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Sample records for mrna secondary structure

  1. Secondary Structure across the Bacterial Transcriptome Reveals Versatile Roles in mRNA Regulation and Function.

    PubMed

    Del Campo, Cristian; Bartholomäus, Alexander; Fedyunin, Ivan; Ignatova, Zoya

    2015-10-01

    Messenger RNA acts as an informational molecule between DNA and translating ribosomes. Emerging evidence places mRNA in central cellular processes beyond its major function as informational entity. Although individual examples show that specific structural features of mRNA regulate translation and transcript stability, their role and function throughout the bacterial transcriptome remains unknown. Combining three sequencing approaches to provide a high resolution view of global mRNA secondary structure, translation efficiency and mRNA abundance, we unraveled structural features in E. coli mRNA with implications in translation and mRNA degradation. A poorly structured site upstream of the coding sequence serves as an additional unspecific binding site of the ribosomes and the degree of its secondary structure propensity negatively correlates with gene expression. Secondary structures within coding sequences are highly dynamic and influence translation only within a very small subset of positions. A secondary structure upstream of the stop codon is enriched in genes terminated by UAA codon with likely implications in translation termination. The global analysis further substantiates a common recognition signature of RNase E to initiate endonucleolytic cleavage. This work determines for the first time the E. coli RNA structurome, highlighting the contribution of mRNA secondary structure as a direct effector of a variety of processes, including translation and mRNA degradation. PMID:26495981

  2. Conservation of mRNA secondary structures may filter out mutations in Escherichia coli evolution.

    PubMed

    Chursov, Andrey; Frishman, Dmitrij; Shneider, Alexander

    2013-09-01

    Recent reports indicate that mutations in viral genomes tend to preserve RNA secondary structure, and those mutations that disrupt secondary structural elements may reduce gene expression levels, thereby serving as a functional knockout. In this article, we explore the conservation of secondary structures of mRNA coding regions, a previously unknown factor in bacterial evolution, by comparing the structural consequences of mutations in essential and nonessential Escherichia coli genes accumulated over 40 000 generations in the course of the 'long-term evolution experiment'. We monitored the extent to which mutations influence minimum free energy (MFE) values, assuming that a substantial change in MFE is indicative of structural perturbation. Our principal finding is that purifying selection tends to eliminate those mutations in essential genes that lead to greater changes of MFE values and, therefore, may be more disruptive for the corresponding mRNA secondary structures. This effect implies that synonymous mutations disrupting mRNA secondary structures may directly affect the fitness of the organism. These results demonstrate that the need to maintain intact mRNA structures imposes additional evolutionary constraints on bacterial genomes, which go beyond preservation of structure and function of the encoded proteins.

  3. Dynamics of tRNA translocation, mRNA translocation and tRNA dissociation during ribosome translation through mRNA secondary structures.

    PubMed

    Xie, Ping

    2014-07-01

    The ribosome can translate through the duplex region or secondary structure of mRNA. Recent single-molecule experimental data showed that downstream mRNA secondary structures have more sensitive effects on deacylated tRNA dissociation from the E site than on tRNA translocation in the 50S subunit. However, it is unclear how the downstream mRNA secondary structure can affect the tRNA dissociation from the E site, which is distant from the secondary structure. Here, based on our proposed ribosomal translocation model, we theoretically study the dynamics of tRNA translocation in the 50S subunit, mRNA translocation and tRNA dissociation, giving quantitative explanations of the single-molecule experimental data. It is shown that the effect of the downstream mRNA secondary structure on tRNA dissociation is via the effect on mRNA translocation, while the mRNA secondary structure has no effect on the rate of deacylated tRNA dissociation from the posttranslocation state. The slow mRNA translocation, which results in slow tRNA dissociation, derives from the occurrence of the futile transition, which is induced by the energy barrier from base pair unwinding to resist the forward translocation. The reduced translation rate through the mRNA secondary structure is induced by the slow mRNA translocation rather than the slow tRNA dissociation.

  4. Leader length and secondary structure modulate mRNA function under conditions of stress

    SciTech Connect

    Kozak, M.

    1988-07-01

    Simina virus 40-based plasmids that direct the synthesis of preproinsulin in cultured monkey cells were used to study the effects of mRNA structure on translational efficiency. Lengthening the leader sequence enhanced translation in this system. The enhancement was most obvious when an unstructured sequence (two, four, or eight copies of the oligonculeotide AGCTAAGTAAGTAAGTA) was inserted upstream from a region of deliberate secondary structure; the degree of enhancement was proportional to the number of copies of the inserted oligonucleotide. Lengthening the leader sequence on the 3' side of a stem-and-loop structure, in contrast, did not offset the potentially inhibitory effect of the hairpin structure. Both the facilitating effect of length and the inhibitory effect of secondary structure were demonstrated most easily under conditions of mRNA competition, which was brought about by an abrupt shift in the tonicity of the culture medium. These experiments suggest a simple structural basis for the long-recognized differential response of viral and cellular mRNAs to hypertonic stress. The fact that the translatability of structure-prone mRNAs varies with changes in the environment may also have general implications for gene expression in eucaryotic cells.

  5. hiCLIP reveals the in vivo atlas of mRNA secondary structures recognized by Staufen 1

    PubMed Central

    Sugimoto, Yoichiro; Vigilante, Alessandra; Darbo, Elodie; Zirra, Alexandra; Militti, Cristina; D’Ambrogio, Andrea; Luscombe, Nicholas M; Ule, Jernej

    2015-01-01

    mRNA structure is important for post-transcriptional regulation, largely because it affects binding of trans-acting factors1. However, little is known about the in vivo structure of full-length mRNAs. Here we present hiCLIP, a high-throughput technique to identify RNA secondary structures interacting with RNA-binding proteins (RBPs) in vivo. Using this technique to investigate RNA structures bound by Staufen 1 (STAU1), we uncover a dominance of intra-molecular RNA duplexes, a depletion of duplexes from coding regions of highly translated mRNAs, an unforeseen prevalence of long-range duplexes in 3′ untranslated regions (UTRs), and a decreased incidence of SNPs in duplex-forming regions. We also discover a duplex spanning 858nts in the 3′ UTR of the X-box binding Protein 1 (XBP1) mRNA that regulates its cytoplasmic splicing and stability. Our study reveals the fundamental role of mRNA secondary structures in gene regulation and introduces hiCLIP as a widely applicable method for discovering novel, especially long-range, RNA duplexes. PMID:25799984

  6. hiCLIP reveals the in vivo atlas of mRNA secondary structures recognized by Staufen 1.

    PubMed

    Sugimoto, Yoichiro; Vigilante, Alessandra; Darbo, Elodie; Zirra, Alexandra; Militti, Cristina; D'Ambrogio, Andrea; Luscombe, Nicholas M; Ule, Jernej

    2015-03-26

    The structure of messenger RNA is important for post-transcriptional regulation, mainly because it affects binding of trans-acting factors. However, little is known about the in vivo structure of full-length mRNAs. Here we present hiCLIP, a biochemical technique for transcriptome-wide identification of RNA secondary structures interacting with RNA-binding proteins (RBPs). Using this technique to investigate RNA structures bound by Staufen 1 (STAU1) in human cells, we uncover a dominance of intra-molecular RNA duplexes, a depletion of duplexes from coding regions of highly translated mRNAs, an unexpected prevalence of long-range duplexes in 3' untranslated regions (UTRs), and a decreased incidence of single nucleotide polymorphisms in duplex-forming regions. We also discover a duplex spanning 858 nucleotides in the 3' UTR of the X-box binding protein 1 (XBP1) mRNA that regulates its cytoplasmic splicing and stability. Our study reveals the fundamental role of mRNA secondary structures in gene expression and introduces hiCLIP as a widely applicable method for discovering new, especially long-range, RNA duplexes.

  7. Compilation of mRNA Polyadenylation Signals in Arabidopsis Revealed a New Signal Element and Potential Secondary Structures1[w

    PubMed Central

    Loke, Johnny C.; Stahlberg, Eric A.; Strenski, David G.; Haas, Brian J.; Wood, Paul Chris; Li, Qingshun Quinn

    2005-01-01

    Using a novel program, SignalSleuth, and a database containing authenticated polyadenylation [poly(A)] sites, we analyzed the composition of mRNA poly(A) signals in Arabidopsis (Arabidopsis thaliana), and reevaluated previously described cis-elements within the 3′-untranslated (UTR) regions, including near upstream elements and far upstream elements. As predicted, there are absences of high-consensus signal patterns. The AAUAAA signal topped the near upstream elements patterns and was found within the predicted location to only approximately 10% of 3′-UTRs. More importantly, we identified a new set, named cleavage elements, of poly(A) signals flanking both sides of the cleavage site. These cis-elements were not previously revealed by conventional mutagenesis and are contemplated as a cluster of signals for cleavage site recognition. Moreover, a single-nucleotide profile scan on the 3′-UTR regions unveiled a distinct arrangement of alternate stretches of U and A nucleotides, which led to a prediction of the formation of secondary structures. Using an RNA secondary structure prediction program, mFold, we identified three main types of secondary structures on the sequences analyzed. Surprisingly, these observed secondary structures were all interrupted in previously constructed mutations in these regions. These results will enable us to revise the current model of plant poly(A) signals and to develop tools to predict 3′-ends for gene annotation. PMID:15965016

  8. Secondary Structure of a Conserved Domain in an Intron of Influenza A M1 mRNA

    PubMed Central

    2014-01-01

    Influenza A virus utilizes RNA throughout infection. Little is known, however, about the roles of RNA structures. A previous bioinformatics survey predicted multiple regions of influenza A virus that are likely to generate evolutionarily conserved and stable RNA structures. One predicted conserved structure is in the pre-mRNA coding for essential proteins, M1 and M2. This structure starts 79 nucleotides downstream of the M2 mRNA 5′ splice site. Here, a combination of biochemical structural mapping, mutagenesis, and NMR confirms the predicted three-way multibranch structure of this RNA. Imino proton NMR spectra reveal no change in secondary structure when 80 mM KCl is supplemented with 4 mM MgCl2. Optical melting curves in 1 M NaCl and in 100 mM KCl with 10 mM MgCl2 are very similar, with melting temperatures ∼14 °C higher than that for 100 mM KCl alone. These results provide a firm basis for designing experiments and potential therapeutics to test for function in cell culture. PMID:25026548

  9. The relationship between third-codon position nucleotide content, codon bias, mRNA secondary structure and gene expression in the drosophilid alcohol dehydrogenase genes Adh and Adhr.

    PubMed Central

    Carlini, D B; Chen, Y; Stephan, W

    2001-01-01

    To gain insights into the relationship between codon bias, mRNA secondary structure, third-codon position nucleotide distribution, and gene expression, we predicted secondary structures in two related drosophilid genes, Adh and Adhr, which differ in degree of codon bias and level of gene expression. Individual structural elements (helices) were inferred using the comparative method. For each gene, four types of randomization simulations were performed to maintain/remove codon bias and/or to maintain or alter third-codon position nucleotide composition (N3). In the weakly expressed, weakly biased gene Adhr, the potential for secondary structure formation was found to be much stronger than in the highly expressed, highly biased gene Adh. This is consistent with the observation of approximately equal G and C percentages in Adhr ( approximately 31% across species), whereas in Adh the N3 distribution is shifted toward C (42% across species). Perturbing the N3 distribution to approximately equal amounts of A, G, C, and T increases the potential for secondary structure formation in Adh, but decreases it in Adhr. On the other hand, simulations that reduce codon bias without changing N3 content indicate that codon bias per se has only a weak effect on the formation of secondary structures. These results suggest that, for these two drosophilid genes, secondary structure is a relatively independent, negative regulator of gene expression. Whereas the degree of codon bias is positively correlated with level of gene expression, strong individual secondary structural elements may be selected for to retard mRNA translation and to decrease gene expression. PMID:11606539

  10. Optimisation of the mRNA secondary structure to improve the expression of interleukin-24 (IL-24) in Escherichia coli.

    PubMed

    Bai, Chaogang; Wang, Xiaojuan; Zhang, Jian; Sun, Aiyou; Wei, Dongzhi; Yang, Shengli

    2014-08-01

    Interleukin-24 (IL-24) is a novel cytokine selectively inhibiting proliferation of cancer cells but with little effect on normal cells. However, IL-24 is difficult to express in Escherichia coli. In this study, we optimised the secondary structure of the translation initiation region using computational approach to obtain non-fusion recombinant IL-24 (nrIL-24). The Gibbs free energy of the region was decreased from -22 to -9.07 kcal mol(-1), potentially promoting a loose secondary structure formation and improving the translation initiation efficiency. As a result, the expression of nrIL-24 was increased to 26 % of the total cellular protein from being barely initially detectable. nrIL-24 showed a concentration-dependent inhibition of A375 cells but had little effect on normal human cells. These results demonstrate that this method in increasing nrIL-24 expression is effective and efficient.

  11. Effect of 3′UTR RET Variants on RET mRNA Secondary Structure and Disease Presentation in Medullary Thyroid Carcinoma

    PubMed Central

    Ceolin, Lucieli; Romitti, Mirian; Rodrigues Siqueira, Débora; Vaz Ferreira, Carla; Oliboni Scapineli, Jessica; Assis-Brazil, Beatriz; Vieira Maximiano, Rodolfo; Dias Amarante, Tauanne; de Souza Nunes, Miriam Celi; Weber, Gerald; Maia, Ana Luiza

    2016-01-01

    Background The RET S836S variant has been associated with early onset and increased risk for metastatic disease in medullary thyroid carcinoma (MTC). However, the mechanism by which this variant modulates MTC pathogenesis is still open to discuss. Of interest, strong linkage disequilibrium (LD) between RET S836S and 3'UTR variants has been reported in Hirschsprung's disease patients. Objective To evaluate the frequency of the RET 3’UTR variants (rs76759170 and rs3026785) in MTC patients and to determine whether these variants are in LD with S836S polymorphism. Methods Our sample comprised 152 patients with sporadic MTC. The RET S836S and 3’UTR (rs76759170 and rs3026785) variants were genotyped using Custom TaqMan Genotyping Assays. Haplotypes were inferred using the phase 2.1 program. RET mRNA structure was assessed by Vienna Package. Results The mean age of MTC diagnosis was 48.5±15.5 years and 57.9% were women. The minor allele frequencies of RET polymorphisms were as follows: S836S, 5.6%; rs76759170, 5.6%; rs3026785, 6.2%. We observed a strong LD among S836S and 3’UTR variants (|D’| = -1, r2 = 1 and |D’| = -1, r2 = 0,967). Patients harboring the S836S/3’UTR variants presented a higher percentage of lymph node and distant metastasis (P = 0.013 and P<0.001, respectively). Accordingly, RNA folding analyses demonstrated different RNA secondary structure predictions for WT(TCCGT), S836S(TTCGT) or 3’UTR(GTCAC) haplotypes. The S836S/3’UTR haplotype presented a greater number of double helices sections and lower levels of minimal free energy when compared to the wild-type haplotype, suggesting that these variants provides the most thermodynamically stable mRNA structure, which may have functional consequences on the rate of mRNA degradation. Conclusion The RET S836S polymorphism is in LD with 3’UTR variants. In silico analysis indicate that the 3’UTR variants may affect the secondary structure of RET mRNA, suggesting that these variants might play a

  12. A molecular beacon-based real time NASBA assay for detection of Listeria monocytogenes in food products: role of target mRNA secondary structure on NASBA design.

    PubMed

    Nadal, Anna; Coll, Anna; Cook, Nigel; Pla, Maria

    2007-03-01

    A molecular beacon-based real-time NASBA (QNASBA) assay for detection and identification of Listeria monocytogenes has been developed. A correlation between targeting highly accessible mRNA sequences and QNASBA efficiency and sensitivity was demonstrated. The assay targets a sequence from the mRNA transcript of the hly gene which is specific for this bacterium; and includes an internal amplification control to disclose failure of the reaction. It was fully selective and consistently detected down to 100 target molecules and 40 L. monocytogenes exponentially growing cells per reaction. In addition, it was capable of accurate quantification of target RNA molecules independently of the presence of DNA in the sample. In combination with a short RNase treatment prior to nucleic acids extraction our QNASBA specifically detected viable L. monocytogenes cells. It was successfully applied to rapid detection of this pathogen in meat and salmon products, and is therefore a useful tool for the study of L. monocytogenes in food samples. We finally discuss considerations of target secondary structure with regard to development of NASBA assays.

  13. Conserved Secondary Structures in Aspergillus

    PubMed Central

    McGuire, Abigail Manson; Galagan, James E.

    2008-01-01

    Background Recent evidence suggests that the number and variety of functional RNAs (ncRNAs as well as cis-acting RNA elements within mRNAs ) is much higher than previously thought; thus, the ability to computationally predict and analyze RNAs has taken on new importance. We have computationally studied the secondary structures in an alignment of six Aspergillus genomes. Little is known about the RNAs present in this set of fungi, and this diverse set of genomes has an optimal level of sequence conservation for observing the correlated evolution of base-pairs seen in RNAs. Methodology/Principal Findings We report the results of a whole-genome search for evolutionarily conserved secondary structures, as well as the results of clustering these predicted secondary structures by structural similarity. We find a total of 7450 predicted secondary structures, including a new predicted ∼60 bp long hairpin motif found primarily inside introns. We find no evidence for microRNAs. Different types of genomic regions are over-represented in different classes of predicted secondary structures. Exons contain the longest motifs (primarily long, branched hairpins), 5′ UTRs primarily contain groupings of short hairpins located near the start codon, and 3′ UTRs contain very little secondary structure compared to other regions. There is a large concentration of short hairpins just inside the boundaries of exons. The density of predicted intronic RNAs increases with the length of introns, and the density of predicted secondary structures within mRNA coding regions increases with the number of introns in a gene. Conclusions/Sigificance There are many conserved, high-confidence RNAs of unknown function in these Aspergillus genomes, as well as interesting spatial distributions of predicted secondary structures. This study increases our knowledge of secondary structure in these aspergillus organisms. PMID:18665251

  14. Expression of phosphinothricin N-acetyltransferase in Escherichia coli and Pseudomonas fluorescens: influence of mRNA secondary structure, host, and other physiological conditions.

    PubMed

    Madduri, Krishna M; Snodderley, Erika M

    2007-10-01

    Expression of a plant codon optimized pat gene encoding phosphinothricin acetyltransferase (PAT) in bacterial expression systems required modification of the 5' end of the pat ORF. Modifications necessary for improving the expression were identified by a coupled in vitro transcription and translation process. The dramatic improvement in the expression of PAT was due to the removal of a potential secondary structure that could have resulted in the inhibition of translational initiation. Therefore, in vitro transcription and translation is a versatile tool to optimize gene sequence for protein overexpression. Additionally, this method was shown to be successful in both Escherichia coli and Pseudomonas fluorescens. Gene sequence optimization and choice of host along with cultivation conditions also had major impact on PAT expression. P. fluorescens was a better host than E. coli resulting in 30-fold more expression of PAT. We were able to recover approximately 95mg of purified PAT from P. fluorescens using a three step chromatographic process.

