the SAM morphology and the expression of CLAVATA3 (CLV3), which is expressed in stem cells and therefore serves as a stem cell marker, are relatively stable against cytokinin treatment regardless of increased WUS expression. These findings indicate the presence of a mechanism to buffer stem cell homeostasis against an increase in cytokinin. Mutant seedlings lacking all ER-family members, which are expressed in the SAM, show an increase in the stem cell population and also the up-regulation of a cytokinin-responsive gene in the SAM. In this mutant, WUS expression is stimulated by cytokinin treatment as efficiently as in wild-type plants. However, in contrast to wild-type plants, SAM morphology and CLV3 expression respond drastically to cytokinin treatment, suggesting that the buffering mechanism to maintain stem cell homeostasis against an increase in cytokinin is severely impaired in this mutant. We suggest that the ER family regulates stem cell homeostasis via buffering its cytokinin responsiveness in the SAM.

Identification of stable fly attractant compounds in vinasse, a byproduct of sugarcane-ethanol distillation

USDA-ARS?s Scientific Manuscript database

The stable fly, Stomoxys calcitrans (Diptera: Muscidae), is a worldwide pest of livestock. Recent outbreaks of stable flies in sugarcane fields in Brazil have become a serious problem for livestock producers. Larvae and pupae found inside sugarcane stems after harvesting may indicate that stable fli...

Kissing-loop interaction between 5′ and 3′ ends of tick-borne Langat virus genome ‘bridges the gap’ between mosquito- and tick-borne flaviviruses in mechanisms of viral RNA cyclization: applications for virus attenuation and vaccine development

PubMed Central

Tsetsarkin, Konstantin A.; Liu, Guangping; Shen, Kui; Pletnev, Alexander G.

2016-01-01

Insertion of microRNA target sequences into the flavivirus genome results in selective tissue-specific attenuation and host-range restriction of live attenuated vaccine viruses. However, previous strategies for miRNA-targeting did not incorporate a mechanism to prevent target elimination under miRNA-mediated selective pressure, restricting their use in vaccine development. To overcome this limitation, we developed a new approach for miRNA-targeting of tick-borne flavivirus (Langat virus, LGTV) in the duplicated capsid gene region (DCGR). Genetic stability of viruses with DCGR was ensured by the presence of multiple cis-acting elements within the N-terminal capsid coding region, including the stem-loop structure (5′SL6) at the 3′ end of the promoter. We found that the 5′SL6 functions as a structural scaffold for the conserved hexanucleotide motif at its tip and engages in a complementary interaction with the region present in the 3′ NCR to enhance viral RNA replication. The resulting kissing-loop interaction, common in tick-borne flaviviruses, supports a single pair of cyclization elements (CYC) and functions as a homolog of the second pair of CYC that is present in the majority of mosquito-borne flaviviruses. Placing miRNA targets into the DCGR results in superior attenuation of LGTV in the CNS and does not interfere with development of protective immunity in immunized mice. PMID:26850640

LoopX: A Graphical User Interface-Based Database for Comprehensive Analysis and Comparative Evaluation of Loops from Protein Structures.

PubMed

Kadumuri, Rajashekar Varma; Vadrevu, Ramakrishna

2017-10-01

Due to their crucial role in function, folding, and stability, protein loops are being targeted for grafting/designing to create novel or alter existing functionality and improve stability and foldability. With a view to facilitate a thorough analysis and effectual search options for extracting and comparing loops for sequence and structural compatibility, we developed, LoopX a comprehensively compiled library of sequence and conformational features of ∼700,000 loops from protein structures. The database equipped with a graphical user interface is empowered with diverse query tools and search algorithms, with various rendering options to visualize the sequence- and structural-level information along with hydrogen bonding patterns, backbone φ, ψ dihedral angles of both the target and candidate loops. Two new features (i) conservation of the polar/nonpolar environment and (ii) conservation of sequence and conformation of specific residues within the loops have also been incorporated in the search and retrieval of compatible loops for a chosen target loop. Thus, the LoopX server not only serves as a database and visualization tool for sequence and structural analysis of protein loops but also aids in extracting and comparing candidate loops for a given target loop based on user-defined search options.

TGF-β1 induces the formation of vascular-like structures in embryoid bodies derived from human embryonic stem cells.

PubMed

Wang, Yan; Qian, DE-Jian; Zhong, Wen-Yu; Lu, Jun-Hong; Guo, Xiang-Kai; Cao, Yi-Lin; Liu, Ju

2014-07-01

Human embryonic stem cells (ESCs) can differentiate into endothelial cells in response to stimuli from extracellular cytokines. Transforming growth factor (TGF)-β1 signaling is involved in stem cell renewal and vascular development. Previously, human ESCs were isolated from inner cell mass and a stable ESC line was developed. In the present study, the effects of extracellular TGF-β1 were investigated on human ESC-derived embryoid bodies (EB) in suspension. The structures of the EBs were analyzed with light and electron microscopy, while the cellular composition of the EBs was examined via the expression levels of specific markers. Vascular-like tubular structures and cardiomyocyte-like beating cells were observed in the EBs at day 3 and 8, respectively. The frequencies of vascular-like structures and beating cells in the TGF-β1 treated group were significantly higher compared with the control group (84.31 vs. 12.77%; P<0.001; 37.25 vs. 8.51%; P<0.001, respectively). Electron microscopy revealed the presence of lumens and gap junctions in the sections of the tubular structures. Semiquantitative polymerase chain reaction revealed elevated expression levels of CD31 and fetal liver kinase-1 in EBs cultured with TGF-β1. In addition, extensive staining of von Willebrand factor was observed in the vascular-like structures of TGF-β1-treated EBs. Therefore, the results of the present study may aid the understanding of the underlying mechanisms of human ESC differentiation and improve the methods of propagating specific cell types for the clinical therapy of cardiovascular diseases.

Modifications modulate anticodon loop dynamics and codon recognition of E. coli tRNA(Arg1,2).

PubMed

Cantara, William A; Bilbille, Yann; Kim, Jia; Kaiser, Rob; Leszczyńska, Grażyna; Malkiewicz, Andrzej; Agris, Paul F

2012-03-02

Three of six arginine codons are read by two tRNA(Arg) isoacceptors in Escherichia coli. The anticodon stem and loop of these isoacceptors (ASL(Arg1,2)) differs only in that the position 32 cytidine of tRNA(Arg1) is posttranscriptionally modified to 2-thiocytidine (s(2)C(32)). The tRNA(Arg1,2) are also modified at positions 34 (inosine, I(34)) and 37 (2-methyladenosine, m(2)A(37)). To investigate the roles of modifications in the structure and function, we analyzed six ASL(Arg1,2) constructs differing in their array of modifications by spectroscopy and codon binding assays. Thermal denaturation and circular dichroism spectroscopy indicated that modifications contribute thermodynamic and base stacking properties, resulting in more order but less stability. NMR-derived structures of the ASL(Arg1,2) showed that the solution structures of the ASLs were nearly identical. Surprisingly, none possessed the U-turn conformation required for effective codon binding on the ribosome. Yet, all ASL(Arg1,2) constructs efficiently bound the cognate CGU codon. Three ASLs with I(34) were able to decode CGC, whereas only the singly modified ASL(Arg1,2)(ICG) with I(34) was able to decode CGA. The dissociation constants for all codon bindings were physiologically relevant (0.4-1.4 μM). However, with the introduction of s(2)C(32) or m(2)A(37) to ASL(Arg1,2)(ICG), the maximum amount of ASL bound to CGU and CGC was significantly reduced. These results suggest that, by allowing loop flexibility, the modifications modulate the conformation of the ASL(Arg1,2), which takes one structure free in solution and two others when bound to the cognate arginyl-tRNA synthetase or to codons on the ribosome where modifications reduce or restrict binding to specific codons. Copyright © 2011 Elsevier Ltd. All rights reserved.