  15. Exon B of human surfactant protein A2 mRNA, alone or within its surrounding sequences, interacts with 14-3-3; role of cis-elements and secondary structure

    PubMed Central

    Noutsios, Georgios T.; Silveyra, Patricia; Bhatti, Faizah

    2013-01-01

    Human surfactant protein A, an innate immunity molecule, is encoded by two genes: SFTPA1 (SP-A1) and SFTPA2 (SP-A2). The 5′ untranslated (5′UTR) splice variant of SP-A2 (ABD), but not of SP-A1 (AD), contains exon B (eB), which is an enhancer for transcription and translation. We investigated whether eB contains cis-regulatory elements that bind trans-acting factors in a sequence-specific manner as well as the role of the eB mRNA secondary structure. Binding of cytoplasmic NCI-H441 proteins to wild-type eB, eB mutant, AD, and ABD 5′UTR mRNAs were studied by RNA electromobility shift assays (REMSAs). The bound proteins were identified by mass spectroscopy and specific antibodies (Abs). We found that 1) proteins bind eB mRNA in a sequence-specific manner, with two cis-elements identified within eB to be important; 2) eB secondary structure is necessary for binding; 3) mass spectroscopy and specific Abs in REMSAs identified 14-3-3 proteins to bind (directly or indirectly) eB and the natural SP-A2 (ABD) splice variant but not the SP-A1 (AD) splice variant; 4) other ribosomal and cytoskeletal proteins, and translation factors, are also present in the eB mRNA-protein complex; 5) knockdown of 14-3-3 β/α isoform resulted in a downregulation of SP-A2 expression. In conclusion, proteins including the 14-3-3 family bind two cis-elements within eB of hSP-A2 mRNA in a sequence- and secondary structure-specific manner. Differential regulation of SP-A1 and SP-A2 is mediated by the 14-3-3 protein family as well as by a number of other proteins that bind UTRs with or without eB mRNA. PMID:23525782

  16. A cis-encoded sRNA, Hfq and mRNA secondary structure act independently to suppress IS200 transposition

    PubMed Central

    Ellis, Michael J.; Trussler, Ryan S.; Haniford, David B.

    2015-01-01

    IS200 is found throughout Enterobacteriaceae and transposes at a notoriously low frequency. In addition to the transposase protein (TnpA), IS200 encodes an uncharacterized Hfq-binding sRNA that is encoded opposite to the tnpA 5'UTR. In the current work we asked if this sRNA represses tnpA expression. We show here that the IS200 sRNA (named art200 for antisense regulator of transposase IS200) basepairs with tnpA to inhibit translation initiation. Unexpectedly, art200-tnpA pairing is limited to 40 bp, despite 90 nt of perfect complementarity. Additionally, we show that Hfq and RNA secondary structure in the tnpA 5'UTR each repress tnpA expression in an art200-independent manner. Finally, we show that disrupting translational control of tnpA expression leads to increased IS200 transposition in E. coli. The current work provides new mechanistic insight into why IS200 transposition is so strongly suppressed. The possibility of art200 acting in trans to regulate a yet-unidentified target is discussed as well as potential applications of the IS200 system for designing novel riboregulators. PMID:26044710

  17. CFTR mRNA expression is regulated by an upstream open reading frame and RNA secondary structure in its 5' untranslated region.

    PubMed

    Lukowski, Samuel W; Rothnagel, Joseph A; Trezise, Ann E O

    2015-02-15

    Post-transcriptional regulation of gene expression through 5' untranslated region (5'UTR)-encoded cis-acting elements is an important mechanism for the control of protein expression levels. Through controlling specific aspects of translation initiation, expression can be tightly regulated while remaining responsive to cellular requirements. With respect to cystic fibrosis (CF), the overexpression of cystic fibrosis transmembrane conductance regulator (CFTR) protein trafficking mutants, such as delta-F508, is of great biological and clinical interest. By understanding the post-transcriptional mechanisms that regulate CFTR expression, new procedures can be developed to enhance CFTR expression in homozygous delta-F508 CF patients. We have identified the key elements of a complex negative regulatory mechanism that is encoded within the human CFTR 5'UTR and show how these elements act in combination to restrict CFTR gene expression to a consistently low level in a transcript-specific manner. This study shows, for the first time, that endogenous human CFTR expression is post-transcriptionally regulated through a 5'UTR-mediated mechanism. We show that the very low levels of endogenous CFTR expression, compared with other low expression genes, are maintained through the co-operative inhibitory effects of an upstream open reading frame and a thermodynamically stable RNA secondary structure. PMID:25274779

  18. Differential accumulation of nif structural gene mRNA in Azotobacter vinelandii.

    PubMed

    Hamilton, Trinity L; Jacobson, Marty; Ludwig, Marcus; Boyd, Eric S; Bryant, Donald A; Dean, Dennis R; Peters, John W

    2011-09-01

    Northern analysis was employed to investigate mRNA produced by mutant strains of Azotobacter vinelandii with defined deletions in the nif structural genes and in the intergenic noncoding regions. The results indicate that intergenic RNA secondary structures effect the differential accumulation of transcripts, supporting the high Fe protein-to-MoFe protein ratio required for optimal diazotrophic growth.

  19. Primary structure of chicken muscle pyruvate kinase mRNA.

    PubMed Central

    Lonberg, N; Gilbert, W

    1983-01-01

    We have determined the cDNA sequence corresponding to chicken muscle pyruvate kinase mRNA; the predicted coding region spans 529 amino acids and establishes the complete amino acid sequence for the vertebrate enzyme. We demonstrate that the level of mRNA for this enzyme is under developmental control and suggest a structural model for the protein kinase-mediated regulation of the mammalian liver isozyme. We report a method for the direct analysis of, and the preparation of cDNA probes from, mRNA which has been fractionated on methylmercury/agarose gels. Images PMID:6574503

  20. Secondary Structure Switch

    ERIC Educational Resources Information Center

    King, Angela G.

    2006-01-01

    Neurogenerative diseases like Alzheimer's disease and Parkinson's disease involve a transformation between two peptide and protein structures of alpha-helices and beta-sheets, where the peptide backbone can also participate in metal ion binding in addition to histidine residues. However, the complete absence of change in conformation of Coiled…

  1. hnRNP A1 controls HIV-1 mRNA splicing through cooperative binding to intron and exon splicing silencers in the context of a conserved secondary structure.

    PubMed Central

    Damgaard, Christian Kroun; Tange, Thomas Ostergaard; Kjems, Jørgen

    2002-01-01

    The removal of the second intron in the HIV-1 rev/tat pre-mRNAs, which involves the joining of splice site SD4 to SA7, is inhibited by hnRNP A1 by a mechanism that requires the intronic splicing silencer (ISS) and the exon splicing silencer (ESS3). In this study, we have determined the RNA secondary structure and the hnRNP A1 binding sites within the 3' splice site region by phylogenetic comparison and chemical/enzymatic probing. A biochemical characterization of the RNA/protein complexes demonstrates that hnRNP A1 binds specifically to primarily three sites, the ISS, a novel UAG motif in the exon splicing enhancer (ESE) and the ESS3 element, which are all situated in experimentally supported stem loop structures. A mutational analysis of the ISS region revealed that the core hnRNP A1 binding site directly overlaps with a major branchpoint used in splicing to SA7, thereby providing a direct explanation for the inhibition of U2 snRNP association with the pre-mRNA by hnRNP A1. Binding of hnRNP A1 to the ISS core site is inhibited by RNA structure but strongly stimulated by the exonic silencer, ESS3. Moreover, the ISS also stimulate binding of hnRNP A1 to the exonic splicing regulators ESS3 and the ESE. Our results suggest a model where a network is formed between hnRNP A1 molecules situated at discrete sites in the intron and exon and that these interactions preclude the recognition of essential splicing signals including the branch point. PMID:12458794

  2. Translational pauses during the synthesis of proteins and mRNA structure.

    PubMed

    Zama, M

    1997-01-01

    Translational pauses are observed during a spider fibroin synthesis (1,2). The spider major ampullate (dragline) silk of the spider Nephila clavipes is composed of multiple proteins. The amino acid sequences of the partial cDNA clones for the two major dragline silk fibroin components (Spidroin 1 and 2) exhibit repetitive motifs (3,4). Our detailed inspection of the nucleotide sequences of the repetitive motifs revealed highly selective site-specific codon usage patterns within a motif, suggesting that the secondary structure of the spider fibroin mRNA is optimized by the nucleotide sequence of the fibroin gene. The results, combined with our preceding results on silk fibroin from Bombyx mori (5) suggest that translational pauses of spider silk are interpreted in terms of the mRNA secondary structure.

  3. Structure of an RNA dimer of a regulatory element from human thymidylate synthase mRNA

    PubMed Central

    Dibrov, Sergey; McLean, Jaime; Hermann, Thomas

    2011-01-01

    A sequence around the start codon of the mRNA of human thymidylate synthase (TS) folds into a secondary-structure motif in which the initiation site is sequestered in a metastable hairpin. Binding of the protein to its own mRNA at the hairpin prevents the production of TS through a translation-repression feedback mechanism. Stabilization of the mRNA hairpin by other ligands has been proposed as a strategy to reduce TS levels in anticancer therapy. Rapidly proliferating cells require high TS activity to maintain the production of thymidine as a building block for DNA synthesis. The crystal structure of a model oligonucleotide (TS1) that represents the TS-binding site of the mRNA has been determined. While fluorescence studies showed that the TS1 RNA preferentially adopts a hairpin structure in solution, even at high RNA concentrations, an asymmetric dimer of two hybridized TS1 strands was obtained in the crystal. The TS1 dimer contains an unusual S-­turn motif that also occurs in the ‘off’ state of the human ribosomal decoding site RNA. PMID:21245530

  4. Structure of an RNA dimer of a regulatory element from human thymidylate synthase mRNA

    SciTech Connect

    Dibrov, Sergey; McLean, Jaime; Hermann, Thomas

    2011-09-27

    A sequence around the start codon of the mRNA of human thymidylate synthase (TS) folds into a secondary-structure motif in which the initiation site is sequestered in a metastable hairpin. Binding of the protein to its own mRNA at the hairpin prevents the production of TS through a translation-repression feedback mechanism. Stabilization of the mRNA hairpin by other ligands has been proposed as a strategy to reduce TS levels in anticancer therapy. Rapidly proliferating cells require high TS activity to maintain the production of thymidine as a building block for DNA synthesis. The crystal structure of a model oligonucleotide (TS1) that represents the TS-binding site of the mRNA has been determined. While fluorescence studies showed that the TS1 RNA preferentially adopts a hairpin structure in solution, even at high RNA concentrations, an asymmetric dimer of two hybridized TS1 strands was obtained in the crystal. The TS1 dimer contains an unusual S-turn motif that also occurs in the 'off' state of the human ribosomal decoding site RNA.

  5. Combinatorics of saturated secondary structures of RNA.

    PubMed

    Clote, P

    2006-11-01

    Following Zuker (1986), a saturated secondary structure for a given RNA sequence is a secondary structure such that no base pair can be added without violating the definition of secondary structure, e.g., without introducing a pseudoknot. In the Nussinov-Jacobson energy model (Nussinov and Jacobson, 1980), where the energy of a secondary structure is -1 times the number of base pairs, saturated secondary structures are local minima in the energy landscape, hence form kinetic traps during the folding process. Here we present recurrence relations and closed form asymptotic limits for combinatorial problems related to the number of saturated secondary structures. In addition, Python source code to compute the number of saturated secondary structures having k base pairs can be found at the web servers link of bioinformatics.bc.edu/clotelab/.

  6. Using DNA sequencing electrophoresis compression artifacts as reporters of stable mRNA structures affecting gene expression.

    PubMed

    Kapoor, Divya; Chandrayan, Sanjeev Kumar; Ahmed, Shubbir; Guptasarma, Purnananda

    2007-11-01

    The formation of secondary structure in oligonucleotide DNA is known to lead to "compression" artifacts in electropherograms produced through DNA sequencing. Separately, the formation of secondary structure in mRNA is known to suppress translation; in particular, when such structures form in a region covered by the ribosome either during, or shortly after, initiation of translation. Here, we demonstrate how a DNA sequencing compression artifact provides important clues to the location(s) of translation-suppressing secondary structural elements in mRNA. Our study involves an engineered version of a gene sourced from Rhodothermus marinus encoding an enzyme called Cel12A. We introduced this gene into Escherichia coli with the intention of overexpressing it, but found that it expressed extremely poorly. Intriguingly, the gene displayed a remarkable compression artifact during DNA sequencing electrophoresis. Selected "designer" silent mutations destroyed the artifact. They also simultaneously greatly enhanced the expression of the cel12A gene, presumably by destroying stable mRNA structures that otherwise suppress translation. We propose that this method of finding problem mRNA sequences is superior to software-based analyses, especially if combined with low-temperature CE.

  7. Systematic Discovery of Structural Elements Governing Mammalian mRNA Stability

    PubMed Central

    Goodarzi, Hani; Najafabadi, Hamed S.; Oikonomou, Panos; Greco, Todd M.; Fish, Lisa; Salavati, Reza; Cristea, Ileana M.; Tavazoie, Saeed

    2012-01-01

    Decoding post-transcriptional regulatory programs in RNA is a critical step in the larger goal to develop predictive dynamical models of cellular behavior. Despite recent efforts1–3, the vast landscape of RNA regulatory elements remain largely uncharacterized. A longstanding obstacle is the contribution of local RNA secondary structure in defining interaction partners in a variety of regulatory contexts, including but not limited to transcript stability3, alternative splicing4 and localization3. There are many documented instances where the presence of a structural regulatory element dictates alternative splicing patterns (e.g. human cardiac troponin T) or affects other aspects of RNA biology5. Thus, a full characterization of post-transcriptional regulatory programs requires capturing information provided by both local secondary structures and the underlying sequence3,6. We have developed a computational framework based on context-free grammars3,7 and mutual information2 that systematically explores the immense space of small structural elements and reveals motifs that are significantly informative of genome-wide measurements of RNA behavior. The application of this framework to genome-wide mammalian mRNA stability data revealed eight highly significant elements with substantial structural information, for the strongest of which we showed a major role in global mRNA regulation. Through biochemistry, mass-spectrometry, and in vivo binding studies, we identified HNRPA2B1 as the key regulator that binds this element and stabilizes a large number of its target genes. Ultimately, we created a global post-transcriptional regulatory map based on the identity of the discovered linear and structural cis-regulatory elements, their regulatory interactions and their target pathways. This approach can also be employed to reveal the structural elements that modulate other aspects of RNA behavior. PMID:22495308

  8. Improving RNA secondary structure prediction with structure mapping data.

    PubMed

    Sloma, Michael F; Mathews, David H

    2015-01-01

    Methods to probe RNA secondary structure, such as small molecule modifying agents, secondary structure-specific nucleases, inline probing, and SHAPE chemistry, are widely used to study the structure of functional RNA. Computational secondary structure prediction programs can incorporate probing data to predict structure with high accuracy. In this chapter, an overview of current methods for probing RNA secondary structure is provided, including modern high-throughput methods. Methods for guiding secondary structure prediction algorithms using these data are explained, and best practices for using these data are provided. This chapter concludes by listing a number of open questions about how to best use probing data, and what these data can provide.

  9. Genetic analysis of bacteriophage lambda cIII gene: mRNA structural requirements for translation initiation.

    PubMed Central

    Kornitzer, D; Teff, D; Altuvia, S; Oppenheim, A B

    1989-01-01

    The bacteriophage lambda cIII gene product regulates the lysogenic pathway. The cIII gene is located in the leftward operon, which is transcribed from the pL promoter. We have previously shown (S. Altuvia and A. B. Oppenheim, J. Bacteriol. 167:415-419, 1986) that mutations that show elevated expression lie within the cIII coding sequence. We isolated mutants that show decreased CIII activity. All the mutations were found to cause a drastic reduction in the rate of initiation of cIII translation. Several mutations were found to be scattered within the first 40 nucleotides of the cIII coding region. Additional mutations affected the AUG initiation codon, the Shine-Dalgarno sequence, and the upstream RNaseIII processing site. Computer folding of the cIII mRNA suggested the presence of two alternative RNA structures. All the mutations within the coding region that reduce expression reduce the stability of one specific mRNA structure (structure B). Mutations that increase expression lie in the loops of this structure and may in fact stabilize it by interfering with the formation of the alternative structure (structure A). Thus, it appears that a specific mRNA secondary structure at the beginning of the cIII coding region is essential for efficient translation, suggesting that changes in mRNA structure regulate cIII expression. Images PMID:2523380

  10. An algebraic representation of RNA secondary structures.

    PubMed

    Magarshak, Y; Benham, C J

    1992-12-01

    This paper develops mathematical methods for describing and analyzing RNA secondary structures. It was motivated by the need to develop rigorous yet efficient methods to treat transitions from one secondary structure to another, which we propose here may occur as motions of loops within RNAs having appropriate sequences. In this approach a molecular sequence is described as a vector of the appropriate length. The concept of symmetries between nucleic acid sequences is developed, and the 48 possible different types of symmetries are described. Each secondary structure possible for a particular nucleotide sequence determines a symmetric, signed permutation matrix. The collection of all possible secondary structures is comprised of all matrices of this type whose left multiplication with the sequence vector leaves that vector unchanged. A transition between two secondary structures is given by the product of the two corresponding structure matrices. This formalism provides an efficient method for describing nucleic acid sequences that allows questions relating to secondary structures and transitions to be addressed using the powerful methods of abstract algebra. In particular, it facilitates the determination of possible secondary structures, including those containing pseudoknots. Although this paper concentrates on RNA structure, this formalism also can be applied to DNA. PMID:1283516

  11. Combinatorics of locally optimal RNA secondary structures.

    PubMed

    Fusy, Eric; Clote, Peter

    2014-01-01

    It is a classical result of Stein and Waterman that the asymptotic number of RNA secondary structures is 1.104366∙n-3/2∙2.618034n. Motivated by the kinetics of RNA secondary structure formation, we are interested in determining the asymptotic number of secondary structures that are locally optimal, with respect to a particular energy model. In the Nussinov energy model, where each base pair contributes -1 towards the energy of the structure, locally optimal structures are exactly the saturated structures, for which we have previously shown that asymptotically, there are 1.07427∙n-3/2∙2.35467n many saturated structures for a sequence of length n. In this paper, we consider the base stacking energy model, a mild variant of the Nussinov model, where each stacked base pair contributes -1 toward the energy of the structure. Locally optimal structures with respect to the base stacking energy model are exactly those secondary structures, whose stems cannot be extended. Such structures were first considered by Evers and Giegerich, who described a dynamic programming algorithm to enumerate all locally optimal structures. In this paper, we apply methods from enumerative combinatorics to compute the asymptotic number of such structures. Additionally, we consider analogous combinatorial problems for secondary structures with annotated single-stranded, stacking nucleotides (dangles).

  12. Combinatorics of locally optimal RNA secondary structures.

    PubMed

    Fusy, Eric; Clote, Peter

    2014-01-01

    It is a classical result of Stein and Waterman that the asymptotic number of RNA secondary structures is 1.104366∙n-3/2∙2.618034n. Motivated by the kinetics of RNA secondary structure formation, we are interested in determining the asymptotic number of secondary structures that are locally optimal, with respect to a particular energy model. In the Nussinov energy model, where each base pair contributes -1 towards the energy of the structure, locally optimal structures are exactly the saturated structures, for which we have previously shown that asymptotically, there are 1.07427∙n-3/2∙2.35467n many saturated structures for a sequence of length n. In this paper, we consider the base stacking energy model, a mild variant of the Nussinov model, where each stacked base pair contributes -1 toward the energy of the structure. Locally optimal structures with respect to the base stacking energy model are exactly those secondary structures, whose stems cannot be extended. Such structures were first considered by Evers and Giegerich, who described a dynamic programming algorithm to enumerate all locally optimal structures. In this paper, we apply methods from enumerative combinatorics to compute the asymptotic number of such structures. Additionally, we consider analogous combinatorial problems for secondary structures with annotated single-stranded, stacking nucleotides (dangles). PMID:23263300

  13. Pairwise amino acid secondary structural propensities

    NASA Astrophysics Data System (ADS)

    Chemmama, Ilan E.; Chapagain, Prem P.; Gerstman, Bernard S.

    2015-04-01

    We investigate the propensities for amino acids to form a specific secondary structure when they are paired with other amino acids. Our investigations use molecular dynamics (MD) computer simulations, and we compare the results to those from the Protein Data Bank (PDB). Proper comparison requires weighting of the MD results in a manner consistent with the relative frequency of appearance in the PDB of each possible pair of amino acids. We find that the propensity for an amino acid to assume a secondary structure varies dramatically depending on the amino acid that is before or after it in the primary sequence. This cooperative effect means that when selecting amino acids to facilitate the formation of a secondary structure in peptide engineering experiments, the adjacent amino acids must be considered. We also examine the preference for a secondary structure in bacterial proteins and compare the results to those of human proteins.

  14. Current perspectives on RNA secondary structure probing.

    PubMed

    Kenyon, Julia; Prestwood, Liam; Lever, Andrew

    2014-08-01

    The range of roles played by structured RNAs in biological systems is vast. At the same time as we are learning more about the importance of RNA structure, recent advances in reagents, methods and technology mean that RNA secondary structural probing has become faster and more accurate. As a result, the capabilities of laboratories that already perform this type of structural analysis have increased greatly, and it has also become more widely accessible. The present review summarizes established and recently developed techniques. The information we can derive from secondary structural analysis is assessed, together with the areas in which we are likely to see exciting developments in the near future. PMID:25110033

  15. Coupling of mRNA Structure Rearrangement to Ribosome Movement during Bypassing of Non-coding Regions.

    PubMed

    Chen, Jin; Coakley, Arthur; O'Connor, Michelle; Petrov, Alexey; O'Leary, Seán E; Atkins, John F; Puglisi, Joseph D

    2015-11-19

    Nearly half of the ribosomes translating a particular bacteriophage T4 mRNA bypass a region of 50 nt, resuming translation 3' of this gap. How this large-scale, specific hop occurs and what determines whether a ribosome bypasses remain unclear. We apply single-molecule fluorescence with zero-mode waveguides to track individual Escherichia coli ribosomes during translation of T4's gene 60 mRNA. Ribosomes that bypass are characterized by a 10- to 20-fold longer pause in a non-canonical rotated state at the take-off codon. During the pause, mRNA secondary structure rearrangements are coupled to ribosome forward movement, facilitated by nascent peptide interactions that disengage the ribosome anticodon-codon interactions for slippage. Close to the landing site, the ribosome then scans mRNA in search of optimal base-pairing interactions. Our results provide a mechanistic and conformational framework for bypassing, highlighting a non-canonical ribosomal state to allow for mRNA structure refolding to drive large-scale ribosome movements. PMID:26590426

  16. RNA Secondary Structure Determination by NMR.

    PubMed

    Chen, Jonathan L; Bellaousov, Stanislav; Turner, Douglas H

    2016-01-01

    Dynamic programming methods for predicting RNA secondary structure often use thermodynamics and experimental restraints and/or constraints to limit folding space. Chemical mapping results typically restrain certain nucleotides not to be in AU or GC pairs. Two-dimensional nuclear magnetic resonance (NMR) spectra can reveal the order of AU, GC, and GU pairs in double helixes. This chapter describes a program, NMR-assisted prediction of secondary structure and chemical shifts (NAPSS-CS), that constrains possible secondary structures on the basis of the NMR determined order and 5'-3' direction of AU, GC, and GU pairs in helixes. NAPSS-CS minimally requires input of the order of base pairs as determined from nuclear Overhauser effect spectroscopy (NOESY) of imino protons. The program deduces the 5'-3' direction of the base pairs if certain chemical shifts are also input. Secondary structures predicted by the program provide assignments of input chemical shifts to particular nucleotides in the sequence, thus facilitating an important step for determination of the three dimensional structure by NMR. The method is particularly useful for revealing pseudoknots and an example is provided. The method may also allow determination of secondary structures when a sequence folds into two structures that exchange slowly. PMID:27665599

  17. RNA Secondary Structure Analysis Using RNAstructure.

    PubMed

    Mathews, David H

    2014-06-17

    RNAstructure is a user-friendly program for the prediction and analysis of RNA secondary structure. It is available as a Web server, as a program with a graphical user interface, or as a set of command-line tools. The programs are available for Microsoft Windows, Macintosh OS X, or Linux. This unit provides protocols for RNA secondary structure prediction (using the Web server or the graphical user interface) and prediction of high-affinity oligonucleotide biding sites to a structured RNA target (using the graphical user interface).

  18. Sequence and structure-specific elements of HERG mRNA determine channel synthesis and trafficking efficiency

    PubMed Central

    Sroubek, Jakub; Krishnan, Yamini; McDonald, Thomas V.

    2013-01-01

    Human ether-á-gogo-related gene (HERG) encodes a potassium channel that is highly susceptible to deleterious mutations resulting in susceptibility to fatal cardiac arrhythmias. Most mutations adversely affect HERG channel assembly and trafficking. Why the channel is so vulnerable to missense mutations is not well understood. Since nothing is known of how mRNA structural elements factor in channel processing, we synthesized a codon-modified HERG cDNA (HERG-CM) where the codons were synonymously changed to reduce GC content, secondary structure, and rare codon usage. HERG-CM produced typical IKr-like currents; however, channel synthesis and processing were markedly different. Translation efficiency was reduced for HERG-CM, as determined by heterologous expression, in vitro translation, and polysomal profiling. Trafficking efficiency to the cell surface was greatly enhanced, as assayed by immunofluorescence, subcellular fractionation, and surface labeling. Chimeras of HERG-NT/CM indicated that trafficking efficiency was largely dependent on 5′ sequences, while translation efficiency involved multiple areas. These results suggest that HERG translation and trafficking rates are independently governed by noncoding information in various regions of the mRNA molecule. Noncoding information embedded within the mRNA may play a role in the pathogenesis of hereditary arrhythmia syndromes and could provide an avenue for targeted therapeutics.—Sroubek, J., Krishnan, Y., McDonald, T V. Sequence- and structure-specific elements of HERG mRNA determine channel synthesis and trafficking efficiency. PMID:23608144

  19. General combinatorics of RNA secondary structure.

    PubMed

    Liao, Bo; Wang, Tian-ming

    2004-09-01

    The total number of RNA secondary structures of a given length with minimal hairpin loop length m(m>0) and with minimal stack length l(l>0) is computed, under the assumption that all base pairs can occur. Asymptotics are derived from the determination of recurrence relations of decomposition properties.

  20. Protein secondary structural types are differentially coded on messenger RNA.

    PubMed Central

    Thanaraj, T. A.; Argos, P.

    1996-01-01

    Tricodon regions on messenger RNAs corresponding to a set of proteins from Escherichia coli were scrutinized for their translation speed. The fractional frequency values of the individual codons as they occur in mRNAs of highly expressed genes from Escherichia coli were taken as an indicative measure of the translation speed. The tricodons were classified by the sum of the frequency values of the constituent codons. Examination of the conformation of the encoded amino acid residues in the corresponding protein tertiary structures revealed a correlation between codon usage in mRNA and topological features of the encoded proteins. Alpha helices on proteins tend to be preferentially coded by translationally fast mRNA regions while the slow segments often code for beta strands and coil regions. Fast regions correspondingly avoid coding for beta strands and coil regions while the slow regions similarly move away from encoding alpha helices. Structural and mechanistic aspects of the ribosome peptide channel support the relevance of sequence fragment translation and subsequent conformation. A discussion is presented relating the observation to the reported kinetic data on the formation and stabilization of protein secondary structural types during protein folding. The observed absence of such strong positive selection for codons in non-highly expressed genes is compatible with existing theories that mutation pressure may well dominate codon selection in non-highly expressed genes. PMID:8897597

  1. Folding and Finding RNA Secondary Structure

    PubMed Central

    Mathews, David H.; Moss, Walter N.; Turner, Douglas H.

    2010-01-01

    SUMMARY Optimal exploitation of the expanding database of sequences requires rapid finding and folding of RNAs. Methods are reviewed that automate folding and discovery of RNAs with algorithms that couple thermodynamics with chemical mapping, NMR, and/or sequence comparison. New functional noncoding RNAs in genome sequences can be found by combining sequence comparison with the assumption that functional noncoding RNAs will have more favorable folding free energies than other RNAs. When a new RNA is discovered, experiments and sequence comparison can restrict folding space so that secondary structure can be rapidly determined with the help of predicted free energies. In turn, secondary structure restricts folding in three dimensions, which allows modeling of three-dimensional structure. An example from a domain of a retrotransposon is described. Discovery of new RNAs and their structures will provide insights into evolution, biology, and design of therapeutics. Applications to studies of evolution are also reviewed. PMID:20685845

  2. Secondary structure formation in peptide amphiphile micelles

    NASA Astrophysics Data System (ADS)

    Tirrell, Matthew

    2012-02-01

    Peptide amphiphiles (PAs) are capable of self-assembly into micelles for use in the targeted delivery of peptide therapeutics and diagnostics. PA micelles exhibit a structural resemblance to proteins by having folded bioactive peptides displayed on the exterior of a hydrophobic core. We have studied two factors that influence PA secondary structure in micellar assemblies: the length of the peptide headgroup and amino acids closest to the micelle core. Peptide length was systematically varied using a heptad repeat PA. For all PAs the addition of a C12 tail induced micellization and secondary structure. PAs with 9 amino acids formed beta-sheet interactions upon aggregation, whereas the 23 and 30 residue peptides were displayed in an apha-helical conformation. The 16 amino acid PA experienced a structural transition from helix to sheet, indicating that kinetics play a role in secondary structure formation. A p53 peptide was conjugated to a C16 tail via various linkers to study the effect of linker chemistry on PA headgroup conformation. With no linker the p53 headgroup was predominantly alpha helix and a four alanine linker drastically changed the structure of the peptide headgroup to beta-sheet, highlighting the importance of hydrogen boding potential near the micelle core.

  3. PSS-SQL: protein secondary structure - structured query language.

    PubMed

    Mrozek, Dariusz; Wieczorek, Dominika; Malysiak-Mrozek, Bozena; Kozielski, Stanislaw

    2010-01-01

    Secondary structure representation of proteins provides important information regarding protein general construction and shape. This representation is often used in protein similarity searching. Since existing commercial database management systems do not offer integrated exploration methods for biological data e.g. at the level of the SQL language, the structural similarity searching is usually performed by external tools. In the paper, we present our newly developed PSS-SQL language, which allows searching a database in order to identify proteins having secondary structure similar to the structure specified by the user in a PSS-SQL query. Therefore, we provide a simple and declarative language for protein structure similarity searching.

  4. Structural perspectives on secondary active transporters

    PubMed Central

    Boudker, Olga; Verdon, Grégory

    2010-01-01

    Secondary active transporters catalyze concentrative transport of substrates across lipid membranes by harnessing the energy of electrochemical ion gradients. These transporters bind their ligands on one side of the membrane, and undergo a global conformational change to release them on the other side of the membrane. Over the last few years, crystal structures have captured several bacterial secondary transporters in different states along their transport cycle, providing insight into possible molecular mechanisms. In this review, we will summarize recent findings focusing on the emerging structural and mechanistic similarities between evolutionary diverse transporters. We will also discuss the structural basis of substrate binding, ion coupling and inhibition viewed from the perspective of these similarities. PMID:20655602

  5. Hfq assists small RNAs in binding to the coding sequence of ompD mRNA and in rearranging its structure

    PubMed Central

    Wroblewska, Zuzanna; Olejniczak, Mikolaj

    2016-01-01

    The bacterial protein Hfq participates in the regulation of translation by small noncoding RNAs (sRNAs). Several mechanisms have been proposed to explain the role of Hfq in the regulation by sRNAs binding to the 5′-untranslated mRNA regions. However, it remains unknown how Hfq affects those sRNAs that target the coding sequence. Here, the contribution of Hfq to the annealing of three sRNAs, RybB, SdsR, and MicC, to the coding sequence of Salmonella ompD mRNA was investigated. Hfq bound to ompD mRNA with tight, subnanomolar affinity. Moreover, Hfq strongly accelerated the rates of annealing of RybB and MicC sRNAs to this mRNA, and it also had a small effect on the annealing of SdsR. The experiments using truncated RNAs revealed that the contributions of Hfq to the annealing of each sRNA were individually adjusted depending on the structures of interacting RNAs. In agreement with that, the mRNA structure probing revealed different structural contexts of each sRNA binding site. Additionally, the annealing of RybB and MicC sRNAs induced specific conformational changes in ompD mRNA consistent with local unfolding of mRNA secondary structure. Finally, the mutation analysis showed that the long AU-rich sequence in the 5′-untranslated mRNA region served as an Hfq binding site essential for the annealing of sRNAs to the coding sequence. Overall, the data showed that the functional specificity of Hfq in the annealing of each sRNA to the ompD mRNA coding sequence was determined by the sequence and structure of the interacting RNAs. PMID:27154968

  6. Prediction of RNA secondary structures with pseudoknots

    NASA Astrophysics Data System (ADS)

    Bon, M.; Orland, H.

    2010-08-01

    We present a new algorithm to predict RNA secondary structures with pseudoknots. The method is based on a classification of RNA structures according to their topological genus. The algorithm utilizes a simplified parametrization of the free energies for pair stacking, loop penalties, etc. and in addition a free energy penalty proportional to the topological genus of the pairing graph. Our method can take into account all pseudoknot topologies and achieves high success rates compared to state-of-the-art methods. This shows that the genus is a promising concept to classify pseudoknots.

  7. RNA secondary structure prediction using soft computing.

    PubMed

    Ray, Shubhra Sankar; Pal, Sankar K

    2013-01-01

    Prediction of RNA structure is invaluable in creating new drugs and understanding genetic diseases. Several deterministic algorithms and soft computing-based techniques have been developed for more than a decade to determine the structure from a known RNA sequence. Soft computing gained importance with the need to get approximate solutions for RNA sequences by considering the issues related with kinetic effects, cotranscriptional folding, and estimation of certain energy parameters. A brief description of some of the soft computing-based techniques, developed for RNA secondary structure prediction, is presented along with their relevance. The basic concepts of RNA and its different structural elements like helix, bulge, hairpin loop, internal loop, and multiloop are described. These are followed by different methodologies, employing genetic algorithms, artificial neural networks, and fuzzy logic. The role of various metaheuristics, like simulated annealing, particle swarm optimization, ant colony optimization, and tabu search is also discussed. A relative comparison among different techniques, in predicting 12 known RNA secondary structures, is presented, as an example. Future challenging issues are then mentioned. PMID:23702539

  8. Automatic display of RNA secondary structures.

    PubMed

    Muller, G; Gaspin, C; Etienne, A; Westhof, E

    1993-10-01

    A set of programs written in C language with the GL library and under UNIX has been developed for generating compact, pleasant and non-overlapping displays of secondary structures of ribonucleic acids. The first program, rnasearch, implements a new search procedure that dynamically rearranges overlapping portions of the two-dimensional drawing while preserving clear and readable displays of the two-dimensional structure. The algorithm is fast (the execution time for the command rnasearch is 38.6 s for the 16S rRNA of Escherichia coli with 1542 bases), accepts outputs from two-dimensional prediction programs and therefore allows for rapid comparison between the various two-dimensional folds generated. A second program, rnadisplay, allows the graphical display of the computed two-dimensional structures on a graphics workstation. Otherwise, it is possible to obtain a paper output of the two-dimensional structure by using the program print2D which builds a Postscript file. Moreover the two-dimensional drawing can be labelled for representing data coming from chemical modifications and/or enzymatic cleavages. Application to a few secondary structures such as RNaseP, 5S rRNA and 16S rRNA are given.

  9. Pattern recognition in nucleic acid sequences. II. An efficient method for finding locally stable secondary structures.

    PubMed Central

    Kanehisa, M I; Goad, W B

    1982-01-01

    We present a method for calculating all possible single hairpin loop secondary structures in a nucleic acid sequence by the order of N2 operations where N is the total number of bases. Each structure may contain any number of bulges and internal loops. Most natural sequences are found to be indistinguishable from random sequences in the potential of forming secondary structures, which is defined by the frequency of possible secondary structures calculated by the method. There is a strong correlation between the higher G+C content and the higher structure forming potential. Interestingly, the removal of intervening sequences in mRNAs is almost always accompanied by an increase in the G+C content, which may suggest an involvement of structural stabilization in the mRNA maturation. PMID:6174936

  10. Secondary flow structures in large rivers

    NASA Astrophysics Data System (ADS)

    Chauvet, H.; Devauchelle, O.; Metivier, F.; Limare, A.; Lajeunesse, E.

    2012-04-01

    Measuring the velocity field in large rivers remains a challenge, even with recent measurement techniques such as Acoustic Doppler Current Profiler (ADCP). Indeed, due to the diverging angle between its ultrasonic beams, an ADCP cannot detect small-scale flow structures. However, when the measurements are limited to a single location for a sufficient period of time, averaging can reveal large, stationary flow structures. Here we present velocity measurements in a straight reach of the Seine river in Paris, France, where the cross-section is close to rectangular. The transverse modulation of the streamwise velocity indicates secondary flow cells, which seem to occupy the entire width of the river. This observation is reminiscent of the longitudinal vortices observed in laboratory experiments (e.g. Blanckaert et al., Advances in Water Resources, 2010, 33, 1062-1074). Although the physical origin of these secondary structures remains unclear, their measured velocity is sufficient to significantly impact the distribution of streamwise momentum. We propose a model for the transverse profile of the depth-averaged velocity based on a crude representation of the longitudinal vortices, with a single free parameter. Preliminary results are in good agreement with field measurements. This model also provides an estimate for the bank shear stress, which controls bank erosion.

  11. Prediction of packing of secondary structure.

    PubMed

    Nagano, K; Ponnuswamy, P K

    1984-01-01

    An improved method of picking up candidates for predicting the packing arrangement of beta-strands and alpha-helices of the alpha/beta type domains is described here. The method of judging whether the region of the protein would fold into the alpha/beta type or not is also described. The folding constraints of globular proteins are analysed and presented in this article for application to the prediction of packing of secondary structure. The analysis of the residue-fluctuations is also applicable for the purpose.

  12. Experiment-Assisted Secondary Structure Prediction with RNAstructure.

    PubMed

    Xu, Zhenjiang Zech; Mathews, David H

    2016-01-01

    Experimental probing data can be used to improve the accuracy of RNA secondary structure prediction. The software package RNAstructure can take advantage of enzymatic cleavage data, FMN cleavage data, traditional chemical modification reactivity data, and SHAPE reactivity data for secondary structure modeling. This chapter provides protocols for using experimental probing data with RNAstructure to restrain or constrain RNA secondary structure prediction. PMID:27665598

  13. The mRNA of the translationally controlled tumor protein P23/TCTP is a highly structured RNA, which activates the dsRNA-dependent protein kinase PKR.