Exact calculation of loop formation probability identifies folding motifs in RNA secondary structures

PubMed Central

Sloma, Michael F.; Mathews, David H.

2016-01-01

RNA secondary structure prediction is widely used to analyze RNA sequences. In an RNA partition function calculation, free energy nearest neighbor parameters are used in a dynamic programming algorithm to estimate statistical properties of the secondary structure ensemble. Previously, partition functions have largely been used to estimate the probability that a given pair of nucleotides form a base pair, the conditional stacking probability, the accessibility to binding of a continuous stretch of nucleotides, or a representative sample of RNA structures. Here it is demonstrated that an RNA partition function can also be used to calculate the exact probability of formation of hairpin loops, internal loops, bulge loops, or multibranch loops at a given position. This calculation can also be used to estimate the probability of formation of specific helices. Benchmarking on a set of RNA sequences with known secondary structures indicated that loops that were calculated to be more probable were more likely to be present in the known structure than less probable loops. Furthermore, highly probable loops are more likely to be in the known structure than the set of loops predicted in the lowest free energy structures. PMID:27852924

Structural features of microRNA (miRNA) precursors and their relevance to miRNA biogenesis and small interfering RNA/short hairpin RNA design.

PubMed

Krol, Jacek; Sobczak, Krzysztof; Wilczynska, Urszula; Drath, Maria; Jasinska, Anna; Kaczynska, Danuta; Krzyzosiak, Wlodzimierz J

2004-10-01

We have established the structures of 10 human microRNA (miRNA) precursors using biochemical methods. Eight of these structures turned out to be different from those that were computer-predicted. The differences localized in the terminal loop region and at the opposite side of the precursor hairpin stem. We have analyzed the features of these structures from the perspectives of miRNA biogenesis and active strand selection. We demonstrated the different thermodynamic stability profiles for pre-miRNA hairpins harboring miRNAs at their 5'- and 3'-sides and discussed their functional implications. Our results showed that miRNA prediction based on predicted precursor structures may give ambiguous results, and the success rate is significantly higher for the experimentally determined structures. On the other hand, the differences between the predicted and experimentally determined structures did not affect the stability of termini produced through "conceptual dicing." This result confirms the value of thermodynamic analysis based on mfold as a predictor of strand section by RNAi-induced silencing complex (RISC).

Selection of stable reference genes for quantitative rt-PCR comparisons of mouse embryonic and extra-embryonic stem cells.

PubMed

Veazey, Kylee J; Golding, Michael C

2011-01-01

Isolation and culture of both embryonic and tissue specific stem cells provide an enormous opportunity to study the molecular processes driving development. To gain insight into the initial events underpinning mammalian embryogenesis, pluripotent stem cells from each of the three distinct lineages present within the preimplantation blastocyst have been derived. Embryonic (ES), trophectoderm (TS) and extraembryonic endoderm (XEN) stem cells possess the developmental potential of their founding lineages and seemingly utilize distinct epigenetic modalities to program gene expression. However, the basis for these differing cellular identities and epigenetic properties remain poorly defined.Quantitative reverse transcription-polymerase chain reaction (qPCR) is a powerful and efficient means of rapidly comparing patterns of gene expression between different developmental stages and experimental conditions. However, careful, empirical selection of appropriate reference genes is essential to accurately measuring transcriptional differences. Here we report the quantitation and evaluation of fourteen commonly used references genes between ES, TS and XEN stem cells. These included: Actb, B2m, Hsp70, Gapdh, Gusb, H2afz, Hk2, Hprt, Pgk1, Ppia, Rn7sk, Sdha, Tbp and Ywhaz. Utilizing three independent statistical analysis, we identify Pgk1, Sdha and Tbp as the most stable reference genes between each of these stem cell types. Furthermore, we identify Sdha, Tbp and Ywhaz as well as Ywhaz, Pgk1 and Hk2 as the three most stable reference genes through the in vitro differentiation of embryonic and trophectoderm stem cells respectively.Understanding the transcriptional and epigenetic regulatory mechanisms controlling cellular identity within these distinct stem cell types provides essential insight into cellular processes controlling both embryogenesis and stem cell biology. Normalizing quantitative RT-PCR measurements using the geometric mean CT values obtained for the identified mRNAs, offers a reliable method to assess differing patterns of gene expression between the three founding stem cell lineages present within the mammalian preimplantation embryo.

Effect of surface site interactions on potentiometric titration of hematite (α-Fe2O3) crystal faces.

PubMed

Chatman, Shawn; Zarzycki, P; Preočanin, T; Rosso, K M

2013-02-01

Time dependent potentiometric pH titrations were used to study the effect of atomic scale surface structure on the protonation behavior of the structurally well-defined hematite/electrolyte interfaces. Our recently proposed thermodynamic model [1,25] was applied to measured acidimetric and alkalimetric titration hysteresis loops, collected from highly organized (001), (012), and (113) crystal face terminations using pH equilibration times ranging from 15 to 30 min. Hysteresis loop areas indicate that (001) faces equilibrate faster than the (012) and (113) faces, consistent with the different expected ensembles of singly-, doubly-, and triply-coordinated surface sites on each face. Strongly non-linear hysteretic pH-potential relationships were found, with slopes exceeding Nernstian, collectively indicating that protonation and deprotonation is much more complex than embodied in present day surface complexation models. The asymmetrical shape of the acidimetric and alkalimetric titration branches were used to illustrate a proposed steric "leaky screen" repulsion/trapping interaction mechanism that stems from high affinity singly-coordinated sites electrostatically and sterically screening lower affinity doubly- and triply-coordinated sites. Our data indicate that site interaction is the dominant phenomenon defining surface potential accumulation behavior on single crystal faces of metal oxide minerals. Copyright © 2012 Elsevier Inc. All rights reserved.

Domain structure of the ribozyme from eubacterial ribonuclease P.

PubMed Central

Loria, A; Pan, T

1996-01-01

Large RNAs can be composed of discrete domains that fold independently. One such "folding domain" has been identified previously in the ribozyme from Bacillus subtilis ribonuclease P (denoted P RNA). This domain contains roughly one-third of all residues. Folding of an RNA construct consisting of the remaining two-thirds of B. subtilis P RNA was examined by Fe(II)-EDTA hydroxyl radical protection. This molecule folds into the proper higher-order structure under identical conditions as the full-length P RNA, suggesting the presence of a second folding domain in B. subtilis P RNA. Folding analysis of the Escherichia coli P RNA by hydroxyl radical protection shows that this P RNA is completely folded at 5-6 mM Mg2+. In order to analyze the structural organization of folding domains in E. coli P RNA, constructs were designed based on the domain structure of B. subtilis P RNA. Fe(II)-EDTA protection indicates that E. coli P RNA also contains two folding domains. Despite the significant differences at the secondary structure level, both P RNAs appear to converge structurally at the folding domain level. The pre-tRNA substrate, localized in previous studies, may bind across the folding domains with the acceptor stem/3'CCA contacting the domain including the active site and the T stem-loop contacting the other. Because all eubacterial P RNAs share considerable homology in secondary structure to either B. subtilis or E. coli P RNA, these results suggest that this domain structure may be applicable for most, if not all, eubacterial P RNAs. Identification of folding domains should be valuable in dissecting structure-function relationship of large RNAs. PMID:8718684

Mechanism of action of chromogranin A on catecholamine release: molecular modeling of the catestatin region reveals a β-strand/loop/β-strand structure secured by hydrophobic interactions and predictive of activity

PubMed Central

Tsigelny, Igor; Mahata, Sushil K.; Taupenot, Laurent; Preece, Nicholas E.; Mahata, Manjula; Khan, Imran; Parmer, Robert J.; O’Connor, Daniel T.