    PubMed Central

    Bommer, Ulrich-Axel; Borovjagin, Anton V; Greagg, Martin A; Jeffrey, Ian W; Russell, Paul; Laing, Kenneth G; Lee, Melanie; Clemens, Michael J

    2002-01-01

    The dsRNA-activated protein kinase PKR is involved in signal transduction pathways that mediate cellular processes as diverse as cell growth and differentiation, the stress response, and apoptosis. PKR was originally described as an interferon-inducible elF2alpha kinase involved in the antiviral defense mechanism of the cell. The interaction of the kinase with specific viral RNAs has been studied in much detail, but information about cellular mRNAs, which are able to bind and activate PKR, is scarce. In search for such cellular mRNAs, we developed a cloning strategy to identify individual mRNA species from the dsRNA-rich fraction of Daudi cell poly(A)+ RNA. Two out of five cDNA clones we obtained contained sequences derived from the mRNA of the translationally controlled tumor protein P23/TCTP, indicating that this mRNA is present in the dsRNA-rich fraction. Secondary structure predictions and gel electrophoretic mobility investigations on P23/TCTP transcripts confirmed the potential of this mRNA to form extensive secondary structure. A full-length P23 transcript, but not a truncated version thereof, was able to bind to PKR in vitro and in vivo. Transient transfection experiments in human 293 cells showed that coexpression of full-length P23 mRNA leads to partial inhibition of the expression of a beta-galactosidase reporter gene in trans. Additional coexpression of a dominant negative mutant of PKR or of adenovirus VA1 RNA suppressed this inhibition, indicating that it is mediated by PKR. Studies on P23/TCTP expression in cells from PKR-knockout mice suggest that P23/TCTP mRNA translation is regulated by PKR. Hence, our results demonstrate that the mRNA of P23/TCTP may both activate PKR and be subject to translational regulation by this kinase. PMID:11991642

  14. Circular dichroism and DNA secondary structure.

    PubMed

    Baase, W A; Johnson, W C

    1979-02-01

    The change in average rotation of the DNA helix has been determined for the transfer from 0.05 M NaCl to 3.0 M CsCl, 6.2 M LiCl and 5.4 M NH4Cl. This work, combined with data at lower salt from other laboratories, allows us to relate the intensity of the CD of DNA at 275 nm directly to the change in the number of base pairs per turn. The change in secondary structure for the transfer of DNA from 0.05 M NaCl (where it is presumably in the B-form) to high salt (where the characteristic CD has been interpreted as corresponding to C-form geometry) is found to be -0.22 (+/- 0.02) base pairs per turn. In the case of mononucleosomes, where the CD indicates the "C-form", the change in secondary structure (including temperature effects) would add -0.31 (+/- 0.03) turns about the histone core to the -1.25 turns estimated from work on SV40 chromatin. Accurate winding angles and molar extinction coefficients were determined for ethidium.

  15. The ribosome structure controls and directs mRNA entry, translocation and exit dynamics

    NASA Astrophysics Data System (ADS)

    Kurkcuoglu, Ozge; Doruker, Pemra; Sen, Taner Z.; Kloczkowski, Andrzej; Jernigan, Robert L.

    2008-12-01

    The protein-synthesizing ribosome undergoes large motions to effect the translocation of tRNAs and mRNA; here, the domain motions of this system are explored with a coarse-grained elastic network model using normal mode analysis. Crystal structures are used to construct various model systems of the 70S complex with/without tRNA, elongation factor Tu and the ribosomal proteins. Computed motions reveal the well-known ratchet-like rotational motion of the large subunits, as well as the head rotation of the small subunit and the high flexibility of the L1 and L7/L12 stalks, even in the absence of ribosomal proteins. This result indicates that these experimentally observed motions during translocation are inherently controlled by the ribosomal shape and only partially dependent upon GTP hydrolysis. Normal mode analysis further reveals the mobility of A- and P-tRNAs to increase in the absence of the E-tRNA. In addition, the dynamics of the E-tRNA is affected by the absence of the ribosomal protein L1. The mRNA in the entrance tunnel interacts directly with helicase proteins S3 and S4, which constrain the mRNA in a clamp-like fashion, as well as with protein S5, which likely orients the mRNA to ensure correct translation. The ribosomal proteins S7, S11 and S18 may also be involved in assuring translation fidelity by constraining the mRNA at the exit site of the channel. The mRNA also interacts with the 16S 3' end forming the Shine-Dalgarno complex at the initiation step; the 3' end may act as a 'hook' to reel in the mRNA to facilitate its exit.

  16. Circumstances and mechanisms of inhibition of translation by secondary structure in eucaryotic mRNAs.

    PubMed Central

    Kozak, M

    1989-01-01

    This paper describes in vitro experiments with two types of intramolecular duplex structures that inhibit translation in cis by preventing the formation of an initiation complex or by causing the complex to be abortive. One stem-loop structure (delta G = -30 kcal/mol) prevented mRNA from engaging 40S subunits when the hairpin occurred 12 nucleotides (nt) from the cap but had no deleterious effect when it was repositioned 52 nt from the cap. This result confirms prior in vivo evidence that the 40S subunit-factor complex, once bound to mRNA, has considerable ability to penetrate secondary structure. Consequently, translation is most sensitive to secondary structure at the entry site for ribosomes, i.e., the 5' end of the mRNA. The second stem-loop structure (hp7; delta G = -61 kcal/mol, located 72 nt from the cap) was too stable to be unwound by 40S ribosomes, hp7 did not prevent a 40S ribosomal subunit from binding but caused the 40S subunit to stall on the 5' side of the hairpin, exactly as the scanning model predicts. Control experiments revealed that 80S elongating ribosomes could disrupt duplex structures, such as hp7, that were too stable to be penetrated by the scanning 40S ribosome-factor complex. A third type of base-paired structure shown to inhibit translation in vivo involves a long-range interaction between the 5' and 3' noncoding sequences. Images PMID:2601712

  17. Maximum expected accuracy structural neighbors of an RNA secondary structure

    PubMed Central

    2012-01-01

    Background Since RNA molecules regulate genes and control alternative splicing by allostery, it is important to develop algorithms to predict RNA conformational switches. Some tools, such as paRNAss, RNAshapes and RNAbor, can be used to predict potential conformational switches; nevertheless, no existent tool can detect general (i.e., not family specific) entire riboswitches (both aptamer and expression platform) with accuracy. Thus, the development of additional algorithms to detect conformational switches seems important, especially since the difference in free energy between the two metastable secondary structures may be as large as 15-20 kcal/mol. It has recently emerged that RNA secondary structure can be more accurately predicted by computing the maximum expected accuracy (MEA) structure, rather than the minimum free energy (MFE) structure. Results Given an arbitrary RNA secondary structure S0 for an RNA nucleotide sequence a = a1,..., an, we say that another secondary structure S of a is a k-neighbor of S0, if the base pair distance between S0 and S is k. In this paper, we prove that the Boltzmann probability of all k-neighbors of the minimum free energy structure S0 can be approximated with accuracy ε and confidence 1 - p, simultaneously for all 0 ≤ k < K, by a relative frequency count over N sampled structures, provided that N>N(ε,p,K)=Φ-1p2K24ε2, where Φ(z) is the cumulative distribution function (CDF) for the standard normal distribution. We go on to describe the algorithm RNAborMEA, which for an arbitrary initial structure S0 and for all values 0 ≤ k < K, computes the secondary structure MEA(k), having maximum expected accuracy over all k-neighbors of S0. Computation time is O(n3 · K2), and memory requirements are O(n2 · K). We analyze a sample TPP riboswitch, and apply our algorithm to the class of purine riboswitches. Conclusions The approximation of RNAbor by sampling, with rigorous bound on accuracy, together with the computation of

  18. Enumeration of Secondary Structure Element Bundles

    SciTech Connect

    Brown, William Michael; Faulon, Jean-Loup

    2004-10-26

    A deterministic algorithm for enumeration of transmembrane protein folds is implemented. Using a set of sparse pairwise atomic distance constraints (such as those obtained from chemical cross-linking, FRET, or dipolar EPR experiments), the algorithm performs an exhaustive search of secondary structure element packing conformations distributed throughout the entire conformational space. The end result is a set of distinct protein conformations which can be scored and refined as part of a process designed for computational elucidation of transmembrane protein structures. Algorithm Overview: The ESSEB algorithm works by dividing the conforrnational space of each secondary structure element (SSE) into a set of cells. For each cell there is a representative conformation and for each atom in the SSE for which a distance restraint is available, there is an associated internal error, The internal error for a distance restraint is the maximum distance that the atom, when positioned in any conformation within a cell, can be from the atom in the representative conformation. The algorithm works recursively by positioning one representative conformation of an SSE. AdI distance restraints are checked with a tolerance that includes both the experimental and internal error. If all restraints are satisfied, every representative conformation of the next SSE is checked, otherwise, the program moves on to the next representative conformation of the current SSE. In addition to the distance restraints, other constraints on protein conformation can be enforced. These include the distance of closest approach between SSE axes, a restraint which prevents the crossover of loops connecting adjacent SSEs, and a restriction on the minimum and maximum distances between axis end-points. Any protein conformation satisfying all of the restraints is enumerated for later scoring and possible refinement. Additionally, in order to make run-times feasible, a divide-and-conquer approach is used in which

  19. Hydrogen bonding in peptide secondary structures

    NASA Astrophysics Data System (ADS)

    Varga, Zoltán; Kovács, Attila

    Hydrogen bonding interactions in various peptide secondary structures (β-sheet, 27-ribbon, 310-helix, α-helix, π-helix, β-turn II, and γ-turn) have been investigated in small oligopeptides by quantum chemical calculations at the B3LYP/6-31G** level. Besides the primary O...HN interactions, the optimized structures revealed the importance of N...HN hydrogen bonding in several structures. The effect of substitution on the energy and structural properties was investigated comparing the properties of glycine, alanine, valine, and serine. The aliphatic substituents generally weaken the hydrogen bonds, the strongest effects being observed in crowded valine conformers. Additional hydrogen bonding interactions introduced by the OH group of serine can both strengthen (by polarizing the amide moiety through N...H interaction) and weaken (constraining the CO oxygen by O...HO interaction) the backbone hydrogen bonds. The effect of water as a polarizable medium on the energy properties was assessed by the COSMO model.

  20. RNA-SSPT: RNA Secondary Structure Prediction Tools.

    PubMed

    Ahmad, Freed; Mahboob, Shahid; Gulzar, Tahsin; Din, Salah U; Hanif, Tanzeela; Ahmad, Hifza; Afzal, Muhammad

    2013-01-01

    The prediction of RNA structure is useful for understanding evolution for both in silico and in vitro studies. Physical methods like NMR studies to predict RNA secondary structure are expensive and difficult. Computational RNA secondary structure prediction is easier. Comparative sequence analysis provides the best solution. But secondary structure prediction of a single RNA sequence is challenging. RNA-SSPT is a tool that computationally predicts secondary structure of a single RNA sequence. Most of the RNA secondary structure prediction tools do not allow pseudoknots in the structure or are unable to locate them. Nussinov dynamic programming algorithm has been implemented in RNA-SSPT. The current studies shows only energetically most favorable secondary structure is required and the algorithm modification is also available that produces base pairs to lower the total free energy of the secondary structure. For visualization of RNA secondary structure, NAVIEW in C language is used and modified in C# for tool requirement. RNA-SSPT is built in C# using Dot Net 2.0 in Microsoft Visual Studio 2005 Professional edition. The accuracy of RNA-SSPT is tested in terms of Sensitivity and Positive Predicted Value. It is a tool which serves both secondary structure prediction and secondary structure visualization purposes. PMID:24250115

  1. Translation with secondary structure: Dynamic blockages in totally asymmetric simple exclusion process

    NASA Astrophysics Data System (ADS)

    Shaw, Leah

    2011-03-01

    The totally asymmetric simple exclusion process (TASEP) is often used as a model for protein synthesis, with the lattice and particles representing the mRNA and ribosomes, respectively. Here we model the effect of secondary structure (folding) of the mRNA by introducing a dynamic blockage region in the lattice. If the region is unoccupied by particles, the blockage can close and prevent upstream particles from moving into it, representing the folding of that section of mRNA. Reopening of the blockage, allowing particles to pass, represents unfolding. We study the effects of the blockage size, closing/opening probabilities, and TASEP parameters on the particle current and blockage switching rates.

  2. Enumeration of Secondary Structure Element Bundles

    2004-10-26

    A deterministic algorithm for enumeration of transmembrane protein folds is implemented. Using a set of sparse pairwise atomic distance constraints (such as those obtained from chemical cross-linking, FRET, or dipolar EPR experiments), the algorithm performs an exhaustive search of secondary structure element packing conformations distributed throughout the entire conformational space. The end result is a set of distinct protein conformations which can be scored and refined as part of a process designed for computational elucidationmore » of transmembrane protein structures. Algorithm Overview: The ESSEB algorithm works by dividing the conforrnational space of each secondary structure element (SSE) into a set of cells. For each cell there is a representative conformation and for each atom in the SSE for which a distance restraint is available, there is an associated internal error, The internal error for a distance restraint is the maximum distance that the atom, when positioned in any conformation within a cell, can be from the atom in the representative conformation. The algorithm works recursively by positioning one representative conformation of an SSE. AdI distance restraints are checked with a tolerance that includes both the experimental and internal error. If all restraints are satisfied, every representative conformation of the next SSE is checked, otherwise, the program moves on to the next representative conformation of the current SSE. In addition to the distance restraints, other constraints on protein conformation can be enforced. These include the distance of closest approach between SSE axes, a restraint which prevents the crossover of loops connecting adjacent SSEs, and a restriction on the minimum and maximum distances between axis end-points. Any protein conformation satisfying all of the restraints is enumerated for later scoring and possible refinement. Additionally, in order to make run-times feasible, a divide-and-conquer approach is used

  3. Secondary structure adventures with Carl Woese

    PubMed Central

    Noller, Harry F

    2014-01-01

    Not long after my arrival at UCSC as an assistant professor, I came across Carl Woese's paper “Molecular Mechanics of Translation: A Reciprocating Ratchet Mechanism.”1 In the days before the crystal structure of tRNA was known, Fuller and Hodgson2 had proposed two alternative conformations for its anticodon loop; one was stacked on the 3′ side (as later found in the crystal structure) and the other on the 5′ side. In an ingenious and elegant model, Woese proposed that the conformation of the loop flips between Fuller and Hodgson's 5′- and 3′-stacked forms during protein synthesis, changing the local direction of the mRNA such that the identities of the tRNA binding sites alternated between binding aminoacyl-tRNA and peptidyl-tRNA. The model predicted that there are no A and P sites, only two binding sites whose identities changed following translation of each codon, and that there would be no translocation of tRNAs in the usual sense—only binding and release. I met Carl in person the following year when he presented a seminar on his ratchet model in Santa Cruz. He was chatting in my colleague Ralph Hinegardner's office in what Carl termed a “Little Jack Horner appointment” (the visitor sits and listens to his host describing “What a good boy am I”). He was of compact stature, and bore a striking resemblance to Oskar Werner in Truffaut's film “Jules and Jim.” He projected the impression of a New-Age guru—a shiny black amulet suspended over the front of his black turtleneck sweater and a crown of prematurely white hair. Ralph asked me to explain to Carl what we were doing with ribosomes. I quickly summarized our early experiments that were pointing to a functional role for 16S rRNA. Carl regarded me silently, with a penetrating stare. He then turned to Ralph and said, in an ominous low voice, “I'm going to have some more tanks made as soon as I get back.” Carl's beautiful model was, unfortunately, wrong—it was simpler and more

  4. Automated discovery of active motifs in multiple RNA secondary structures

    SciTech Connect

    Wang, J.T.L.; Chang, Chia-Yo; Shapiro, B.A.

    1996-12-31

    In this paper we present a method for discovering approximately common motifs (also known as active motifs) in multiple RNA secondary structures. The secondary structures can be represented as ordered trees (i.e., the order among siblings matters). Motifs in these trees are connected subgraphs that can differ in both substitutions and deletions/insertions. The proposed method consists of two steps: (1) find candidate motifs in a small sample of the secondary structures; (2) search all of the secondary structures to determine how frequently these motifs occur (within the allowed approximation) in the secondary structures. To reduce the running time, we develop two optimization heuristics based on sampling and pattern matching techniques. Experimental results obtained by running these algorithms on both generated data and RNA secondary structures show the good performance of the algorithms. To demonstrate the utility of our algorithms, we discuss their applications to conducting the phylogenetic study of RNA sequences obtained from GenBank.

  5. An intronic RNA structure modulates expression of the mRNA biogenesis factor Sus1.

    PubMed

    AbuQattam, Ali; Gallego, José; Rodríguez-Navarro, Susana

    2016-01-01

    Sus1 is a conserved protein involved in chromatin remodeling and mRNA biogenesis. Unlike most yeast genes, the SUS1 pre-mRNA of Saccharomyces cerevisiae contains two introns and is alternatively spliced, retaining one or both introns in response to changes in environmental conditions. SUS1 splicing may allow the cell to control Sus1 expression, but the mechanisms that regulate this process remain unknown. Using in silico analyses together with NMR spectroscopy, gel electrophoresis, and UV thermal denaturation experiments, we show that the downstream intron (I2) of SUS1 forms a weakly stable, 37-nucleotide stem-loop structure containing the branch site near its apical loop and the 3' splice site after the stem terminus. A cellular assay revealed that two of four mutants containing altered I2 structures had significantly impaired SUS1 expression. Semiquantitative RT-PCR experiments indicated that all mutants accumulated unspliced SUS1 pre-mRNA and/or induced distorted levels of fully spliced mRNA relative to wild type. Concomitantly, Sus1 cellular functions in histone H2B deubiquitination and mRNA export were affected in I2 hairpin mutants that inhibited splicing. This work demonstrates that I2 structure is relevant for SUS1 expression, and that this effect is likely exerted through modulation of splicing.

  6. An intronic RNA structure modulates expression of the mRNA biogenesis factor Sus1

    PubMed Central

    AbuQattam, Ali; Gallego, José; Rodríguez-Navarro, Susana

    2016-01-01

    Sus1 is a conserved protein involved in chromatin remodeling and mRNA biogenesis. Unlike most yeast genes, the SUS1 pre-mRNA of Saccharomyces cerevisiae contains two introns and is alternatively spliced, retaining one or both introns in response to changes in environmental conditions. SUS1 splicing may allow the cell to control Sus1 expression, but the mechanisms that regulate this process remain unknown. Using in silico analyses together with NMR spectroscopy, gel electrophoresis, and UV thermal denaturation experiments, we show that the downstream intron (I2) of SUS1 forms a weakly stable, 37-nucleotide stem–loop structure containing the branch site near its apical loop and the 3′ splice site after the stem terminus. A cellular assay revealed that two of four mutants containing altered I2 structures had significantly impaired SUS1 expression. Semiquantitative RT-PCR experiments indicated that all mutants accumulated unspliced SUS1 pre-mRNA and/or induced distorted levels of fully spliced mRNA relative to wild type. Concomitantly, Sus1 cellular functions in histone H2B deubiquitination and mRNA export were affected in I2 hairpin mutants that inhibited splicing. This work demonstrates that I2 structure is relevant for SUS1 expression, and that this effect is likely exerted through modulation of splicing. PMID:26546116

  7. Co-evolution of Bacterial Ribosomal Protein S15 with Diverse mRNA Regulatory Structures

    PubMed Central

    Slinger, Betty L.; Newman, Hunter; Lee, Younghan; Pei, Shermin; Meyer, Michelle M.

    2015-01-01

    RNA-protein interactions are critical in many biological processes, yet how such interactions affect the evolution of both partners is still unknown. RNA and protein structures are impacted very differently by mechanisms of genomic change. While most protein families are identifiable at the nucleotide level across large phylogenetic distances, RNA families display far less nucleotide similarity and are often only shared by closely related bacterial species. Ribosomal protein S15 has two RNA binding functions. First, it is a ribosomal protein responsible for organizing the rRNA during ribosome assembly. Second, in many bacterial species S15 also interacts with a structured portion of its own transcript to negatively regulate gene expression. While the first interaction is conserved in most bacteria, the second is not. Four distinct mRNA structures interact with S15 to enable regulation, each of which appears to be independently derived in different groups of bacteria. With the goal of understanding how protein-binding specificity may influence the evolution of such RNA regulatory structures, we examine whether examples of these mRNA structures are able to interact with, and regulate in response to, S15 homologs from organisms containing distinct mRNA structures. We find that despite their shared RNA binding function in the rRNA, S15 homologs have distinct RNA recognition profiles. We present a model to explain the specificity patterns observed, and support this model by with further mutagenesis. After analyzing the patterns of conservation for the S15 protein coding sequences, we also identified amino acid changes that alter the binding specificity of an S15 homolog. In this work we demonstrate that homologous RNA-binding proteins have different specificity profiles, and minor changes to amino acid sequences, or to RNA structural motifs, can have large impacts on RNA-protein recognition. PMID:26675164

  8. Metastable structures and refolding kinetics in hok mRNA of plasmid R1.

    PubMed Central

    Nagel, J H; Gultyaev, A P; Gerdes, K; Pleij, C W

    1999-01-01

    Programmed cell death by hok/sok of plasmid R1 and pnd/pndB of R483 mediates plasmid maintenance by killing of plasmid-free cells. It has been previously suggested that premature translation of the plasmid-mediated toxin is prevented during transcription of the hok and pnd mRNAs by the formation of metastable hairpins in the mRNA at the 5' end. Here, experimental evidence is presented for the existence of metastable structures in the 5' leader of the hok and pnd mRNAs in vitro. The kinetics of refolding from the metastable to the stable structure in the isolated fragments of the 5' ends of both the hok and pnd mRNAs could be estimated, in agreement with the structural rearrangement in this region, as predicted to occur during transcription and mRNA activation. The refolding rates of hok and pnd structures are slow enough to allow for the formation of downstream hairpin structures during elongation of the mRNAs, which thereby helps to stabilize the metastable structures. Thus, the kinetic refolding parameters of the hok and pnd mRNAs are consistent with the proposal that the metastable structures prevent premature translation and/or antisense RNA binding during transcription. PMID:10580469

  9. RNA Secondary Structure Prediction by Using Discrete Mathematics: An Interdisciplinary Research Experience for Undergraduate Students

    PubMed Central

    Ellington, Roni; Wachira, James

    2010-01-01

    The focus of this Research Experience for Undergraduates (REU) project was on RNA secondary structure prediction by using a lattice walk approach. The lattice walk approach is a combinatorial and computational biology method used to enumerate possible secondary structures and predict RNA secondary structure from RNA sequences. The method uses discrete mathematical techniques and identifies specified base pairs as parameters. The goal of the REU was to introduce upper-level undergraduate students to the principles and challenges of interdisciplinary research in molecular biology and discrete mathematics. At the beginning of the project, students from the biology and mathematics departments of a mid-sized university received instruction on the role of secondary structure in the function of eukaryotic RNAs and RNA viruses, RNA related to combinatorics, and the National Center for Biotechnology Information resources. The student research projects focused on RNA secondary structure prediction on a regulatory region of the yellow fever virus RNA genome and on an untranslated region of an mRNA of a gene associated with the neurological disorder epilepsy. At the end of the project, the REU students gave poster and oral presentations, and they submitted written final project reports to the program director. The outcome of the REU was that the students gained transferable knowledge and skills in bioinformatics and an awareness of the applications of discrete mathematics to biological research problems. PMID:20810968

  10. RNA secondary structure prediction by using discrete mathematics: an interdisciplinary research experience for undergraduate students.

    PubMed

    Ellington, Roni; Wachira, James; Nkwanta, Asamoah

    2010-01-01

    The focus of this Research Experience for Undergraduates (REU) project was on RNA secondary structure prediction by using a lattice walk approach. The lattice walk approach is a combinatorial and computational biology method used to enumerate possible secondary structures and predict RNA secondary structure from RNA sequences. The method uses discrete mathematical techniques and identifies specified base pairs as parameters. The goal of the REU was to introduce upper-level undergraduate students to the principles and challenges of interdisciplinary research in molecular biology and discrete mathematics. At the beginning of the project, students from the biology and mathematics departments of a mid-sized university received instruction on the role of secondary structure in the function of eukaryotic RNAs and RNA viruses, RNA related to combinatorics, and the National Center for Biotechnology Information resources. The student research projects focused on RNA secondary structure prediction on a regulatory region of the yellow fever virus RNA genome and on an untranslated region of an mRNA of a gene associated with the neurological disorder epilepsy. At the end of the project, the REU students gave poster and oral presentations, and they submitted written final project reports to the program director. The outcome of the REU was that the students gained transferable knowledge and skills in bioinformatics and an awareness of the applications of discrete mathematics to biological research problems.

  11. Neural network definitions of highly predictable protein secondary structure classes

    SciTech Connect

    Lapedes, A. |; Steeg, E.; Farber, R.

    1994-02-01

    We use two co-evolving neural networks to determine new classes of protein secondary structure which are significantly more predictable from local amino sequence than the conventional secondary structure classification. Accurate prediction of the conventional secondary structure classes: alpha helix, beta strand, and coil, from primary sequence has long been an important problem in computational molecular biology. Neural networks have been a popular method to attempt to predict these conventional secondary structure classes. Accuracy has been disappointingly low. The algorithm presented here uses neural networks to similtaneously examine both sequence and structure data, and to evolve new classes of secondary structure that can be predicted from sequence with significantly higher accuracy than the conventional classes. These new classes have both similarities to, and differences with the conventional alpha helix, beta strand and coil.

  12. ncRNA consensus secondary structure derivation using grammar strings.

    PubMed

    Achawanantakun, Rujira; Sun, Yanni; Takyar, Seyedeh Shohreh

    2011-04-01

    Many noncoding RNAs (ncRNAs) function through both their sequences and secondary structures. Thus, secondary structure derivation is an important issue in today's RNA research. The state-of-the-art structure annotation tools are based on comparative analysis, which derives consensus structure of homologous ncRNAs. Despite promising results from existing ncRNA aligning and consensus structure derivation tools, there is a need for more efficient and accurate ncRNA secondary structure modeling and alignment methods. In this work, we introduce a consensus structure derivation approach based on grammar string, a novel ncRNA secondary structure representation that encodes an ncRNA's sequence and secondary structure in the parameter space of a context-free grammar (CFG) and a full RNA grammar including pseudoknots. Being a string defined on a special alphabet constructed from a grammar, grammar string converts ncRNA alignment into sequence alignment. We derive consensus secondary structures from hundreds of ncRNA families from BraliBase 2.1 and 25 families containing pseudoknots using grammar string alignment. Our experiments have shown that grammar string-based structure derivation competes favorably in consensus structure quality with Murlet and RNASampler. Source code and experimental data are available at http://www.cse.msu.edu/~yannisun/grammar-string. PMID:21523935

  13. ncRNA consensus secondary structure derivation using grammar strings.

    PubMed

    Achawanantakun, Rujira; Sun, Yanni; Takyar, Seyedeh Shohreh

    2011-04-01

    Many noncoding RNAs (ncRNAs) function through both their sequences and secondary structures. Thus, secondary structure derivation is an important issue in today's RNA research. The state-of-the-art structure annotation tools are based on comparative analysis, which derives consensus structure of homologous ncRNAs. Despite promising results from existing ncRNA aligning and consensus structure derivation tools, there is a need for more efficient and accurate ncRNA secondary structure modeling and alignment methods. In this work, we introduce a consensus structure derivation approach based on grammar string, a novel ncRNA secondary structure representation that encodes an ncRNA's sequence and secondary structure in the parameter space of a context-free grammar (CFG) and a full RNA grammar including pseudoknots. Being a string defined on a special alphabet constructed from a grammar, grammar string converts ncRNA alignment into sequence alignment. We derive consensus secondary structures from hundreds of ncRNA families from BraliBase 2.1 and 25 families containing pseudoknots using grammar string alignment. Our experiments have shown that grammar string-based structure derivation competes favorably in consensus structure quality with Murlet and RNASampler. Source code and experimental data are available at http://www.cse.msu.edu/~yannisun/grammar-string.

  14. Thermodynamic and kinetic characterization of antisense oligodeoxynucleotide binding to a structured mRNA.

    PubMed Central

    Walton, S Patrick; Stephanopoulos, Gregory N; Yarmush, Martin L; Roth, Charles M

    2002-01-01

    Antisense oligonucleotides act as exogenous inhibitors of gene expression by binding to a complementary sequence on the target mRNA, preventing translation into protein. Antisense technology is being applied successfully as a research tool and as a molecular therapeutic. However, a quantitative understanding of binding energetics between short oligonucleotides and longer mRNA targets is lacking, and selecting a high-affinity antisense oligonucleotide sequence from the many possibilities complementary to a particular RNA is a critical step in designing an effective antisense inhibitor. Here, we report measurements of the thermodynamics and kinetics of hybridization for a number of oligodeoxynucleotides (ODNs) complementary to the rabbit beta-globin (RBG) mRNA using a binding assay that facilitates rapid separation of bound from free species in solution. A wide range of equilibrium dissociation constants were observed, and association rate constants within the measurable range correlated strongly with binding affinity. In addition, a significant correlation was observed of measured binding affinities with binding affinity values predicted using a thermodynamic model involving DNA and RNA unfolding, ODN hybridization, and RNA restructuring to a final free energy minimum. In contrast to the behavior observed for hybridization of short strands, the association rate constant increased with temperature, suggesting that the kinetics of association are related to disrupting the native structure of the target RNA. The rate of cleavage of the RBG mRNA in the presence of ribonuclease H and ODNs of varying association kinetics displayed apparent first-order kinetics, with the rate constant exhibiting binding-limited behavior at low association rates and reaction-limited behavior at higher rates. Implications for the rational design of effective antisense reagents are discussed. PMID:11751323

  15. A dynamic programming algorithm for finding alternative RNA secondary structures.

    PubMed

    Williams, A L; Tinoco, I

    1986-01-10

    Dynamic programming algorithms that predict RNA secondary structure by minimizing the free energy have had one important limitation. They were able to predict only one optimal structure. Given the uncertainties of the thermodynamic data and the effects of proteins and other environmental factors on structure, the optimal structure predicted by these methods may not have biological significance. We present a dynamic programming algorithm that can determine optimal and suboptimal secondary structures for an RNA. The power and utility of the method is demonstrated in the folding of the intervening sequence of the rRNA of Tetrahymena. By first identifying the major secondary structures corresponding to the lowest free energy minima, a secondary structure of possible biological significance is derived.

  16. Widespread signatures of local mRNA folding structure selection in four Dengue virus serotypes

    PubMed Central

    2015-01-01

    Background It is known that mRNA folding can affect and regulate various gene expression steps both in living organisms and in viruses. Previous studies have recognized functional RNA structures in the genome of the Dengue virus. However, these studies usually focused either on the viral untranslated regions or on very specific and limited regions at the beginning of the coding sequences, in a limited number of strains, and without considering evolutionary selection. Results Here we performed the first large scale comprehensive genomics analysis of selection for local mRNA folding strength in the Dengue virus coding sequences, based on a total of 1,670 genomes and 4 serotypes. Our analysis identified clusters of positions along the coding regions that may undergo a conserved evolutionary selection for strong or weak local folding maintained across different viral variants. Specifically, 53-66 clusters for strong folding and 49-73 clusters for weak folding (depending on serotype) aggregated of positions with a significant conservation of folding energy signals (related to partially overlapping local genomic regions) were recognized. In addition, up to 7% of these positions were found to be conserved in more than 90% of the viral genomes. Although some of the identified positions undergo frequent synonymous / non-synonymous substitutions, the selection for folding strength therein is preserved, and thus cannot be trivially explained based on sequence conservation alone. Conclusions The fact that many of the positions with significant folding related signals are conserved among different Dengue variants suggests that a better understanding of the mRNA structures in the corresponding regions may promote the development of prospective anti- Dengue vaccination strategies. The comparative genomics approach described here can be employed in the future for detecting functional regions in other pathogens with very high mutations rates. PMID:26449467

  17. The structure of a ribosomal protein S8/spc operon mRNA complex.

    PubMed

    Merianos, Helen J; Wang, Jimin; Moore, Peter B

    2004-06-01

    In bacteria, translation of all the ribosomal protein cistrons in the spc operon mRNA is repressed by the binding of the product of one of them, S8, to an internal sequence at the 5' end of the L5 cistron. The way in which the first two genes of the spc operon are regulated, retroregulation, is mechanistically distinct from translational repression by S8 of the genes from L5 onward. A 2.8 A resolution crystal structure has been obtained of Escherichia coli S8 bound to this site. Despite sequence differences, the structure of this complex is almost identical to that of the S8/helix 21 complex seen in the small ribosomal subunit, consistent with the hypothesis that autogenous regulation of ribosomal protein synthesis results from conformational similarities between mRNAs and rRNAs. S8 binding must repress the translation of its own mRNA by inhibiting the formation of a ribosomal initiation complex at the start of the L5 cistron.

  18. Unified approach to partition functions of RNA secondary structures.

    PubMed

    Bundschuh, Ralf

    2014-11-01

    RNA secondary structure formation is a field of considerable biological interest as well as a model system for understanding generic properties of heteropolymer folding. This system is particularly attractive because the partition function and thus all thermodynamic properties of RNA secondary structure ensembles can be calculated numerically in polynomial time for arbitrary sequences and homopolymer models admit analytical solutions. Such solutions for many different aspects of the combinatorics of RNA secondary structure formation share the property that the final solution depends on differences of statistical weights rather than on the weights alone. Here, we present a unified approach to a large class of problems in the field of RNA secondary structure formation. We prove a generic theorem for the calculation of RNA folding partition functions. Then, we show that this approach can be applied to the study of the molten-native transition, denaturation of RNA molecules, as well as to studies of the glass phase of random RNA sequences.

  19. Unified approach to partition functions of RNA secondary structures.

    PubMed

    Bundschuh, Ralf

    2014-11-01

    RNA secondary structure formation is a field of considerable biological interest as well as a model system for understanding generic properties of heteropolymer folding. This system is particularly attractive because the partition function and thus all thermodynamic properties of RNA secondary structure ensembles can be calculated numerically in polynomial time for arbitrary sequences and homopolymer models admit analytical solutions. Such solutions for many different aspects of the combinatorics of RNA secondary structure formation share the property that the final solution depends on differences of statistical weights rather than on the weights alone. Here, we present a unified approach to a large class of problems in the field of RNA secondary structure formation. We prove a generic theorem for the calculation of RNA folding partition functions. Then, we show that this approach can be applied to the study of the molten-native transition, denaturation of RNA molecules, as well as to studies of the glass phase of random RNA sequences. PMID:24177391

  20. Dynamics in Sequence Space for RNA Secondary Structure Design.

    PubMed

    Matthies, Marco C; Bienert, Stefan; Torda, Andrew E

    2012-10-01

    We have implemented a method for the design of RNA sequences that should fold to arbitrary secondary structures. A popular energy model allows one to take the derivative with respect to composition, which can then be interpreted as a force and used for Newtonian dynamics in sequence space. Combined with a negative design term, one can rapidly sample sequences which are compatible with a desired secondary structure via simulated annealing. Results for 360 structures were compared with those from another nucleic acid design program using measures such as the probability of the target structure and an ensemble-weighted distance to the target structure.

  1. Combinatorics of RNA Secondary Structures with Base Triples.

    PubMed

    Müller, Robert; Nebel, Markus E

    2015-07-01

    The structure of RNA has been the subject of intense research over the last decades due to its importance for the correct functioning of RNA molecules in biological processes. Hence, a large number of models for RNA folding and corresponding algorithms for structure prediction have been developed. However, previous models often only consider base pairs, although every base is capable of up to three edge-to-edge interactions with other bases. Recently, Höner zu Siederdissen et al. presented an extended model of RNA secondary structure, including base triples together with a folding algorithm-the first thermodynamics-based algorithm that allows the prediction of secondary structures with base triples. In this article, we investigate the search space processed by this new algorithm, that is, the combinatorics of extended RNA secondary structures with base triples. We present generalized definitions for structural motifs like hairpins, stems, bulges, or interior loops occurring in structures with base triples. Furthermore, we prove precise asymptotic results for the number of different structures (size of search space) and expectations for various parameters associated with structural motifs (typical shape of folding). Our analysis shows that the asymptotic number of secondary structures of size n increases exponentially to [Formula: see text] compared to the classic model by Stein and Waterman for which [Formula: see text] structures exist. A comparison with the classic model reveals large deviations in the expected structural appearance, too. The inclusion of base triples constitutes a significant refinement of the combinatorial model of RNA secondary structure, which, by our findings, is quantitatively characterized. Our results are of special theoretical interest, because a closer look at the numbers involved suggests that extended RNA secondary structures constitute a new combinatorial class not bijective with any other combinatorial objects studied so far.

  2. Combinatorics of RNA Secondary Structures with Base Triples.

    PubMed

    Müller, Robert; Nebel, Markus E

    2015-07-01

    The structure of RNA has been the subject of intense research over the last decades due to its importance for the correct functioning of RNA molecules in biological processes. Hence, a large number of models for RNA folding and corresponding algorithms for structure prediction have been developed. However, previous models often only consider base pairs, although every base is capable of up to three edge-to-edge interactions with other bases. Recently, Höner zu Siederdissen et al. presented an extended model of RNA secondary structure, including base triples together with a folding algorithm-the first thermodynamics-based algorithm that allows the prediction of secondary structures with base triples. In this article, we investigate the search space processed by this new algorithm, that is, the combinatorics of extended RNA secondary structures with base triples. We present generalized definitions for structural motifs like hairpins, stems, bulges, or interior loops occurring in structures with base triples. Furthermore, we prove precise asymptotic results for the number of different structures (size of search space) and expectations for various parameters associated with structural motifs (typical shape of folding). Our analysis shows that the asymptotic number of secondary structures of size n increases exponentially to [Formula: see text] compared to the classic model by Stein and Waterman for which [Formula: see text] structures exist. A comparison with the classic model reveals large deviations in the expected structural appearance, too. The inclusion of base triples constitutes a significant refinement of the combinatorial model of RNA secondary structure, which, by our findings, is quantitatively characterized. Our results are of special theoretical interest, because a closer look at the numbers involved suggests that extended RNA secondary structures constitute a new combinatorial class not bijective with any other combinatorial objects studied so far. PMID

  3. Crystal Structure of the Lysine Riboswitch Regulatory mRNA Element*S⃞

    PubMed Central

    Garst, Andrew D.; Héroux, Annie; Rambo, Robert P.; Batey, Robert T.