2009-01-01

A novel fragment of chromogranin A, known as ‘catestatin’ (bovine chromogranin A344–364), inhibits catecholamine release from chromaffin cells and noradrenergic neurons by acting as a non-competitive nicotinic cholinergic antagonist, and may therefore constitute an endogenous autocrine feedback regulator of sympathoadrenal activity. To characterize how this activity depends on the peptide’s structure, we searched for common 3-dimensional motifs for this primary structure or its homologs. Catestatin’s primary structure bore significant (29–35.5% identity, general alignment score 44–57) sequence homology to fragment sequences within three homologs of known 3-dimensional structures, based on solved X-ray crystals: 8FAB, 1PKM, and 2IG2. Each of these sequences exists in nature as a β-strand/loop/β-strand structure, stabilized by hydrophobic interactions between the β-strands. The catestatin structure was stable during molecular dynamics simulations. The catestatin loop contains three Arg residues, whose electropositive side chains form the terminus of the structure, and give rise to substantial uncompensated charge asymmetry in the molecule. A hydrophobic moment plot revealed that catestatin is the only segment of chromogranin A predicted to contain amphiphilic β-strand. Circular dichroism in the far ultraviolet showed substantial (63%) β-sheet structure, especially in a hydrophobic environment. Alanine-substitution mutants of catestatin established a crucial role for the three central arginine residues in the loop (Arg351, Arg353, and Arg358), though not for two arginine residues in the strand region toward the amino-terminus. [125I]Catestatin bound to Torpedo membranes at a site other than the nicotinic agonist binding site. When the catestatin structure was ‘docked’ with the extracellular domain of the Torpedo nicotinic cholinergic receptor, it interacted principally with the β and δ subunits, in a relatively hydrophobic region of the cation pore extracellular orifice, and the complex of ligand and receptor largely occluded the cation pore, providing a structural basis for the non-competitive nicotinic cholinergic antagonist properties of the peptide. We conclude that a homology model of catestatin correctly predicts actual features of the peptide, both physical and biological. The model suggests particular spatial and charge features of the peptide which may serve as starting points in the development of non-peptide mimetics of this endogenous nicotinic cholinergic antagonist. PMID:9809795

Maintenance of a functional higher order chromatin structure: The role of the nuclear matrix in normal and disease states

PubMed Central

Linnemann, Amelia K.; Krawetz, Stephen A.

2010-01-01

Summary The ordered packaging of DNA within the nucleus of somatic cells reflects a dynamic supportive structure that facilitates stable transcription interrupted by intermittent cycles of extreme condensation. This dynamic mode of packing and unpacking chromatin is intimately linked to the ability of the genome to specifically complex with both histones and non-histone proteins. Understanding the underlying mechanism that governs the formation of higher order chromatin structures is a key to understanding how local architecture modulates transcription. In part, the formation of these structures appears to be regulated through genomic looping that is dynamically mediated by attachment to the nuclear scaffold/matrix at S/MARs, i.e., Scaffold/Matrix Attachment Regions. Although the mechanism guiding the formation and use of these higher-ordered structures remains unknown, S/MARs continue to reveal a multitude of roles in development and the pathogenesis of disease. PMID:20948980

Maintenance of a functional higher order chromatin structure: The role of the nuclear matrix in normal and disease states.

PubMed

Linnemann, Amelia K; Krawetz, Stephen A

2009-01-01

The ordered packaging of DNA within the nucleus of somatic cells reflects a dynamic supportive structure that facilitates stable transcription interrupted by intermittent cycles of extreme condensation. This dynamic mode of packing and unpacking chromatin is intimately linked to the ability of the genome to specifically complex with both histones and non-histone proteins. Understanding the underlying mechanism that governs the formation of higher order chromatin structures is a key to understanding how local architecture modulates transcription. In part, the formation of these structures appears to be regulated through genomic looping that is dynamically mediated by attachment to the nuclear scaffold/matrix at S/MARs, i.e., Scaffold/Matrix Attachment Regions. Although the mechanism guiding the formation and use of these higher-ordered structures remains unknown, S/MARs continue to reveal a multitude of roles in development and the pathogenesis of disease.

NMR studies of the backbone flexibility and structure of human growth hormone: a comparison of high and low pH conformations.

PubMed

Kasimova, Marina R; Kristensen, Søren M; Howe, Peter W A; Christensen, Thorkild; Matthiesen, Finn; Petersen, Jørgen; Sørensen, Hans H; Led, Jens J

2002-05-03

(15)N NMR relaxation parameters and amide (1)H/(2)H-exchange rates have been used to characterize the structural flexibility of human growth hormone (rhGH) at neutral and acidic pH. Our results show that the rigidity of the molecule is strongly affected by the solution conditions. At pH 7.0 the backbone dynamics parameters of rhGH are uniform along the polypeptide chain and their values are similar to those of other folded proteins. In contrast, at pH 2.7 the overall backbone flexibility increases substantially compared to neutral pH and the average order parameter approaches the lower limit expected for a folded protein. However, a significant variation of the backbone dynamics through the molecule indicates that under acidic conditions the mobility of the residues becomes more dependent on their location within the secondary structure units. In particular, the order parameters of certain loop regions decrease dramatically and become comparable to those found in unfolded proteins. Furthermore, the HN-exchange rates at low pH reveal that the residues most protected from exchange are clustered at one end of the helical bundle, forming a stable nucleus. We suggest that this nucleus maintains the overall fold of the protein under destabilizing conditions. We therefore conclude that the acid state of rhGH consists of a structurally conserved, but dynamically more flexible helical core surrounded by an aura of highly mobile, unstructured loops. However, in spite of its prominent flexibility the acid state of rhGH cannot be considered a "molten globule" state because of its high stability. It appears from our work that under certain conditions, a protein can tolerate a considerable increase in flexibility of its backbone, along with an increased penetration of water into its core, while still maintaining a stable folded conformation.

HER2 in Breast Cancer Stemness: A Negative Feedback Loop towards Trastuzumab Resistance

PubMed Central

Nami, Babak; Wang, Zhixiang

2017-01-01

HER2 receptor tyrosine kinase that is overexpressed in approximately 20% of all breast cancers (BCs) is a poor prognosis factor and a precious target for BC therapy. Trastuzumab is approved by FDA to specifically target HER2 for treating HER2+ BC. However, about 60% of patients with HER2+ breast tumor develop de novo resistance to trastuzumab, partially due to the loss of expression of HER2 extracellular domain on their tumor cells. This is due to shedding/cleavage of HER2 by metalloproteinases (ADAMs and MMPs). HER2 shedding results in the accumulation of intracellular carboxyl-terminal HER2 (p95HER2), which is a common phenomenon in trastuzumab-resistant tumors and is suggested as a predictive marker for trastuzumab resistance. Up-regulation of the metalloproteinases is a poor prognosis factor and is commonly seen in mesenchymal-like cancer stem cells that are risen during epithelial to mesenchymal transition (EMT) of tumor cells. HER2 cleavage during EMT can explain why secondary metastatic tumors with high percentage of mesenchymal-like cancer stem cells are mostly resistant to trastuzumab but still sensitive to lapatinib. Importantly, many studies report HER2 interaction with oncogenic/stemness signaling pathways including TGF-β/Smad, Wnt/β-catenin, Notch, JAK/STAT and Hedgehog. HER2 overexpression promotes EMT and the emergence of cancer stem cell properties in BC. Increased expression and activation of metalloproteinases during EMT leads to proteolytic cleavage and shedding of HER2 receptor, which downregulates HER2 extracellular domain and eventually increases trastuzumab resistance. Here, we review the hypothesis that a negative feedback loop between HER2 and stemness signaling drives resistance of BC to trastuzumab. PMID:28445439

Backbone dynamics of the antifungal Psd1 pea defensin and its correlation with membrane interaction by NMR spectroscopy.