    2008-01-01

    Riboswitches are metabolite-sensitive elements found in mRNAs that control gene expression through a regulatory secondary structural switch. Along with regulation of lysine biosynthetic genes, mutations within the lysine-responsive riboswitch (L-box) play a role in the acquisition of resistance to antimicrobial lysine analogs. To understand the structural basis for lysine binding, we have determined the 2.8Å resolution crystal structure of lysine bound to the Thermotoga maritima asd lysine riboswitch ligand-binding domain. The structure reveals a complex architecture scaffolding a binding pocket completely enveloping lysine. Mutations conferring antimicrobial resistance cluster around this site as well as highly conserved long range interactions, indicating that they disrupt lysine binding or proper folding of the RNA. Comparison of the free and bound forms by x-ray crystallography, small angle x-ray scattering, and chemical probing reveals almost identical structures, indicating that lysine induces only limited and local conformational changes upon binding. PMID:18593706

  4. Crystal structure of the lysine riboswitch regulatory mRNA element.

    PubMed

    Garst, Andrew D; Héroux, Annie; Rambo, Robert P; Batey, Robert T

    2008-08-15

    Riboswitches are metabolite-sensitive elements found in mRNAs that control gene expression through a regulatory secondary structural switch. Along with regulation of lysine biosynthetic genes, mutations within the lysine-responsive riboswitch (L-box) play a role in the acquisition of resistance to antimicrobial lysine analogs. To understand the structural basis for lysine binding, we have determined the 2.8 angstroms resolution crystal structure of lysine bound to the Thermotoga maritima asd lysine riboswitch ligand-binding domain. The structure reveals a complex architecture scaffolding a binding pocket completely enveloping lysine. Mutations conferring antimicrobial resistance cluster around this site as well as highly conserved long range interactions, indicating that they disrupt lysine binding or proper folding of the RNA. Comparison of the free and bound forms by x-ray crystallography, small angle x-ray scattering, and chemical probing reveals almost identical structures, indicating that lysine induces only limited and local conformational changes upon binding. PMID:18593706

  5. Crystal Structure of the Lysine Riboswitch Regulatory mRNA Element

    SciTech Connect

    Garst, A.; Heroux, A; Rambo, R; Batey, R

    2008-01-01

    Riboswitches are metabolite-sensitive elements found in mRNAs that control gene expression through a regulatory secondary structural switch. Along with regulation of lysine biosynthetic genes, mutations within the lysine-responsive riboswitch (L-box) play a role in the acquisition of resistance to antimicrobial lysine analogs. To understand the structural basis for lysine binding, we have determined the 2.8{angstrom} resolution crystal structure of lysine bound to the Thermotoga maritima asd lysine riboswitch ligand-binding domain. The structure reveals a complex architecture scaffolding a binding pocket completely enveloping lysine. Mutations conferring antimicrobial resistance cluster around this site as well as highly conserved long range interactions, indicating that they disrupt lysine binding or proper folding of the RNA. Comparison of the free and bound forms by x-ray crystallography, small angle x-ray scattering, and chemical probing reveals almost identical structures, indicating that lysine induces only limited and local conformational changes upon binding.

  6. Computing the Partition Function for Kinetically Trapped RNA Secondary Structures

    PubMed Central

    Lorenz, William A.; Clote, Peter

    2011-01-01

    An RNA secondary structure is locally optimal if there is no lower energy structure that can be obtained by the addition or removal of a single base pair, where energy is defined according to the widely accepted Turner nearest neighbor model. Locally optimal structures form kinetic traps, since any evolution away from a locally optimal structure must involve energetically unfavorable folding steps. Here, we present a novel, efficient algorithm to compute the partition function over all locally optimal secondary structures of a given RNA sequence. Our software, RNAlocopt runs in time and space. Additionally, RNAlocopt samples a user-specified number of structures from the Boltzmann subensemble of all locally optimal structures. We apply RNAlocopt to show that (1) the number of locally optimal structures is far fewer than the total number of structures – indeed, the number of locally optimal structures approximately equal to the square root of the number of all structures, (2) the structural diversity of this subensemble may be either similar to or quite different from the structural diversity of the entire Boltzmann ensemble, a situation that depends on the type of input RNA, (3) the (modified) maximum expected accuracy structure, computed by taking into account base pairing frequencies of locally optimal structures, is a more accurate prediction of the native structure than other current thermodynamics-based methods. The software RNAlocopt constitutes a technical breakthrough in our study of the folding landscape for RNA secondary structures. For the first time, locally optimal structures (kinetic traps in the Turner energy model) can be rapidly generated for long RNA sequences, previously impossible with methods that involved exhaustive enumeration. Use of locally optimal structure leads to state-of-the-art secondary structure prediction, as benchmarked against methods involving the computation of minimum free energy and of maximum expected accuracy. Web server

  7. Secondary Structure Prediction of Single Sequences Using RNAstructure.

    PubMed

    Xu, Zhenjiang Zech; Mathews, David H

    2016-01-01

    RNA secondary structure is often predicted using folding thermodynamics. RNAstructure is a software package that includes structure prediction by free energy minimization, prediction of base pairing probabilities, prediction of structures composed of highly probably base pairs, and prediction of structures with pseudoknots. A user-friendly graphical user interface is provided, and this interface works on Windows, Apple OS X, and Linux. This chapter provides protocols for using RNAstructure for structure prediction. PMID:27665590

  8. Computing the partition function for kinetically trapped RNA secondary structures.

    PubMed

    Lorenz, William A; Clote, Peter

    2011-01-01

    An RNA secondary structure is locally optimal if there is no lower energy structure that can be obtained by the addition or removal of a single base pair, where energy is defined according to the widely accepted Turner nearest neighbor model. Locally optimal structures form kinetic traps, since any evolution away from a locally optimal structure must involve energetically unfavorable folding steps. Here, we present a novel, efficient algorithm to compute the partition function over all locally optimal secondary structures of a given RNA sequence. Our software, RNAlocopt runs in O(n3) time and O(n2) space. Additionally, RNAlocopt samples a user-specified number of structures from the Boltzmann subensemble of all locally optimal structures. We apply RNAlocopt to show that (1) the number of locally optimal structures is far fewer than the total number of structures--indeed, the number of locally optimal structures approximately equal to the square root of the number of all structures, (2) the structural diversity of this subensemble may be either similar to or quite different from the structural diversity of the entire Boltzmann ensemble, a situation that depends on the type of input RNA, (3) the (modified) maximum expected accuracy structure, computed by taking into account base pairing frequencies of locally optimal structures, is a more accurate prediction of the native structure than other current thermodynamics-based methods. The software RNAlocopt constitutes a technical breakthrough in our study of the folding landscape for RNA secondary structures. For the first time, locally optimal structures (kinetic traps in the Turner energy model) can be rapidly generated for long RNA sequences, previously impossible with methods that involved exhaustive enumeration. Use of locally optimal structure leads to state-of-the-art secondary structure prediction, as benchmarked against methods involving the computation of minimum free energy and of maximum expected accuracy

  9. Novel and efficient RNA secondary structure prediction using hierarchical folding.

    PubMed

    Jabbari, Hosna; Condon, Anne; Zhao, Shelly

    2008-03-01

    Algorithms for prediction of RNA secondary structure-the set of base pairs that form when an RNA molecule folds-are valuable to biologists who aim to understand RNA structure and function. Improving the accuracy and efficiency of prediction methods is an ongoing challenge, particularly for pseudoknotted secondary structures, in which base pairs overlap. This challenge is biologically important, since pseudoknotted structures play essential roles in functions of many RNA molecules, such as splicing and ribosomal frameshifting. State-of-the-art methods, which are based on free energy minimization, have high run-time complexity (typically Theta(n(5)) or worse), and can handle (minimize over) only limited types of pseudoknotted structures. We propose a new approach for prediction of pseudoknotted structures, motivated by the hypothesis that RNA structures fold hierarchically, with pseudoknot-free (non-overlapping) base pairs forming first, and pseudoknots forming later so as to minimize energy relative to the folded pseudoknot-free structure. Our HFold algorithm uses two-phase energy minimization to predict hierarchically formed secondary structures in O(n(3)) time, matching the complexity of the best algorithms for pseudoknot-free secondary structure prediction via energy minimization. Our algorithm can handle a wide range of biological structures, including kissing hairpins and nested kissing hairpins, which have previously required Theta(n(6)) time.

  10. Predicting RNA secondary structures from sequence and probing data.

    PubMed

    Lorenz, Ronny; Wolfinger, Michael T; Tanzer, Andrea; Hofacker, Ivo L

    2016-07-01

    RNA secondary structures have proven essential for understanding the regulatory functions performed by RNA such as microRNAs, bacterial small RNAs, or riboswitches. This success is in part due to the availability of efficient computational methods for predicting RNA secondary structures. Recent advances focus on dealing with the inherent uncertainty of prediction by considering the ensemble of possible structures rather than the single most stable one. Moreover, the advent of high-throughput structural probing has spurred the development of computational methods that incorporate such experimental data as auxiliary information.

  11. Resistance of mRNAs with AUG-proximal nonsense mutations to nonsense-mediated decay reflects variables of mRNA structure and translational activity.

    PubMed

    Pereira, Francisco J C; Teixeira, Alexandre; Kong, Jian; Barbosa, Cristina; Silva, Ana Luísa; Marques-Ramos, Ana; Liebhaber, Stephen A; Romão, Luísa

    2015-07-27

    Nonsense-mediated mRNA decay (NMD) is a surveillance pathway that recognizes and selectively degrades mRNAs carrying premature termination codons (PTCs). The level of sensitivity of a PTC-containing mRNA to NMD is multifactorial. We have previously shown that human β-globin mRNAs carrying PTCs in close proximity to the translation initiation AUG codon escape NMD. This was called the 'AUG-proximity effect'. The present analysis of nonsense codons in the human α-globin mRNA illustrates that the determinants of the AUG-proximity effect are in fact quite complex, reflecting the ability of the ribosome to re-initiate translation 3' to the PTC and the specific sequence and secondary structure of the translated ORF. These data support a model in which the time taken to translate the short ORF, impacted by distance, sequence, and structure, not only modulates translation re-initiation, but also impacts on the exact boundary of AUG-proximity protection from NMD.

  12. Principles for Predicting RNA Secondary Structure Design Difficulty.

    PubMed

    Anderson-Lee, Jeff; Fisker, Eli; Kosaraju, Vineet; Wu, Michelle; Kong, Justin; Lee, Jeehyung; Lee, Minjae; Zada, Mathew; Treuille, Adrien; Das, Rhiju

    2016-02-27

    Designing RNAs that form specific secondary structures is enabling better understanding and control of living systems through RNA-guided silencing, genome editing and protein organization. Little is known, however, about which RNA secondary structures might be tractable for downstream sequence design, increasing the time and expense of design efforts due to inefficient secondary structure choices. Here, we present insights into specific structural features that increase the difficulty of finding sequences that fold into a target RNA secondary structure, summarizing the design efforts of tens of thousands of human participants and three automated algorithms (RNAInverse, INFO-RNA and RNA-SSD) in the Eterna massive open laboratory. Subsequent tests through three independent RNA design algorithms (NUPACK, DSS-Opt and MODENA) confirmed the hypothesized importance of several features in determining design difficulty, including sequence length, mean stem length, symmetry and specific difficult-to-design motifs such as zigzags. Based on these results, we have compiled an Eterna100 benchmark of 100 secondary structure design challenges that span a large range in design difficulty to help test future efforts. Our in silico results suggest new routes for improving computational RNA design methods and for extending these insights to assess "designability" of single RNA structures, as well as of switches for in vitro and in vivo applications. PMID:26902426

  13. Principles for Predicting RNA Secondary Structure Design Difficulty.

    PubMed

    Anderson-Lee, Jeff; Fisker, Eli; Kosaraju, Vineet; Wu, Michelle; Kong, Justin; Lee, Jeehyung; Lee, Minjae; Zada, Mathew; Treuille, Adrien; Das, Rhiju

    2016-02-27

    Designing RNAs that form specific secondary structures is enabling better understanding and control of living systems through RNA-guided silencing, genome editing and protein organization. Little is known, however, about which RNA secondary structures might be tractable for downstream sequence design, increasing the time and expense of design efforts due to inefficient secondary structure choices. Here, we present insights into specific structural features that increase the difficulty of finding sequences that fold into a target RNA secondary structure, summarizing the design efforts of tens of thousands of human participants and three automated algorithms (RNAInverse, INFO-RNA and RNA-SSD) in the Eterna massive open laboratory. Subsequent tests through three independent RNA design algorithms (NUPACK, DSS-Opt and MODENA) confirmed the hypothesized importance of several features in determining design difficulty, including sequence length, mean stem length, symmetry and specific difficult-to-design motifs such as zigzags. Based on these results, we have compiled an Eterna100 benchmark of 100 secondary structure design challenges that span a large range in design difficulty to help test future efforts. Our in silico results suggest new routes for improving computational RNA design methods and for extending these insights to assess "designability" of single RNA structures, as well as of switches for in vitro and in vivo applications.

  14. DNA Secondary Structure at Chromosomal Fragile Sites in Human Disease

    PubMed Central

    Thys, Ryan G; Lehman, Christine E; Pierce, Levi C. T; Wang, Yuh-Hwa

    2015-01-01

    DNA has the ability to form a variety of secondary structures that can interfere with normal cellular processes, and many of these structures have been associated with neurological diseases and cancer. Secondary structure-forming sequences are often found at chromosomal fragile sites, which are hotspots for sister chromatid exchange, chromosomal translocations, and deletions. Structures formed at fragile sites can lead to instability by disrupting normal cellular processes such as DNA replication and transcription. The instability caused by disruption of replication and transcription can lead to DNA breakage, resulting in gene rearrangements and deletions that cause disease. In this review, we discuss the role of DNA secondary structure at fragile sites in human disease. PMID:25937814

  15. Different structure and mRNA expression of Entamoeba invadens chitinases in the encystation and excystation.

    PubMed

    Makioka, Asao; Kumagai, Masahiro; Hiranuka, Kazushi; Kobayashi, Seiki; Takeuchi, Tsutomu

    2011-08-01

    Entamoeba histolytica forms chitin-walled cysts during encystation process, where formation of the cyst wall needs not only chitin synthase but also chitinase. During excystation, quadruplet amoebae emerge from the chitin-walled cysts by dissolving the wall, so that chitinase may be necessary for excystation process as well. There is, however, no report on chitinase expression during excystation. In this study, we used Entamoeba invadens, a reptilian amoeba, as a model for encystation and excystation of E. histolytica, and studied chitinase mRNA expression in those processes. Although expression of three E. invadens chitinases designated EiChit1, EiChit2, and EiChit3 during encystation has been reported, we identified another enzyme named as EiChit4 in the E. invadens genome database. Therefore, we investigated the primary structure and mRNA expression of these four chitinases of Ei in the excystation as well as the encystation by real-time reverse transcription polymerase chain reaction (RT-PCR). Like EiChit1, EiChit4 had an 8 × Cys chitin-binding domain (CBD) and a hydrophilic spacer between the CBD and catalytic domain, and was also closer to EiChit1 than EiChit2 and EiChit3 in the phylogenetic tree. During encystation, the expression of all four chitinases increased in the early phase; the increase in EiChit1 and EiChit4 was much higher than in EiChit2 and EiChit3. Then, the expression of all four chitinases sharply decreased in the later phase. In cysts, EiChit1 was most abundantly expressed and EiChit4 was at a lower level, while the expressions of EiChit2 and EiChit3 were virtually absent. Following the induction of excystation, mRNA levels of EiChit1 and EiChit4 in cysts 5 h after induction were significantly lower than those in cysts before induction, while those of EiChit2 and EiChit3 were remarkably higher than before induction. The mRNAs of only EiChit2 and EiChit3 remarkably increased when the excystation was induced in the presence of cytochalasin D

  16. Binding of protein synthesis initiation factor 4E to oligoribonucleotides: effects of cap accessibility and secondary structure.

    PubMed

    Carberry, S E; Friedland, D E; Rhoads, R E; Goss, D J

    1992-02-11

    The binding of rabbit globin mRNA to the 25-kDa cap binding protein eIF-4E from human erythrocytes was found to be 5.3-fold stronger than the binding of the cap analogue m7GpppG to eIF-4E [Gross et al. (1990) Biochemistry 29, 5008-5012]. In order to investigate whether this effect is due to the longer sequence of nucleotides in globin mRNA or to other features such as cap accessibility or secondary structure, oligoribonucleotide analogues of rabbit alpha-globin mRNA were synthesized by T7 RNA polymerase from a synthetic oligodeoxynucleotide template in the presence of m7GpppG; these oligoribonucleotide analogues possess varying degrees of cap accessibility and secondary structure. Equilibrium association constants for the interaction of these oligoribonucleotides and purified human erythrocyte eIF-4E were obtained from direct fluorescence titration experiments. The data indicate that while the presence of the m7G cap is required for efficient recognition by eIF-4E, the cap need not be completely sterically accessible, since other structural features within the mRNA also influence binding.

  17. Prediction of Secondary Structures Conserved in Multiple RNA Sequences.

    PubMed

    Xu, Zhenjiang Zech; Mathews, David H

    2016-01-01

    RNA structure is conserved by evolution to a greater extent than sequence. Predicting the conserved structure for multiple homologous sequences can be much more accurate than predicting the structure for a single sequence. RNAstructure is a software package that includes the programs Dynalign, Multilign, TurboFold, and PARTS for predicting conserved RNA secondary structure. This chapter provides protocols for using these programs. PMID:27665591

  18. Secondary structural analyses of ITS1 in Paramecium.

    PubMed

    Hoshina, Ryo

    2010-01-01

    The nuclear ribosomal RNA gene operon is interrupted by internal transcribed spacer (ITS) 1 and ITS2. Although the secondary structure of ITS2 has been widely investigated, less is known about ITS1 and its structure. In this study, the secondary structure of ITS1 sequences for Paramecium and other ciliates was predicted. Each Paramecium ITS1 forms an open loop with three helices, A through C. Helix B was highly conserved among Paramecium, and similar helices were found in other ciliates. A phylogenetic analysis using the ITS1 sequences showed high-resolution, implying that ITS1 is a good tool for species-level analyses.

  19. The RNA Structure of cis-acting Translational Elements of the Chloroplast psbC mRNA in Chlamydomonas reinhardtii

    PubMed Central

    Rahim, Mir Munir A.; Vigneault, Frederic; Zerges, William

    2016-01-01

    Photosystem II is the first of two light-driven oxidoreductase complexes in oxygenic photosynthesis. The biogenesis of photosystem II requires the synthesis of polypeptide subunits encoded by the genomes in the chloroplast and the nucleus. In the chloroplast of the green alga Chlamydomonas reinhardtii, the synthesis of each subunit requires interactions between the 5′ UTR of the mRNA encoding it and gene-specific translation factors. Here, we analyze the sequences and structures in the 5′ UTR of the psbC mRNA, which are known to be required to promote translation and genetic interaction with TBC1, a nuclear gene required specifically for psbC translation. Results of enzymatic probing in vitro and chemical probing in vivo and in vitro support three secondary structures and reveal that one participates in a pseudoknot structure. Analyses of the effects of mutations affecting pseudoknot sequences, by structural mapping and thermal gradient gel electrophoresis, reveal that flexibility at the base of the major stem-loop is required for translation and higher order RNA conformation, and suggest that this conformation is stabilized by TBC1. This RNA pseudoknot tertiary structure is analogous to the internal ribosome entry sites that promote translation of certain viruses and cellular mRNAs in the nuclear-cytoplasmic systems of eukaryotes. PMID:27379123

  20. The RNA Structure of cis-acting Translational Elements of the Chloroplast psbC mRNA in Chlamydomonas reinhardtii.

    PubMed

    Rahim, Mir Munir A; Vigneault, Frederic; Zerges, William

    2016-01-01

    Photosystem II is the first of two light-driven oxidoreductase complexes in oxygenic photosynthesis. The biogenesis of photosystem II requires the synthesis of polypeptide subunits encoded by the genomes in the chloroplast and the nucleus. In the chloroplast of the green alga Chlamydomonas reinhardtii, the synthesis of each subunit requires interactions between the 5' UTR of the mRNA encoding it and gene-specific translation factors. Here, we analyze the sequences and structures in the 5' UTR of the psbC mRNA, which are known to be required to promote translation and genetic interaction with TBC1, a nuclear gene required specifically for psbC translation. Results of enzymatic probing in vitro and chemical probing in vivo and in vitro support three secondary structures and reveal that one participates in a pseudoknot structure. Analyses of the effects of mutations affecting pseudoknot sequences, by structural mapping and thermal gradient gel electrophoresis, reveal that flexibility at the base of the major stem-loop is required for translation and higher order RNA conformation, and suggest that this conformation is stabilized by TBC1. This RNA pseudoknot tertiary structure is analogous to the internal ribosome entry sites that promote translation of certain viruses and cellular mRNAs in the nuclear-cytoplasmic systems of eukaryotes. PMID:27379123

  1. A New Secondary Structure Assignment Algorithm Using Cα Backbone Fragments

    PubMed Central

    Cao, Chen; Wang, Guishen; Liu, An; Xu, Shutan; Wang, Lincong; Zou, Shuxue

    2016-01-01

    The assignment of secondary structure elements in proteins is a key step in the analysis of their structures and functions. We have developed an algorithm, SACF (secondary structure assignment based on Cα fragments), for secondary structure element (SSE) assignment based on the alignment of Cα backbone fragments with central poses derived by clustering known SSE fragments. The assignment algorithm consists of three steps: First, the outlier fragments on known SSEs are detected. Next, the remaining fragments are clustered to obtain the central fragments for each cluster. Finally, the central fragments are used as a template to make assignments. Following a large-scale comparison of 11 secondary structure assignment methods, SACF, KAKSI and PROSS are found to have similar agreement with DSSP, while PCASSO agrees with DSSP best. SACF and PCASSO show preference to reducing residues in N and C cap regions, whereas KAKSI, P-SEA and SEGNO tend to add residues to the terminals when DSSP assignment is taken as standard. Moreover, our algorithm is able to assign subtle helices (310-helix, π-helix and left-handed helix) and make uniform assignments, as well as to detect rare SSEs in β-sheets or long helices as outlier fragments from other programs. The structural uniformity should be useful for protein structure classification and prediction, while outlier fragments underlie the structure–function relationship. PMID:26978354

  2. NMR Methods for Characterization of RNA Secondary Structure.

    PubMed

    Kennedy, Scott D

    2016-01-01

    Knowledge of RNA secondary structure is often sufficient to identify relationships between the structure of RNA and processing pathways, and the design of therapeutics. Nuclear magnetic resonance (NMR) can identify types of nucleotide base pairs and the sequence, thus limiting possible secondary structures. Because NMR experiments, like chemical mapping, are performed in solution, not in single crystals, experiments can be initiated as soon as the biomolecule is expressed and purified. This chapter summarizes NMR methods that permit rapid identification of RNA secondary structure, information that can be used as supplements to chemical mapping, and/or as preliminary steps required for 3D structure determination. The primary aim is to provide guidelines to enable a researcher with minimal knowledge of NMR to quickly extract secondary structure information from basic datasets. Instrumental and sample considerations that can maximize data quality are discussed along with some details for optimal data acquisition and processing parameters. Approaches for identifying base pair types in both unlabeled and isotopically labeled RNA are covered. Common problems, such as missing signals and overlaps, and approaches to address them are considered. Programs under development for merging NMR data with structure prediction algorithms are briefly discussed. PMID:27665604

  3. Feedback regulation of ribosomal protein gene expression in Escherichia coli: structural homology of ribosomal RNA and ribosomal protein MRNA.

    PubMed Central

    Nomura, M; Yates, J L; Dean, D; Post, L E

    1980-01-01

    Certain ribosomal proteins (r proteins) in Escherichia coli, such as S4 and S7, function as feedback repressors in the regulation of r-protein synthesis. These proteins inhibit the translation of their own mRNA. The repressor r proteins so far identified are also known to bind specifically to rRNA at an initial stage in ribosome assembly. We have found structural homology between the S7 binding region on 16S rRNA and a region of the mRNA where S7 acts as a translational repressor. Similarly, there is structural homology between one of the reported S4 binding regions on 16S rRNA and the mRNA target site for S4. The observed homology supports the concept that regulation by repressor r proteins is based on competition between rRNA and mRNA for these proteins and that the same structural features and of the r proteins are used in their interactions with both rRNA and mRNA. PMID:7012833

  4. Feedback regulation of ribosomal protein gene expression in Escherichia coli: structural homology of ribosomal RNA and ribosomal protein MRNA.

    PubMed

    Nomura, M; Yates, J L; Dean, D; Post, L E

    1980-12-01

    Certain ribosomal proteins (r proteins) in Escherichia coli, such as S4 and S7, function as feedback repressors in the regulation of r-protein synthesis. These proteins inhibit the translation of their own mRNA. The repressor r proteins so far identified are also known to bind specifically to rRNA at an initial stage in ribosome assembly. We have found structural homology between the S7 binding region on 16S rRNA and a region of the mRNA where S7 acts as a translational repressor. Similarly, there is structural homology between one of the reported S4 binding regions on 16S rRNA and the mRNA target site for S4. The observed homology supports the concept that regulation by repressor r proteins is based on competition between rRNA and mRNA for these proteins and that the same structural features and of the r proteins are used in their interactions with both rRNA and mRNA.

  5. Identification of Secondary Structure Elements in Intermediate Resolution Density Maps

    PubMed Central

    Baker, Matthew L.; Ju, Tao; Chiu, Wah

    2007-01-01

    An increasing number of structural studies of large macromolecular complexes, both in X-ray crystallography and electron cryomicroscopy, have resulted in intermediate resolution (5–10 Å) structures. Despite being limited in resolution, significant structural and functional information may be extractable from these maps. To aid in the analysis and annotation of these complexes, we have developed SSEhunter, a tool for the quantitative detection of α-helices and β-sheets. Based on density skeletonization, local geometry calculations and a template-based search, SSEhunter has been tested and validated on a variety of simulated and authentic subnanometer resolution density maps. The result is a robust, user-friendly approach that allows users to quickly visualize, assess and annotate intermediate resolution density maps. Beyond secondary structure element identification, the skeletonization algorithm in SSEhunter provides secondary structure topology, potentially useful in leading to structural models of individual molecular components directly from the density. PMID:17223528

  6. Structural basis for specific recognition of multiple mRNA targets by a PUF regulatory protein

    SciTech Connect

    Wang, Yeming; Opperman, Laura; Wickens, Marvin; Tanaka Hall, Traci M.

    2011-11-02

    Caenorhabditis elegans fem-3 binding factor (FBF) is a founding member of the PUMILIO/FBF (PUF) family of mRNA regulatory proteins. It regulates multiple mRNAs critical for stem cell maintenance and germline development. Here, we report crystal structures of FBF in complex with 6 different 9-nt RNA sequences, including elements from 4 natural mRNAs. These structures reveal that FBF binds to conserved bases at positions 1-3 and 7-8. The key specificity determinant of FBF vs. other PUF proteins lies in positions 4-6. In FBF/RNA complexes, these bases stack directly with one another and turn away from the RNA-binding surface. A short region of FBF is sufficient to impart its unique specificity and lies directly opposite the flipped bases. We suggest that this region imposes a flattened curvature on the protein; hence, the requirement for the additional nucleotide. The principles of FBF/RNA recognition suggest a general mechanism by which PUF proteins recognize distinct families of RNAs yet exploit very nearly identical atomic contacts in doing so.

  7. Probing dimensionality beyond the linear sequence of mRNA.

    PubMed

    Del Campo, Cristian; Ignatova, Zoya

    2016-05-01

    mRNA is a nexus entity between DNA and translating ribosomes. Recent developments in deep sequencing technologies coupled with structural probing have revealed new insights beyond the classic role of mRNA and place it more centrally as a direct effector of a variety of processes, including translation, cellular localization, and mRNA degradation. Here, we highlight emerging approaches to probe mRNA secondary structure on a global transcriptome-wide level and compare their potential and resolution. Combined approaches deliver a richer and more complex picture. While our understanding on the effect of secondary structure for various cellular processes is quite advanced, the next challenge is to unravel more complex mRNA architectures and tertiary interactions. PMID:26650615

  8. Structure-function Studies of Nucleocytoplasmic Transport of Retroviral Genomic RNA by mRNA Export Factor TAP

    SciTech Connect

    M Teplova; L Wohlbold; N Khin; E Izaurralde; D Patel

    2011-12-31

    mRNA export is mediated by the TAP-p15 heterodimer, which belongs to the family of NTF2-like export receptors. TAP-p15 heterodimers also bind to the constitutive transport element (CTE) present in simian type D retroviral RNAs, and they mediate the export of viral unspliced RNAs to the host cytoplasm. We have solved the crystal structure of the RNA recognition and leucine-rich repeat motifs of TAP bound to one symmetrical half of the CTE RNA. L-shaped conformations of protein and RNA are involved in a mutual molecular embrace on complex formation. We have monitored the impact of structure-guided mutations on binding affinities in vitro and transport assays in vivo. Our studies define the principles by which CTE RNA subverts the mRNA export receptor TAP, thereby facilitating the nuclear export of viral genomic RNAs, and, more generally, provide insights on cargo RNA recognition by mRNA export receptors.

  9. The Structural Basis for mRNA Recognition and Cleavage by the Ribosome-Dependent Endonuclease RelE

    PubMed Central

    Neubauer, Cajetan; Gao, Yong-Gui; Andersen, Kasper R.; Dunham, Christine M.; Kelley, Ann C.; Hentschel, Jendrik; Gerdes, Kenn; Ramakrishnan, V.; Brodersen, Ditlev E.

    2009-01-01

    Summary Translational control is widely used to adjust gene expression levels. During the stringent response in bacteria, mRNA is degraded on the ribosome by the ribosome-dependent endonuclease, RelE. The molecular basis for recognition of the ribosome and mRNA by RelE and the mechanism of cleavage are unknown. Here, we present crystal structures of E. coli RelE in isolation (2.5 Å) and bound to programmed Thermus thermophilus 70S ribosomes before (3.3 Å) and after (3.6 Å) cleavage. RelE occupies the A site and causes cleavage of mRNA after the second nucleotide of the codon by reorienting and activating the mRNA for 2′-OH-induced hydrolysis. Stacking of A site codon bases with conserved residues in RelE and 16S rRNA explains the requirement for the ribosome in catalysis and the subtle sequence specificity of the reaction. These structures provide detailed insight into the translational regulation on the bacterial ribosome by mRNA cleavage. PMID:20005802

  10. Functional and Structural Analysis of the Internal Ribosome Entry Site Present in the mRNA of Natural Variants of the HIV-1

    PubMed Central

    Vallejos, Maricarmen; Carvajal, Felipe; Pino, Karla; Navarrete, Camilo; Ferres, Marcela; Huidobro-Toro, Juan Pablo; Sargueil, Bruno; López-Lastra, Marcelo

    2012-01-01

    The 5′untranslated regions (UTR) of the full length mRNA of the HIV-1 proviral clones pNL4.3 and pLAI, harbor an internal ribosomal entry site (IRES). In this study we extend this finding by demonstrating that the mRNA 5′UTRs of natural variants of HIV-1 also exhibit IRES-activity. Cap-independent translational activity was demonstrated using bicistronic mRNAs in HeLa cells and in Xenopus laevis oocytes. The possibility that expression of the downstream cistron in these constructs was due to alternative splicing or to cryptic promoter activity was ruled out. The HIV-1 variants exhibited significant 5′UTR nucleotide diversity with respect to the control sequence recovered from pNL4.3. Interestingly, translational activity from the 5′UTR of some of the HIV-1 variants was enhanced relative to that observed for the 5′UTR of pNL4.3. In an attempt to explain these findings we probed the secondary structure of the variant HIV-1 5′UTRs using enzymatic and chemical approaches. Yet subsequent structural analyses did not reveal significant variations when compared to the pNL4.3-5′UTR. Thus, the increased IRES-activity observed for some of the HIV-1 variants cannot be ascribed to a specific structural modification. A model to explain these findings is proposed. PMID:22496887

  11. Drosophila germ granules are structured and contain homotypic mRNA clusters

    PubMed Central

    Trcek, Tatjana; Grosch, Markus; York, Andrew; Shroff, Hari; Lionnet, Timothée; Lehmann, Ruth

    2015-01-01

    Germ granules, specialized ribonucleoprotein particles, are a hallmark of all germ cells. In Drosophila, an estimated 200 mRNAs are enriched in the germ plasm, and some of these have important, often conserved roles in germ cell formation, specification, survival and migration. How mRNAs are spatially distributed within a germ granule and whether their position defines functional properties is unclear. Here we show, using single-molecule FISH and structured illumination microscopy, a super-resolution approach, that mRNAs are spatially organized within the granule whereas core germ plasm proteins are distributed evenly throughout the granule. Multiple copies of single mRNAs organize into ‘homotypic clusters' that occupy defined positions within the center or periphery of the granule. This organization, which is maintained during embryogenesis and independent of the translational or degradation activity of mRNAs, reveals new regulatory mechanisms for germ plasm mRNAs that may be applicable to other mRNA granules. PMID:26242323

  12. JPred4: a protein secondary structure prediction server.

    PubMed

    Drozdetskiy, Alexey; Cole, Christian; Procter, James; Barton, Geoffrey J

    2015-07-01

    JPred4 (http://www.compbio.dundee.ac.uk/jpred4) is the latest version of the popular JPred protein secondary structure prediction server which provides predictions by the JNet algorithm, one of the most accurate methods for secondary structure prediction. In addition to protein secondary structure, JPred also makes predictions of solvent accessibility and coiled-coil regions. The JPred service runs up to 94 000 jobs per month and has carried out over 1.5 million predictions in total for users in 179 countries. The JPred4 web server has been re-implemented in the Bootstrap framework and JavaScript to improve its design, usability and accessibility from mobile devices. JPred4 features higher accuracy, with a blind three-state (α-helix, β-strand and coil) secondary structure prediction accuracy of 82.0% while solvent accessibility prediction accuracy has been raised to 90% for residues <5% accessible. Reporting of results is enhanced both on the website and through the optional email summaries and batch submission results. Predictions are now presented in SVG format with options to view full multiple sequence alignments with and without gaps and insertions. Finally, the help-pages have been updated and tool-tips added as well as step-by-step tutorials. PMID:25883141

  13. JPred4: a protein secondary structure prediction server

    PubMed Central

    Drozdetskiy, Alexey; Cole, Christian; Procter, James; Barton, Geoffrey J.

    2015-01-01

    JPred4 (http://www.compbio.dundee.ac.uk/jpred4) is the latest version of the popular JPred protein secondary structure prediction server which provides predictions by the JNet algorithm, one of the most accurate methods for secondary structure prediction. In addition to protein secondary structure, JPred also makes predictions of solvent accessibility and coiled-coil regions. The JPred service runs up to 94 000 jobs per month and has carried out over 1.5 million predictions in total for users in 179 countries. The JPred4 web server has been re-implemented in the Bootstrap framework and JavaScript to improve its design, usability and accessibility from mobile devices. JPred4 features higher accuracy, with a blind three-state (α-helix, β-strand and coil) secondary structure prediction accuracy of 82.0% while solvent accessibility prediction accuracy has been raised to 90% for residues <5% accessible. Reporting of results is enhanced both on the website and through the optional email summaries and batch submission results. Predictions are now presented in SVG format with options to view full multiple sequence alignments with and without gaps and insertions. Finally, the help-pages have been updated and tool-tips added as well as step-by-step tutorials. PMID:25883141

  14. RNA Movies 2: sequential animation of RNA secondary structures.

    PubMed

    Kaiser, Alexander; Krüger, Jan; Evers, Dirk J

    2007-07-01

    RNA Movies is a simple, yet powerful visualization tool in likeness to a media player application, which enables to browse sequential paths through RNA secondary structure landscapes. It can be used to visualize structural rearrangement processes of RNA, such as folding pathways and conformational switches, or to browse lists of alternative structure candidates. Besides extending the feature set, retaining and improving usability and availability in the web is the main aim of this new version. RNA Movies now supports the DCSE and RNAStructML input formats besides its own RNM format. Pseudoknots and 'entangled helices' can be superimposed on the RNA secondary structure layout. Publication quality output is provided through the Scalable Vector Graphics output format understood by most current drawing programs. The software has been completely re-implemented in Java to enable pure client-side operation as applet and web-start application available at the Bielefeld Bioinformatics Server http://bibiserv.techfak.uni-bielefeld.de/rnamovies. PMID:17567618

  15. Refinement by shifting secondary structure elements improves sequence alignments.

    PubMed

    Tong, Jing; Pei, Jimin; Otwinowski, Zbyszek; Grishin, Nick V

    2015-03-01

    Constructing a model of a query protein based on its alignment to a homolog with experimentally determined spatial structure (the template) is still the most reliable approach to structure prediction. Alignment errors are the main bottleneck for homology modeling when the query is distantly related to the template. Alignment methods often misalign secondary structural elements by a few residues. Therefore, better alignment solutions can be found within a limited set of local shifts of secondary structures. We present a refinement method to improve pairwise sequence alignments by evaluating alignment variants generated by local shifts of template-defined secondary structures. Our method SFESA is based on a novel scoring function that combines the profile-based sequence score and the structure score derived from residue contacts in a template. Such a combined score frequently selects a better alignment variant among a set of candidate alignments generated by local shifts and leads to overall increase in alignment accuracy. Evaluation of several benchmarks shows that our refinement method significantly improves alignments made by automatic methods such as PROMALS, HHpred and CNFpred. The web server is available at http://prodata.swmed.edu/sfesa. PMID:25546158

  16. Refinement by shifting secondary structure elements improves sequence alignments

    PubMed Central

    Tong, Jing; Pei, Jimin; Otwinowski, Zbyszek; Grishin, Nick V.

    2015-01-01

    Constructing a model of a query protein based on its alignment to a homolog with experimentally determined spatial structure (the template) is still the most reliable approach to structure prediction. Alignment errors are the main bottleneck for homology modeling when the query is distantly related to the template. Alignment methods often misalign secondary structural elements by a few residues. Therefore, better alignment solutions can be found within a limited set of local shifts of secondary structures. We present a refinement method to improve pairwise sequence alignments by evaluating alignment variants generated by local shifts of template-defined secondary structures. Our method SFESA is based on a novel scoring function that combines the profile-based sequence score and the structure score derived from residue contacts in a template. Such a combined score frequently selects a better alignment variant among a set of candidate alignments generated by local shifts and leads to overall increase in alignment accuracy. Evaluation of several benchmarks shows that our refinement method significantly improves alignments made by automatic methods such as PROMALS, HHpred and CNFpred. The web server is available at http://prodata.swmed.edu/sfesa. PMID:25546158

  17. Control of mammalian translation by mRNA structure near caps.

    PubMed

    Babendure, Jeremy R; Babendure, Jennie L; Ding, Jian-Hua; Tsien, Roger Y

    2006-05-01

    The scanning model of RNA translation proposes that highly stable secondary structures within mRNAs can inhibit translation, while structures of lower thermal stability also affect translation if close enough to the 5' methyl G cap. However, only fragmentary information is available about the dependence of translation efficiency in live mammalian cells on the thermodynamic stability, location, and GC content of RNA structures in the 5'-untranslated region. We devised a two-color fluorescence assay for translation efficiency in single live cells and compared a wide range of hairpins with predicted thermal stabilities ranging from -10 to -50 kcal/mol and 5' G cap-to-hairpin distances of 1-46 bases. Translation efficiency decreased abruptly as hairpin stabilities increased from deltaG = -25 to -35 kcal/mol. Shifting a hairpin as little as nine bases relative to the 5' cap could modulate translation more than 50-fold. Increasing GC content diminished translation efficiency when predicted thermal stability and cap-to-hairpin distances were held constant. We additionally found naturally occurring 5'-untranslated regions affected translation differently in live cells compared with translation in in vitro lysates. Our study will assist scientists in designing experiments that deliberately modulate mammalian translation with designed 5' UTRs.