PubMed

de Medeiros, Luciano Neves; Angeli, Renata; Sarzedas, Carolina G; Barreto-Bergter, Eliana; Valente, Ana Paula; Kurtenbach, Eleonora; Almeida, Fabio C L

2010-02-01

Plant defensins are cysteine-rich cationic peptides, components of the innate immune system. The antifungal sensitivity of certain exemplars was correlated to the level of complex glycosphingolipids in the membrane of fungi strains. Psd1 is a 46 amino acid residue defensin isolated from pea seeds which exhibit antifungal activity. Its structure is characterized by the so-called cysteine-stabilized alpha/beta motif linked by three loops as determined by two-dimensional NMR. In the present work we explored the measurement of heteronuclear Nuclear Overhauser Effects, R1 and R2 (15)N relaxation ratios, and chemical shift to probe the backbone dynamics of Psd1 and its interaction with membrane mimetic systems with phosphatidylcholine (PC) or dodecylphosphocholine (DPC) with glucosylceramide (CMH) isolated from Fusarium solani. The calculated R2 values predicted a slow motion around the highly conserved among Gly12 residue and also in the region of the Turn3 His36-Trp38. The results showed that Psd1 interacts with vesicles of PC or PC:CMH in slightly different forms. The interaction was monitored by chemical shift perturbation and relaxation properties. Using this approach we could map the loops as the binding site of Psd1 with the membrane. The major binding epitope showed conformation exchange properties in the mus-ms timescale supporting the conformation selection as the binding mechanism. Moreover, the peptide corresponding to part of Loop1 (pepLoop1: Gly12 to Ser19) is also able to interact with DPC micelles acquiring a stable structure and in the presence of DPC:CMH the peptide changes to an extended conformation, exhibiting NOE mainly with the carbohydrate and ceramide parts of CMH. Copyright 2009 Elsevier B.V. All rights reserved.

Application of phase matching autofocus in airborne long-range oblique photography camera

NASA Astrophysics Data System (ADS)

Petrushevsky, Vladimir; Guberman, Asaf

2014-06-01

The Condor2 long-range oblique photography (LOROP) camera is mounted in an aerodynamically shaped pod carried by a fast jet aircraft. Large aperture, dual-band (EO/MWIR) camera is equipped with TDI focal plane arrays and provides high-resolution imagery of extended areas at long stand-off ranges, at day and night. Front Ritchey-Chretien optics is made of highly stable materials. However, the camera temperature varies considerably in flight conditions. Moreover, a composite-material structure of the reflective objective undergoes gradual dehumidification in dry nitrogen atmosphere inside the pod, causing some small decrease of the structure length. The temperature and humidity effects change a distance between the mirrors by just a few microns. The distance change is small but nevertheless it alters the camera's infinity focus setpoint significantly, especially in the EO band. To realize the optics' resolution potential, the optimal focus shall be constantly maintained. In-flight best focus calibration and temperature-based open-loop focus control give mostly satisfactory performance. To get even better focusing precision, a closed-loop phase-matching autofocus method was developed for the camera. The method makes use of an existing beamsharer prism FPA arrangement where aperture partition exists inherently in an area of overlap between the adjacent detectors. The defocus is proportional to an image phase shift in the area of overlap. Low-pass filtering of raw defocus estimate reduces random errors related to variable scene content. Closed-loop control converges robustly to precise focus position. The algorithm uses the temperature- and range-based focus prediction as an initial guess for the closed-loop phase-matching control. The autofocus algorithm achieves excellent results and works robustly in various conditions of scene illumination and contrast.

Microbial Abundances in Salt Marsh Soils: A Molecular Approach for Small Spatial Scales

NASA Astrophysics Data System (ADS)

Granse, Dirk; Mueller, Peter; Weingartner, Magdalena; Hoth, Stefan; Jensen, Kai

2016-04-01

The rate of biological decomposition greatly determines the carbon sequestration capacity of salt marshes. Microorganisms are involved in the decomposition of biomass and the rate of decomposition is supposed to be related to microbial abundance. Recent studies quantified microbial abundance by means of quantitative polymerase chain reaction (QPCR), a method that also allows determining the microbial community structure by applying specific primers. The main microbial community structure can be determined by using primers specific for 16S rRNA (Bacteria) and 18S rRNA (Fungi) of the microbial DNA. However, the investigation of microbial abundance pattern at small spatial scales, such as locally varying abiotic conditions within a salt-marsh system, requires high accuracy in DNA extraction and QPCR methods. Furthermore, there is evidence that a single extraction may not be sufficient to reliably quantify rRNA gene copies. The aim of this study was to establish a suitable DNA extraction method and stable QPCR conditions for the measurement of microbial abundances in semi-terrestrial environments. DNA was extracted from two soil samples (top WE{5}{cm}) by using the PowerSoil DNA Extraction Kit (Mo Bio Laboratories, Inc., Carlsbad, CA) and applying a modified extraction protocol. The DNA extraction was conducted in four consecutive DNA extraction loops from three biological replicates per soil sample by reusing the PowerSoil bead tube. The number of Fungi and Bacteria rRNA gene copies of each DNA extraction loop and a pooled DNA solution (extraction loop 1 - 4) was measured by using the QPCR method with taxa specific primer pairs (Bacteria: B341F, B805R; Fungi: FR1, FF390). The DNA yield of the replicates varied at DNA extraction loop 1 between WE{25 and 85}{ng

Critical chemical features in trans-acting-responsive RNA are required for interaction with human immunodeficiency virus type 1 Tat protein.

PubMed Central

Sumner-Smith, M; Roy, S; Barnett, R; Reid, L S; Kuperman, R; Delling, U; Sonenberg, N

1991-01-01

The human immunodeficiency virus type 1 Tat protein binds to an RNA stem-loop structure called TAR which is present at the 5' end of all human immunodeficiency virus type 1 transcripts. This binding is centered on a bulge within the stem of TAR and is an essential step in the trans-activation process which results in a dramatic increase in viral gene expression. By analysis of a series of TAR derivatives produced by transcription or direct chemical synthesis, we determined the structural and chemical requirements for Tat binding. Tat binds well to structures which have a bulge of two to at least five unpaired bases bounded on both sides by a double-stranded RNA stem. This apparent flexibility in bulge size is in contrast to an absolute requirement for an unpaired uridine (U) in the 5'-most position of the bulge (+23). Substitution of the U with either natural bases or chemical analogs demonstrated that the imido group at the N-3 position and, possibly, the carbonyl group at the C-4 position of U are critical for Tat binding. Cytosine (C), which differs from U at only these positions, is not an acceptable substitute. Furthermore, methylation at N-3 abolishes binding. While methylation of U at the C-5 position has little effect on binding, fluorination reduces it, possibly because of its effects on relative tautomer stability at the N-3 and C-4 positions. Thus, we have identified key moieties in the U residue that are of importance for the binding of Tat to TAR RNA. We hypothesize that the invariant U is involved in hydrogen bond interactions with either another part of TAR or the TAR-binding domain in Tat. Images PMID:1895380

Frost Grape Polysaccharide (FGP), an Emulsion-Forming Arabinogalactan Gum from the Stems of Native North American Grape Species Vitis riparia Michx.

PubMed

Price, Neil P J; Vermillion, Karl E; Eller, Fred J; Vaughn, Steven F

2015-08-19

A new arabinogalactan is described that is produced in large quantity from the cut stems of the North American grape species Vitis riparia (Frost grape). The sugar composition consists of l-arabinofuranose (l-Araf, 55.2%) and d-galactopyranose (d-Galp 30.1%), with smaller components of d-xylose (11.2%), d-mannose (3.5%), and glucuronic acid (GlcA, ∼2%), the latter linked via a galactosyl residue. Permethylation identified 3-linked Galp residues, some substituted at the 2-position with Galp or Manp, terminal Araf and Xylp, and an internal 3-substituted Araf. NMR (HSQC, TOCSY, HMBC, DOSY) identified βGalp and three αAraf spin systems, in an Araf-α1,3-Araf-α1,2-Araf-α1,2-Galp structural motif. Diffusion-ordered NMR showed that the FGP has a molecular weight of 1-10 MDa. Unlike gum arabic, the FGP does not contain a hydroxyproline-rich protein (HPRP). FGP forms stable gels at >15% w/v and at 1-12% solutions are viscous and are excellent emulsifiers of flavoring oils (grapefruit, clove, and lemongrass), giving stable emulsions for ≥72 h. Lower concentrations (0.1% w/v) were less viscous, yet still gave stable grapefruit oil/water emulsions. Hence, FGP is a β1,3-linked arabinogalactan with potential as a gum arabic replacement in the food and beverage industries.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Walden, William E.; Selezneva, Anna I.; Dupuy, Jérôme

Iron regulatory protein 1 (IRP1) binds iron-responsive elements (IREs) in messenger RNAs (mRNAs), to repress translation or degradation, or binds an iron-sulfur cluster, to become a cytosolic aconitase enzyme. The 2.8 angstrom resolution crystal structure of the IRP1:ferritin H IRE complex shows an open protein conformation compared with that of cytosolic aconitase. The extended, L-shaped IRP1 molecule embraces the IRE stem-loop through interactions at two sites separated by {approx}30 angstroms, each involving about a dozen protein:RNA bonds. Extensive conformational changes related to binding the IRE or an iron-sulfur cluster explain the alternate functions of IRP1 as an mRNA regulator ormore » enzyme.« less

Highly divergent cyclo-like virus in a great roundleaf bat (Hipposideros armiger) in Vietnam.

PubMed

Kemenesi, Gábor; Kurucz, Kornélia; Zana, Brigitta; Tu, Vuong Tan; Görföl, Tamás; Estók, Péter; Földes, Fanni; Sztancsik, Katalin; Urbán, Péter; Fehér, Enikő; Jakab, Ferenc

2017-08-01

Members of the viral family Circoviridae are increasingly recognized worldwide. Bats seem to be natural reservoirs or dietary-related dispensers of these viruses. Here, we report a distantly related member of the genus Cyclovirus detected in the faeces of a great roundleaf bat (Hipposideros armiger). Interestingly, the novel virus lacks a Circoviridae-specific stem-loop structure, although a Geminiviridae-like nonamer sequence was detected in the large intergenic region. Based on these differences and its phylogenetic position, we propose that our new virus represents a distant and highly divergent member of the genus Cyclovirus. However it is lacking several characteristics of members of the genus, which raises a challenge in its taxonomic classification.

D19S Mutation of the Cationic, Cysteine-Rich Protein PAF: Novel Insights into Its Structural Dynamics, Thermal Unfolding and Antifungal Function

PubMed Central

Burtscher, Laura; Hajdu, Dorottya; Muñoz, Alberto; Gáspári, Zoltán; Read, Nick D.; Batta, Gyula; Marx, Florentine

2017-01-01

The cysteine-rich, cationic, antifungal protein PAF is abundantly secreted into the culture supernatant of the filamentous Ascomycete Penicillium chrysogenum. The five β-strands of PAF form a compact β-barrel that is stabilized by three disulphide bonds. The folding of PAF allows the formation of four surface-exposed loops and distinct charged motifs on the protein surface that might regulate the interaction of PAF with the sensitive target fungus. The growth inhibitory activity of this highly stable protein against opportunistic fungal pathogens provides great potential in antifungal drug research. To understand its mode of action, we started to investigate the surface-exposed loops of PAF and replaced one aspartic acid at position 19 in loop 2 that is potentially involved in PAF active or binding site, with a serine (Asp19 to Ser19). We analysed the overall effects, such as unfolding, electrostatic changes, sporadic conformers and antifungal activity when substituting this specific amino acid to the fairly indifferent amino acid serine. Structural analyses revealed that the overall 3D solution structure is virtually identical with that of PAF. However, PAFD19S showed slightly increased dynamics and significant differences in the surface charge distribution. Thermal unfolding identified PAFD19S to be rather a two-state folder in contrast to the three-state folder PAF. Functional comparison of PAFD19S and PAF revealed that the exchange at residue 19 caused a dramatic loss of antifungal activity: the binding and internalization of PAFD19S by target cells was reduced and the protein failed to trigger an intracellular Ca2+ response, all of which are closely linked to the antifungal toxicity of PAF. We conclude that the negatively charged residue Asp19 in loop 2 is essential for full function of the cationic protein PAF. PMID:28072824

Crystal structures of CaSiO3 polymorphs control growth and osteogenic differentiation of human mesenchymal stem cells on bioceramic surfaces.

PubMed

Zhang, Nianli; Molenda, James A; Mankoci, Steven; Zhou, Xianfeng; Murphy, William L; Sahai, Nita

2013-10-01

The repair and replacement of damaged or diseased human bone tissue requires a stable interface between the orthopedic implant and living tissue. The ideal material should be both osteoconductive (promote bonding to bone) and osteoinductive (induce osteogenic differentiation of cells and generate new bone). Partially resorbable bioceramic materials with both properties are developed by expensive trial-and-error methods. Structure-reactivity relationships for predicting the osteoinductive properties of ceramics would significantly increase the efficiency of developing materials for bone tissue engineering. Here we propose the novel hypothesis that the crystal structure of a bioceramic controls the release rates, subsequent surface modifications due to precipitation of new phases, and thus, the concentrations of soluble factors, and ultimately, the attachment, viability and osteogenic differentiation of human Mesenchymal Stem Cells (hMSCs). To illustrate our hypothesis, we used two CaSiO 3 polymorphs, pseudo-wollastonite (psw, β-CaSiO 3 ) and wollastonite (wol, α-CaSiO 3 ) as scaffolds for hMSC culture. Polymorphs are materials which have identical chemical composition and stoichiometry, but different crystal structures. We combined the results of detailed surface characterizations, including environmental Scanning Electron Microscopy (SEM) back-scattered imaging, and spot-analysis and 2D elemental mapping by SEM-Energy Dispersive X-ray (SEM-EDX), High Resolution Transmission Electron Microscopy (HRTEM) and surface roughness analysis; culture medium solution analyses; and molecular/genetic assays from cell culture. Our results confirmed the hypothesis that the psw polymorph, which has a strained silicate ring structure, is more osteoinductive than the wol polymorph, which has a more stable, open silicate chain structure. The observations could be attributed to easier dissolution (resorption) of psw compared to wol, which resulted in concentration profiles that were more osteoinductive for the former. Thus, we showed that crystal structure is a fundamental parameter to be considered in the intelligent design of pro-osteogenic, partially resorbable bioceramics.

DNA nanostructure-based fluorescence thermometer with silver nanoclusters

NASA Astrophysics Data System (ADS)

Bu, Congcong; Mu, Lixuan; Cao, Xingxing; Chen, Min; She, Guangwei; Shi, Wensheng

2018-07-01

DNA nanostructure-based fluorescence thermometers were fabricated by linking fluorescent silver nanoclusters (AgNCs) and guanine-rich（G-rich）DNA chains via a thermally sensitive DNA stem-loop at terminals 5‧ and 3‧. Variations of temperature alter the distance between the AgNCs and G-rich DNA chain, affecting the interaction between them. As a result, the intensity of fluorescence emission from the AgNCs at 636 nm can be sensitively modulated. It was found that the intensity of such red emission is more temperature sensitive than the equivalent green emission at 543 nm; sensitivity of ‑3.6%/°C was achieved. Through variation of the melting temperature of the DNA stem-loop, the response temperature range of the thermometers could be readily adjusted. Novel DNA nanostructure-based fluorescence thermometers as described in this work are anticipated to be able to measure the temperature of biological systems at small scales—even a single cell.

DNA nanostructure-based fluorescence thermometer with silver nanoclusters.

PubMed

Bu, Congcong; Mu, Lixuan; Cao, XIngxing; Chen, Min; She, Guangwei; Shi, Wensheng

2018-04-27

Linking the fluorescent silver nanoclusters (AgNCs) and guanine-rich（G-rich）DNA chains by the thermal sensitive DNA stem-loop at teminal 5' and 3', DNA nanostructure-based fluorescence thermometers were fabricated. The variations of the temperature alter the distance between AgNCs and G-rich DNA chain, which could affect the interaction between them. As a result, the intensity of fluorescence emission from AgNCs at 636 nm can be sensitively modulated. It was found that such red emission is more sensitive to the temperature comparing with its intrinsic green emission at 543 nm, and sensitivity of -3.6%/℃ was achieved. Varying the melting temperature of the DNA stem-loop could readily adjust the response temperature range of thermometers. Novel DNA nanostructure-based fluorescence thermometers in this work could be anticipated to measure the temperature of biological system, even a single cell. © 2018 IOP Publishing Ltd.

Mixture models for protein structure ensembles.

PubMed

Hirsch, Michael; Habeck, Michael

2008-10-01

Protein structure ensembles provide important insight into the dynamics and function of a protein and contain information that is not captured with a single static structure. However, it is not clear a priori to what extent the variability within an ensemble is caused by internal structural changes. Additional variability results from overall translations and rotations of the molecule. And most experimental data do not provide information to relate the structures to a common reference frame. To report meaningful values of intrinsic dynamics, structural precision, conformational entropy, etc., it is therefore important to disentangle local from global conformational heterogeneity. We consider the task of disentangling local from global heterogeneity as an inference problem. We use probabilistic methods to infer from the protein ensemble missing information on reference frames and stable conformational sub-states. To this end, we model a protein ensemble as a mixture of Gaussian probability distributions of either entire conformations or structural segments. We learn these models from a protein ensemble using the expectation-maximization algorithm. Our first model can be used to find multiple conformers in a structure ensemble. The second model partitions the protein chain into locally stable structural segments or core elements and less structured regions typically found in loops. Both models are simple to implement and contain only a single free parameter: the number of conformers or structural segments. Our models can be used to analyse experimental ensembles, molecular dynamics trajectories and conformational change in proteins. The Python source code for protein ensemble analysis is available from the authors upon request.

Ultra-Stable Oscillators for Probe Radio Science Investigations

NASA Technical Reports Server (NTRS)

Asmar, Sami

2012-01-01

An Ultra-Stable Oscillator (USO) is: A frequency reference, and A clock It is stable, small, and sensitive. It is a science and an art form. It is flown on spacecraft/probes. It]is utilized at ground stations alone or as a cleanup loop. It eliminates lock-up time on uplink for occultation egress & effect of media on uplink signal. It has enabled significant planetary science investigations.

Implementing Audio Digital Feedback Loop Using the National Instruments RIO System

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huang, G.; Byrd, J. M.

2006-11-20

Development of system for high precision RF distribution and laser synchronization at Berkeley Lab has been ongoing for several years. Successful operation of these systems requires multiple audio bandwidth feedback loops running at relatively high gains. Stable operation of the feedback loops requires careful design of the feedback transfer function. To allow for flexible and compact implementation, we have developed digital feedback loops on the National Instruments Reconfigurable Input/Output (RIO) platform. This platform uses an FPGA and multiple I/Os that can provide eight parallel channels running different filters. We present the design and preliminary experimental results of this system.

The helix-loop-helix protein id1 controls stem cell proliferation during regenerative neurogenesis in the adult zebrafish telencephalon.

PubMed

Rodriguez Viales, Rebecca; Diotel, Nicolas; Ferg, Marco; Armant, Olivier; Eich, Julia; Alunni, Alessandro; März, Martin; Bally-Cuif, Laure; Rastegar, Sepand; Strähle, Uwe

2015-03-01

The teleost brain has the remarkable ability to generate new neurons and to repair injuries during adult life stages. Maintaining life-long neurogenesis requires careful management of neural stem cell pools. In a genome-wide expression screen for transcription regulators, the id1 gene, encoding a negative regulator of E-proteins, was found to be upregulated in response to injury. id1 expression was mapped to quiescent type I neural stem cells in the adult telencephalic stem cell niche. Gain and loss of id1 function in vivo demonstrated that Id1 promotes stem cell quiescence. The increased id1 expression observed in neural stem cells in response to injury appeared independent of inflammatory signals, suggesting multiple antagonistic pathways in the regulation of reactive neurogenesis. Together, we propose that Id1 acts to maintain the neural stem cell pool by counteracting neurogenesis-promoting signals. © 2014 AlphaMed Press.

Exact calculation of loop formation probability identifies folding motifs in RNA secondary structures.

PubMed

Sloma, Michael F; Mathews, David H

2016-12-01

RNA secondary structure prediction is widely used to analyze RNA sequences. In an RNA partition function calculation, free energy nearest neighbor parameters are used in a dynamic programming algorithm to estimate statistical properties of the secondary structure ensemble. Previously, partition functions have largely been used to estimate the probability that a given pair of nucleotides form a base pair, the conditional stacking probability, the accessibility to binding of a continuous stretch of nucleotides, or a representative sample of RNA structures. Here it is demonstrated that an RNA partition function can also be used to calculate the exact probability of formation of hairpin loops, internal loops, bulge loops, or multibranch loops at a given position. This calculation can also be used to estimate the probability of formation of specific helices. Benchmarking on a set of RNA sequences with known secondary structures indicated that loops that were calculated to be more probable were more likely to be present in the known structure than less probable loops. Furthermore, highly probable loops are more likely to be in the known structure than the set of loops predicted in the lowest free energy structures. © 2016 Sloma and Mathews; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Long-Range Chromosome Interactions Mediated by Cohesin Shape Circadian Gene Expression

PubMed Central

Xu, Yichi; Guo, Weimin; Li, Ping; Zhang, Yan; Zhao, Meng; Fan, Zenghua; Zhao, Zhihu; Yan, Jun

2016-01-01

Mammalian circadian rhythm is established by the negative feedback loops consisting of a set of clock genes, which lead to the circadian expression of thousands of downstream genes in vivo. As genome-wide transcription is organized under the high-order chromosome structure, it is largely uncharted how circadian gene expression is influenced by chromosome architecture. We focus on the function of chromatin structure proteins cohesin as well as CTCF (CCCTC-binding factor) in circadian rhythm. Using circular chromosome conformation capture sequencing, we systematically examined the interacting loci of a Bmal1-bound super-enhancer upstream of a clock gene Nr1d1 in mouse liver. These interactions are largely stable in the circadian cycle and cohesin binding sites are enriched in the interactome. Global analysis showed that cohesin-CTCF co-binding sites tend to insulate the phases of circadian oscillating genes while cohesin-non-CTCF sites are associated with high circadian rhythmicity of transcription. A model integrating the effects of cohesin and CTCF markedly improved the mechanistic understanding of circadian gene expression. Further experiments in cohesin knockout cells demonstrated that cohesin is required at least in part for driving the circadian gene expression by facilitating the enhancer-promoter looping. This study provided a novel insight into the relationship between circadian transcriptome and the high-order chromosome structure. PMID:27135601

Foot-and-mouth disease virus 5'-terminal S fragment is required for replication and modulation of the innate immune response in host cells.

PubMed

Kloc, Anna; Diaz-San Segundo, Fayna; Schafer, Elizabeth A; Rai, Devendra K; Kenney, Mary; de Los Santos, Teresa; Rieder, Elizabeth

2017-12-01

The S fragment of the FMDV 5' UTR is predicted to fold into a long stem-loop structure and it has been implicated in virus-host protein interactions. In this study, we report the minimal S fragment sequence required for virus viability and show a direct correlation between the extent of the S fragment deletion mutations and attenuated phenotypes. Furthermore, we provide novel insight into the role of the S fragment in modulating the host innate immune response. Importantly, in an FMDV mouse model system, all animals survive the inoculation with the live A 24 FMDV-S 4 mutant, containing a 164 nucleotide deletion in the upper S fragment loop, at a dose 1000 higher than the one causing lethality by parental A 24 FMDV, indicating that the A 24 FMDV-S 4 virus is highly attenuated in vivo. Additionally, mice exposed to high doses of live A 24 FMDV-S 4 virus are fully protected when challenged with parental A 24 FMDV virus. Published by Elsevier Inc.

Autoregulatory mechanisms controlling the Microprocessor.

PubMed

Triboulet, Robinson; Gregory, Richard I

2010-01-01

The Microprocessor, comprising the ribonuclease Drosha and its essential cofactor, the double-stranded RNA-binding protein, DGCR8, is essential for the first step of the miRNA biogenesis pathway. It specifically cleaves double-stranded RNA within stem-loop structures of primary miRNA transcripts (pri-miRNAs) to generate precursor (pre-miRNA) intermediates. Pre-miRNAs are subsequently processed by Dicer to their mature 22 nt form. Thus, Microprocessor is essential for miRNA maturation, and pri-miRNA cleavage by this complex defines one end of the mature miRNA. Moreover, it is emerging that dysregulation of the Microprocessor is associated with various human diseases. It is therefore important to understand the mechanisms by which the expression of the subunits of the Microprocessor is regulated. Recent findings have uncovered a post-transcriptional mechanism that maintains the integrity of the Microprocessor. These studies revealed that the Microprocessor is involved in the processing of the messenger RNA (mRNA) that encodes DGCR8. This regulatory feedback loop, along with the reported role played by DGCR8 in the stabilization of Drosha protein, is part ofa newly identified regulatory mechanism controlling Microprocessor activity.

Can one generate stable hyaline cartilage from adult mesenchymal stem cells? A developmental approach.

PubMed

Hellingman, Catharine A; Koevoet, Wendy; van Osch, Gerjo J V M

2012-11-01

Chondrogenically differentiating bone marrow-derived mesenchymal stem cells (BMSCs) display signs of chondrocyte hypertrophy, such as production of collagen type X, MMP13 and alkaline phosphatase (ALPL). For cartilage reconstructions this is undesirable, as terminally differentiated cartilage produced by BMSCs mineralizes when implanted in vivo. Terminal differentiation is not restricted to BMSCs but is also encountered in chondrogenic differentiation of adipose-derived mesenchymal stem cells (MSCs) as well as embryonic stem cells, which by definition should be able to generate all types of tissues, including stable cartilage. Therefore, we propose that the currently used culture conditions may drive the cells towards terminal differentiation. In this manuscript we aim to review the literature, supplemented by our own data to answer the question, is it possible to generate stable hyaline cartilage from adult MSCs? We demonstrate that recently published methods for inhibiting terminal differentiation (through PTHrP, MMP13 or blocking phosphorylation of Smad1/5/8) result in cartilage formation with reduction of hypertrophic markers, although this does not reach the low level of stable chondrocytes. A set of hypertrophy markers should be included in future studies to characterize the phenotype more precisely. Finally, we used what is currently known in developmental biology about the differential development of hyaline and terminally differentiated cartilage to provide thought and insights to change current culture models for creating hyaline cartilage. Inhibiting terminal differentiation may not result in stable hyaline cartilage if the right balance of signals has not been created from the start of culture onwards. Copyright © 2011 John Wiley & Sons, Ltd.

Advancing viral RNA structure prediction: measuring the thermodynamics of pyrimidine-rich internal loops

PubMed Central

Phan, Andy; Mailey, Katherine; Saeki, Jessica; Gu, Xiaobo

2017-01-01

Accurate thermodynamic parameters improve RNA structure predictions and thus accelerate understanding of RNA function and the identification of RNA drug binding sites. Many viral RNA structures, such as internal ribosome entry sites, have internal loops and bulges that are potential drug target sites. Current models used to predict internal loops are biased toward small, symmetric purine loops, and thus poorly predict asymmetric, pyrimidine-rich loops with >6 nucleotides (nt) that occur frequently in viral RNA. This article presents new thermodynamic data for 40 pyrimidine loops, many of which can form UU or protonated CC base pairs. Uracil and protonated cytosine base pairs stabilize asymmetric internal loops. Accurate prediction rules are presented that account for all thermodynamic measurements of RNA asymmetric internal loops. New loop initiation terms for loops with >6 nt are presented that do not follow previous assumptions that increasing asymmetry destabilizes loops. Since the last 2004 update, 126 new loops with asymmetry or sizes greater than 2 × 2 have been measured. These new measurements significantly deepen and diversify the thermodynamic database for RNA. These results will help better predict internal loops that are larger, pyrimidine-rich, and occur within viral structures such as internal ribosome entry sites. PMID:28213527

Impact of primer dimers and self-amplifying hairpins on reverse transcription loop-mediated isothermal amplification detection of viral RNA

DOE PAGES

Meagher, Robert J.; Priye, Aashish; Light, Yooli K.; ...

2018-03-27

Loop-mediated isothermal amplification (LAMP), coupled with reverse transcription (RT), has become a popular technique for detection of viral RNA due to several desirable characteristics for use in point-of-care or low-resource settings. The large number of primers in LAMP (six per target) leads to an increased likelihood of primer-dimer interactions, and the inner primers in particular are prone to formation of stable hairpin structures due to their length (typically 40-45 bases). Although primer-dimers and hairpin structures are known features to avoid in nucleic acid amplification techniques, there is little quantitative information in literature regarding the impact of these structures on LAMPmore » or RT-LAMP assays. In this study, we examine the impact of primer-dimers and hairpins on previously-published primer sets for dengue virus and yellow fever virus. We demonstrate that minor changes to the primers to eliminate amplifiable primer dimers and hairpins improves the performance of the assays when monitored in real time with intercalating dyes, and when monitoring a fluorescent endpoint using the QUASR technique. We also discuss the thermodynamic implications of these minor changes on the overall stability of amplifiable secondary structures, and we present a single thermodynamic parameter to predict the probability of non-specific amplification associated with LAMP primers.« less

Impact of primer dimers and self-amplifying hairpins on reverse transcription loop-mediated isothermal amplification detection of viral RNA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Meagher, Robert J.; Priye, Aashish; Light, Yooli K.

Loop-mediated isothermal amplification (LAMP), coupled with reverse transcription (RT), has become a popular technique for detection of viral RNA due to several desirable characteristics for use in point-of-care or low-resource settings. The large number of primers in LAMP (six per target) leads to an increased likelihood of primer-dimer interactions, and the inner primers in particular are prone to formation of stable hairpin structures due to their length (typically 40-45 bases). Although primer-dimers and hairpin structures are known features to avoid in nucleic acid amplification techniques, there is little quantitative information in literature regarding the impact of these structures on LAMPmore » or RT-LAMP assays. In this study, we examine the impact of primer-dimers and hairpins on previously-published primer sets for dengue virus and yellow fever virus. We demonstrate that minor changes to the primers to eliminate amplifiable primer dimers and hairpins improves the performance of the assays when monitored in real time with intercalating dyes, and when monitoring a fluorescent endpoint using the QUASR technique. We also discuss the thermodynamic implications of these minor changes on the overall stability of amplifiable secondary structures, and we present a single thermodynamic parameter to predict the probability of non-specific amplification associated with LAMP primers.« less

Structural basis for recognition of human 7SK long noncoding RNA by the La-related protein Larp7.

PubMed

Eichhorn, Catherine D; Yang, Yuan; Repeta, Lucas; Feigon, Juli

2018-06-26

The La and the La-related protein (LARP) superfamily is a diverse class of RNA binding proteins involved in RNA processing, folding, and function. Larp7 binds to the abundant long noncoding 7SK RNA and is required for 7SK ribonucleoprotein (RNP) assembly and function. The 7SK RNP sequesters a pool of the positive transcription elongation factor b (P-TEFb) in an inactive state; on release, P-TEFb phosphorylates RNA Polymerase II to stimulate transcription elongation. Despite its essential role in transcription, limited structural information is available for the 7SK RNP, particularly for protein-RNA interactions. Larp7 contains an N-terminal La module that binds UUU-3'OH and a C-terminal atypical RNA recognition motif (xRRM) required for specific binding to 7SK and P-TEFb assembly. Deletion of the xRRM is linked to gastric cancer in humans. We report the 2.2-Å X-ray crystal structure of the human La-related protein group 7 (hLarp7) xRRM bound to the 7SK stem-loop 4, revealing a unique binding interface. Contributions of observed interactions to binding affinity were investigated by mutagenesis and isothermal titration calorimetry. NMR 13 C spin relaxation data and comparison of free xRRM, RNA, and xRRM-RNA structures show that the xRRM is preordered to bind a flexible loop 4. Combining structures of the hLarp7 La module and the xRRM-7SK complex presented here, we propose a structural model for Larp7 binding to the 7SK 3' end and mechanism for 7SK RNP assembly. This work provides insight into how this domain contributes to 7SK recognition and assembly of the core 7SK RNP.

Protein Loop Structure Prediction Using Conformational Space Annealing.

PubMed

Heo, Seungryong; Lee, Juyong; Joo, Keehyoung; Shin, Hang-Cheol; Lee, Jooyoung

2017-05-22

We have developed a protein loop structure prediction method by combining a new energy function, which we call E PLM (energy for protein loop modeling), with the conformational space annealing (CSA) global optimization algorithm. The energy function includes stereochemistry, dynamic fragment assembly, distance-scaled finite ideal gas reference (DFIRE), and generalized orientation- and distance-dependent terms. For the conformational search of loop structures, we used the CSA algorithm, which has been quite successful in dealing with various hard global optimization problems. We assessed the performance of E PLM with two widely used loop-decoy sets, Jacobson and RAPPER, and compared the results against the DFIRE potential. The accuracy of model selection from a pool of loop decoys as well as de novo loop modeling starting from randomly generated structures was examined separately. For the selection of a nativelike structure from a decoy set, E PLM was more accurate than DFIRE in the case of the Jacobson set and had similar accuracy in the case of the RAPPER set. In terms of sampling more nativelike loop structures, E PLM outperformed E DFIRE for both decoy sets. This new approach equipped with E PLM and CSA can serve as the state-of-the-art de novo loop modeling method.

Cerebral vascular structure in the motor cortex of adult mice is stable and is not altered by voluntary exercise.

PubMed

Cudmore, Robert H; Dougherty, Sarah E; Linden, David J

2017-12-01

The cerebral vasculature provides blood flow throughout the brain, and local changes in blood flow are regulated to match the metabolic demands of the active brain regions. This neurovascular coupling is mediated by real-time changes in vessel diameter and depends on the underlying vascular network structure. Neurovascular structure is configured during development by genetic and activity-dependent factors. In adulthood, it can be altered by experiences such as prolonged hypoxia, sensory deprivation and seizure. Here, we have sought to determine whether exercise could alter cerebral vascular structure in the adult mouse. We performed repeated in vivo two-photon imaging in the motor cortex of adult transgenic mice expressing membrane-anchored green fluorescent protein in endothelial cells (tyrosine endothelial kinase 2 receptor (Tie2)-Cre:mTmG). This strategy allows for high-resolution imaging of the vessel walls throughout the lifespan. Vascular structure, as measured by capillary branch point number and position, segment diameter and length remained stable over a time scale of months as did pericyte number and position. Furthermore, we compared the vascular structure before, during, and after periods of voluntary wheel running and found no alterations in these same parameters. In both running and control mice, we observed a low rate of capillary segment subtraction. Interestingly, these rare subtraction events preferentially remove short vascular loops.

The conserved CAAGAAAGA spacer sequence is an essential element for the formation of 3' termini of the sea urchin H3 histone mRNA by RNA processing.

PubMed Central

Georgiev, O; Birnstiel, M L

1985-01-01

Analysis of cDNA sequences obtained from the small nuclear RNA U7 has previously suggested specific contacts, by base pairing, between the conserved stem-loop structure and CAAGAAAGA sequence of the histone pre-mRNA and the 5'-terminal sequence of the U7 RNA during RNA processing. In order to test some aspects of the model we have created a series of linker scan, deletion and insertion mutants of the 3' terminus of a sea urchin H3 histone gene and have injected mutant DNAs or in vitro synthesized precursors into frog oocyte nuclei for interpretation. We find that, in addition to the stem-loop structure of the mRNA, the CAAGAAAGA spacer transcript within the histone pre-mRNA is required absolutely for RNA processing, as predicted from our model. Spacer sequences immediately downstream of the CAAGAAAGA motif are not complementary to U7 RNA. Nevertheless, they are necessary for obtaining a maximal rate of RNA processing, as is the ACCA sequence coding for the 3' terminus of the mature mRNA. An increase of distance between the mRNA palindrome and the CAAGAAAGA by as little as six nucleotides abolishes all processing. It may, therefore, be useful to regard both these sequence motifs as part of one and the same RNA processing signal with narrowly defined topologies. Interestingly, U7 RNA-dependent 3' processing of histone pre-mRNA can occur in RNA injection experiments only when the in vitro synthesized pre-mRNA contains sequence extensions well beyond the region of sequence complementarities to the U7 RNA. In addition to directing 3' processing the terminal mRNA sequences may have a role in histone mRNA stabilization in the cytoplasmic compartment. Images Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. PMID:2410259

Novel poly-uridine insertion in the 3'UTR and E2 amino acid substitutions in a low virulent classical swine fever virus.

PubMed

Coronado, Liani; Liniger, Matthias; Muñoz-González, Sara; Postel, Alexander; Pérez, Lester Josue; Pérez-Simó, Marta; Perera, Carmen Laura; Frías-Lepoureau, Maria Teresa; Rosell, Rosa; Grundhoff, Adam; Indenbirken, Daniela; Alawi, Malik; Fischer, Nicole; Becher, Paul; Ruggli, Nicolas; Ganges, Llilianne

2017-03-01

In this study, we compared the virulence in weaner pigs of the Pinar del Rio isolate and the virulent Margarita strain. The latter caused the Cuban classical swine fever (CSF) outbreak of 1993. Our results showed that the Pinar del Rio virus isolated during an endemic phase is clearly of low virulence. We analysed the complete nucleotide sequence of the Pinar del Rio virus isolated after persistence in newborn piglets, as well as the genome sequence of the inoculum. The consensus genome sequence of the Pinar del Rio virus remained completely unchanged after 28days of persistent infection in swine. More importantly, a unique poly-uridine tract was discovered in the 3'UTR of the Pinar del Rio virus, which was not found in the Margarita virus or any other known CSFV sequences. Based on RNA secondary structure prediction, the poly-uridine tract results in a long single-stranded intervening sequence (SS) between the stem-loops I and II of the 3'UTR, without major changes in the stem- loop structures when compared to the Margarita virus. The possible implications of this novel insertion on persistence and attenuation remain to be investigated. In addition, comparison of the amino acid sequence of the viral proteins E rns , E1, E2 and p7 of the Margarita and Pinar del Rio viruses showed that all non-conservative amino acid substitutions acquired by the Pinar del Rio isolate clustered in E2, with two of them being located within the B/C domain. Immunisation and cross-neutralisation experiments in pigs and rabbits suggest differences between these two viruses, which may be attributable to the amino acid differences observed in E2. Altogether, these data provide fresh insights into viral molecular features which might be associated with the attenuation and adaptation of CSFV for persistence in the field. Copyright © 2017 Elsevier B.V. All rights reserved.

Switchable dual-wavelength fiber laser based on PCF Sagnac loop and broadband FBG

NASA Astrophysics Data System (ADS)

Chen, Weiguo; Lou, Shuqin; Feng, Suchun; Wang, Liwen; Li, Honglei; Guo, Tieying; Jian, Shuisheng

2009-11-01

Switchable dual-wavelength fiber laser with photonic crystal fiber (PCF) Sagnac loop and broadband fiber Bragg grating (BFBG) at room temperature is demonstrated. By adjusting the polarization controller (PC) appropriately, the laser can be switched between the stable single- and dual-wavelength lasing operations by exploiting polarization hole burning (PHB) and spectral hole burning effects (SHB).