  18. Computation of statistical secondary structure of nucleic acids.

    PubMed Central

    Yamamoto, K; Kitamura, Y; Yoshikura, H

    1984-01-01

    This paper presents a computer analysis of statistical secondary structure of nucleic acids. For a given single stranded nucleic acid, we generated "structure map" which included all the annealing structures in the sequence. The map was transformed into "energy map" by rough approximation; here, the energy level of every pairing structure consisting of more than 2 successive nucleic acid pairs was calculated. By using the "energy map", the probability of occurrence of each annealed structure was computed, i.e., the structure was computed statistically. The basis of computation was the 8-queen problem in the chess game. The validity of our computer programme was checked by computing tRNA structure which has been well established. Successful application of this programme to small nuclear RNAs of various origins is demonstrated. PMID:6198622

  19. Data-directed RNA secondary structure prediction using probabilistic modeling.

    PubMed

    Deng, Fei; Ledda, Mirko; Vaziri, Sana; Aviran, Sharon

    2016-08-01

    Structure dictates the function of many RNAs, but secondary RNA structure analysis is either labor intensive and costly or relies on computational predictions that are often inaccurate. These limitations are alleviated by integration of structure probing data into prediction algorithms. However, existing algorithms are optimized for a specific type of probing data. Recently, new chemistries combined with advances in sequencing have facilitated structure probing at unprecedented scale and sensitivity. These novel technologies and anticipated wealth of data highlight a need for algorithms that readily accommodate more complex and diverse input sources. We implemented and investigated a recently outlined probabilistic framework for RNA secondary structure prediction and extended it to accommodate further refinement of structural information. This framework utilizes direct likelihood-based calculations of pseudo-energy terms per considered structural context and can readily accommodate diverse data types and complex data dependencies. We use real data in conjunction with simulations to evaluate performances of several implementations and to show that proper integration of structural contexts can lead to improvements. Our tests also reveal discrepancies between real data and simulations, which we show can be alleviated by refined modeling. We then propose statistical preprocessing approaches to standardize data interpretation and integration into such a generic framework. We further systematically quantify the information content of data subsets, demonstrating that high reactivities are major drivers of SHAPE-directed predictions and that better understanding of less informative reactivities is key to further improvements. Finally, we provide evidence for the adaptive capability of our framework using mock probe simulations.

  20. Integrating chemical footprinting data into RNA secondary structure prediction.

    PubMed

    Zarringhalam, Kourosh; Meyer, Michelle M; Dotu, Ivan; Chuang, Jeffrey H; Clote, Peter

    2012-01-01

    Chemical and enzymatic footprinting experiments, such as shape (selective 2'-hydroxyl acylation analyzed by primer extension), yield important information about RNA secondary structure. Indeed, since the [Formula: see text]-hydroxyl is reactive at flexible (loop) regions, but unreactive at base-paired regions, shape yields quantitative data about which RNA nucleotides are base-paired. Recently, low error rates in secondary structure prediction have been reported for three RNAs of moderate size, by including base stacking pseudo-energy terms derived from shape data into the computation of minimum free energy secondary structure. Here, we describe a novel method, RNAsc (RNA soft constraints), which includes pseudo-energy terms for each nucleotide position, rather than only for base stacking positions. We prove that RNAsc is self-consistent, in the sense that the nucleotide-specific probabilities of being unpaired in the low energy Boltzmann ensemble always become more closely correlated with the input shape data after application of RNAsc. From this mathematical perspective, the secondary structure predicted by RNAsc should be 'correct', in as much as the shape data is 'correct'. We benchmark RNAsc against the previously mentioned method for eight RNAs, for which both shape data and native structures are known, to find the same accuracy in 7 out of 8 cases, and an improvement of 25% in one case. Furthermore, we present what appears to be the first direct comparison of shape data and in-line probing data, by comparing yeast asp-tRNA shape data from the literature with data from in-line probing experiments we have recently performed. With respect to several criteria, we find that shape data appear to be more robust than in-line probing data, at least in the case of asp-tRNA.

  1. COOLAIR Antisense RNAs Form Evolutionarily Conserved Elaborate Secondary Structures.

    PubMed

    Hawkes, Emily J; Hennelly, Scott P; Novikova, Irina V; Irwin, Judith A; Dean, Caroline; Sanbonmatsu, Karissa Y

    2016-09-20

    There is considerable debate about the functionality of long non-coding RNAs (lncRNAs). Lack of sequence conservation has been used to argue against functional relevance. We investigated antisense lncRNAs, called COOLAIR, at the A. thaliana FLC locus and experimentally determined their secondary structure. The major COOLAIR variants are highly structured, organized by exon. The distally polyadenylated transcript has a complex multi-domain structure, altered by a single non-coding SNP defining a functionally distinct A. thaliana FLC haplotype. The A. thaliana COOLAIR secondary structure was used to predict COOLAIR exons in evolutionarily divergent Brassicaceae species. These predictions were validated through chemical probing and cloning. Despite the relatively low nucleotide sequence identity, the structures, including multi-helix junctions, show remarkable evolutionary conservation. In a number of places, the structure is conserved through covariation of a non-contiguous DNA sequence. This structural conservation supports a functional role for COOLAIR transcripts rather than, or in addition to, antisense transcription. PMID:27653675

  2. Using Circular Dichroism Spectra to Estimate Protein Secondary Structure

    SciTech Connect

    Greenfield, N.

    2006-01-01

    Circular dichroism (CD) is an excellent tool for rapid determination of the secondary structure and folding properties of proteins that have been obtained using recombinant techniques or purified from tissues. The most widely used applications of protein CD are to determine whether an expressed, purified protein is folded, or if a mutation affects its conformation or stability. In addition, it can be used to study protein interactions. This protocol details the basic steps of obtaining and interpreting CD data and methods for analyzing spectra to estimate the secondary structural composition of proteins. CD has the advantage that it is that measurements may be made on multiple samples containing 20 {mu}g or less of proteins in physiological buffers in a few hours. However, it does not give the residue-specific information that can be obtained by X-ray crystallography or NMR.

  3. Using circular dichroism spectra to estimate protein secondary structure

    PubMed Central

    Greenfield, Norma J.

    2009-01-01

    Circular dichroism (CD) is an excellent tool for rapid determination of the secondary structure and folding properties of proteins that have been obtained using recombinant techniques or purified from tissues. The most widely used applications of protein CD are to determine whether an expressed, purified protein is folded, or if a mutation affects its conformation or stability. In addition, it can be used to study protein interactions. This protocol details the basic steps of obtaining and interpreting CD data and methods for analyzing spectra to estimate the secondary structural composition of proteins. CD has the advantage that it is that measurements may be made on multiple samples containing 20 µg or less of proteins in physiological buffers in a few hours. However, it does not give the residue-specific information that can be obtained by X-ray crystallography or NMR. PMID:17406547

  4. Secondary Fast Magnetoacoustic Waves Trapped in Randomly Structured Plasmas

    NASA Astrophysics Data System (ADS)

    Yuan, Ding; Li, Bo; Walsh, Robert W.

    2016-09-01

    Fast magnetoacoustic waves are an important tool for inferring parameters of the solar atmosphere. We numerically simulate the propagation of fast wave pulses in randomly structured plasmas that mimic the highly inhomogeneous solar corona. A network of secondary waves is formed by a series of partial reflections and transmissions. These secondary waves exhibit quasi-periodicities in both time and space. Since the temporal and spatial periods are related simply through the speed of the fast wave, we quantify the properties of secondary waves by examining the dependence of the average temporal period (\\bar{p}) on the initial pulse width (w 0) and studying the density contrast ({δ }ρ ) and correlation length (L c ) that characterize the randomness of the equilibrium density profiles. For small-amplitude pulses, {δ }ρ does not alter \\bar{p} significantly. Large-amplitude pulses, on the other hand, enhance the density contrast when {δ }ρ is small but have a smoothing effect when {δ }ρ is sufficiently large. We found that \\bar{p} scales linearly with L c and that the scaling factor is larger for a narrower pulse. However, in terms of the absolute values of \\bar{p}, broader pulses generate secondary waves with longer periods, and this effect is stronger in random plasmas with shorter correlation lengths. Secondary waves carry the signatures of both the leading wave pulse and the background plasma. Our study may find applications in magnetohydrodynamic seismology by exploiting the secondary waves detected in the dimming regions after coronal mass ejections or extreme ultraviolet waves.

  5. Distinct circular dichroism spectroscopic signatures of polyproline II and unordered secondary structures: Applications in secondary structure analyses

    PubMed Central

    Lopes, Jose L S; Miles, Andrew J; Whitmore, Lee; Wallace, B A

    2014-01-01

    Circular dichroism (CD) spectroscopy is a valuable method for defining canonical secondary structure contents of proteins based on empirically-defined spectroscopic signatures derived from proteins with known three-dimensional structures. Many proteins identified as being “Intrinsically Disordered Proteins” have a significant amount of their structure that is neither sheet, helix, nor turn; this type of structure is often classified by CD as “other”, “random coil”, “unordered”, or “disordered”. However the “other” category can also include polyproline II (PPII)-type structures, whose spectral properties have not been well-distinguished from those of unordered structures. In this study, synchrotron radiation circular dichroism spectroscopy was used to investigate the spectral properties of collagen and polyproline, which both contain PPII-type structures. Their native spectra were compared as representatives of PPII structures. In addition, their spectra before and after treatment with various conditions to produce unfolded or denatured structures were also compared, with the aim of defining the differences between CD spectra of PPII and disordered structures. We conclude that the spectral features of collagen are more appropriate than those of polyproline for use as the representative spectrum for PPII structures present in typical amino acid-containing proteins, and that the single most characteristic spectroscopic feature distinguishing a PPII structure from a disordered structure is the presence of a positive peak around 220nm in the former but not in the latter. These spectra are now available for inclusion in new reference data sets used for CD analyses of the secondary structures of soluble proteins. PMID:25262612

  6. Coating concrete secondary containment structures exposed to agrichemicals

    SciTech Connect

    Broder, M.F.; Nguyen, D.T.

    1995-06-01

    Concrete has traditionally been the material of choice for building secondary containment structures because it is relatively inexpensive and has structural properties which make it ideal for supporting the loads of vehicles and large tanks. However, concrete`s chemical properties make it susceptible to corrosion by some common fertilizers. Though fairly impervious to water movement, concrete is easily penetrated by vapors and solvents. It is also prone to cracking. For these reasons, the Environmental Protection Agency (EPA) believes that concrete alone may not provide an effective barrier to pesticide movement and has proposed that concrete in pesticide secondary containment structures be sealed or coated to reduce its permeability. Some state secondary containment regulations require that concrete exposed to fertilizers and pesticides be sealed or protected with a coating. Lacking guidelines, some retailers have used penetrating sealants to satisfy the law, even though these products provide little protection from chemical attack nor do they prevent pesticide egress. Other retailers who have applied thick film coatings which were properly selected have had disastrous results because the application was poorly done. Consequently, much skepticism exists regarding the performance and benefit of protective coatings.

  7. Identification of local variations within secondary structures of proteins.

    PubMed

    Kumar, Prasun; Bansal, Manju

    2015-05-01

    Secondary-structure elements (SSEs) play an important role in the folding of proteins. Identification of SSEs in proteins is a common problem in structural biology. A new method, ASSP (Assignment of Secondary Structure in Proteins), using only the path traversed by the C(α) atoms has been developed. The algorithm is based on the premise that the protein structure can be divided into continuous or uniform stretches, which can be defined in terms of helical parameters, and depending on their values the stretches can be classified into different SSEs, namely α-helices, 310-helices, π-helices, extended β-strands and polyproline II (PPII) and other left-handed helices. The methodology was validated using an unbiased clustering of these parameters for a protein data set consisting of 1008 protein chains, which suggested that there are seven well defined clusters associated with different SSEs. Apart from α-helices and extended β-strands, 310-helices and π-helices were also found to occur in substantial numbers. ASSP was able to discriminate non-α-helical segments from flanking α-helices, which were often identified as part of α-helices by other algorithms. ASSP can also lead to the identification of novel SSEs. It is believed that ASSP could provide a better understanding of the finer nuances of protein secondary structure and could make an important contribution to the better understanding of comparatively less frequently occurring structural motifs. At the same time, it can contribute to the identification of novel SSEs. A standalone version of the program for the Linux as well as the Windows operating systems is freely downloadable and a web-server version is also available at http://nucleix.mbu.iisc.ernet.in/assp/index.php.

  8. Identification of local variations within secondary structures of proteins.

    PubMed

    Kumar, Prasun; Bansal, Manju

    2015-05-01

    Secondary-structure elements (SSEs) play an important role in the folding of proteins. Identification of SSEs in proteins is a common problem in structural biology. A new method, ASSP (Assignment of Secondary Structure in Proteins), using only the path traversed by the C(α) atoms has been developed. The algorithm is based on the premise that the protein structure can be divided into continuous or uniform stretches, which can be defined in terms of helical parameters, and depending on their values the stretches can be classified into different SSEs, namely α-helices, 310-helices, π-helices, extended β-strands and polyproline II (PPII) and other left-handed helices. The methodology was validated using an unbiased clustering of these parameters for a protein data set consisting of 1008 protein chains, which suggested that there are seven well defined clusters associated with different SSEs. Apart from α-helices and extended β-strands, 310-helices and π-helices were also found to occur in substantial numbers. ASSP was able to discriminate non-α-helical segments from flanking α-helices, which were often identified as part of α-helices by other algorithms. ASSP can also lead to the identification of novel SSEs. It is believed that ASSP could provide a better understanding of the finer nuances of protein secondary structure and could make an important contribution to the better understanding of comparatively less frequently occurring structural motifs. At the same time, it can contribute to the identification of novel SSEs. A standalone version of the program for the Linux as well as the Windows operating systems is freely downloadable and a web-server version is also available at http://nucleix.mbu.iisc.ernet.in/assp/index.php. PMID:25945573

  9. How well are protein structures annotated in secondary databases?

    PubMed

    Rother, Kristian; Michalsky, Elke; Leser, Ulf

    2005-09-01

    We investigated to what extent Protein Data Bank (PDB) entries are annotated with second-party information based on existing cross-references between PDB and 15 other databases. We report 2 interesting findings. First, there is a clear "annotation gap" for structures less than 7 years old for secondary databases that are manually curated. Second, the examined databases overlap with each other quite well, dividing the PDB into 2 well-annotated thirds and one poorly annotated third. Both observations should be taken into account in any study depending on the selection of protein structures by their annotation.

  10. Protein backbone torsion angle-based structure comparison and secondary structure database web server.

    PubMed

    Jung, Sunghoon; Bae, Se-Eun; Ahn, Insung; Son, Hyeon S

    2013-09-01

    Structural information has been a major concern for biological and pharmaceutical studies for its intimate relationship to the function of a protein. Three-dimensional representation of the positions of protein atoms is utilized among many structural information repositories that have been published. The reliability of the torsional system, which represents the native processes of structural change in the structural analysis, was partially proven with previous structural alignment studies. Here, a web server providing structural information and analysis based on the backbone torsional representation of a protein structure is newly introduced. The web server offers functions of secondary structure database search, secondary structure calculation, and pair-wise protein structure comparison, based on a backbone torsion angle representation system. Application of the implementation in pair-wise structural alignment showed highly accurate results. The information derived from this web server might be further utilized in the field of ab initio protein structure modeling or protein homology-related analyses.

  11. Beta-integrin of Anopheles gambiae: mRNA cloning and analysis of structure and expression.

    PubMed

    Mahairaki, V; Lycett, G; Blass, C; Louis, C

    2001-06-01

    We have isolated an mRNA encoding a beta integrin subunit of the malaria mosquito Anopheles gambiae. Our analysis predicts a protein that is very similar to betaPS, the fruitfly orthologue. The gene is expressed during all developmental stages and it is found in all body parts, including the midgut. Finally, the expression of the gene does not seem to be modulated during blood meals, except for a substantial increase 48 h posthaematophagy, when digestion is nearly complete. PMID:11437913

  12. HOTAIR forms an intricate and modular secondary structure

    PubMed Central

    Somarowthu, Srinivas; Legiewicz, Michal; Chillón, Isabel; Marcia, Marco; Liu, Fei; Pyle, Anna Marie

    2015-01-01

    SUMMARY Long non-coding RNAs (lncRNAs) have recently emerged as key players in fundamental cellular processes and diseases, but their functions are poorly understood. HOTAIR is a 2,148-nucleotide-long lncRNA molecule involved in physiological epidermal development and in pathogenic cancer progression, where it has been demonstrated to repress tumor and metastasis suppressor genes. To gain insights into the molecular mechanisms of HOTAIR, we purified it in a stable and homogenous form in vitro and we determined its functional secondary structure through chemical probing and phylogenetic analysis. The HOTAIR structure reveals a degree of structural organization comparable to well-folded RNAs, like the group II intron, rRNA or lncRNA steroid receptor activator. It is composed of four independently-folding modules, two of which correspond to predicted protein-binding domains. Secondary structure elements that surround protein-binding motifs are evolutionarily conserved. Our work serves as a guide for “navigating” through the lncRNA HOTAIR and ultimately for understanding its function. PMID:25866246

  13. Structural basis for binding the TREX2 complex to nuclear pores, GAL1 localisation and mRNA export.

    PubMed

    Jani, Divyang; Valkov, Eugene; Stewart, Murray

    2014-06-01

    The conserved Sac3:Thp1:Sem1:Sus1:Cdc31 (TREX2) complex binds to nuclear pore complexes (NPCs) and, in addition to integrating mRNA nuclear export with preceding steps in the gene expression pathway, facilitates re-positioning of highly regulated actively transcribing genes (such as GAL1) to NPCs. Although TREX2 is thought to bind NPC protein Nup1, defining the precise role of this interaction has been frustrated by the complex pleiotropic phenotype exhibited by nup1Δ strains. To provide a structural framework for understanding the binding of TREX2 to NPCs and its function in the gene expression pathway, we have determined the structure of the Nup1:TREX2 interaction interface and used this information to engineer a Sac3 variant that impairs NPC binding while not compromising TREX2 assembly. This variant inhibited the NPC association of both de-repressed and activated GAL1 and also produced mRNA export and growth defects. These results indicate that the TREX2:Nup1 interaction facilitates the efficient nuclear export of bulk mRNA together with the re-positioning of GAL1 to NPCs that is required for transcriptional control that is mediated by removal of SUMO from repressors by NPC-bound Ulp1.

  14. Structural basis for binding the TREX2 complex to nuclear pores, GAL1 localisation and mRNA export.

    PubMed

    Jani, Divyang; Valkov, Eugene; Stewart, Murray

    2014-06-01

    The conserved Sac3:Thp1:Sem1:Sus1:Cdc31 (TREX2) complex binds to nuclear pore complexes (NPCs) and, in addition to integrating mRNA nuclear export with preceding steps in the gene expression pathway, facilitates re-positioning of highly regulated actively transcribing genes (such as GAL1) to NPCs. Although TREX2 is thought to bind NPC protein Nup1, defining the precise role of this interaction has been frustrated by the complex pleiotropic phenotype exhibited by nup1Δ strains. To provide a structural framework for understanding the binding of TREX2 to NPCs and its function in the gene expression pathway, we have determined the structure of the Nup1:TREX2 interaction interface and used this information to engineer a Sac3 variant that impairs NPC binding while not compromising TREX2 assembly. This variant inhibited the NPC association of both de-repressed and activated GAL1 and also produced mRNA export and growth defects. These results indicate that the TREX2:Nup1 interaction facilitates the efficient nuclear export of bulk mRNA together with the re-positioning of GAL1 to NPCs that is required for transcriptional control that is mediated by removal of SUMO from repressors by NPC-bound Ulp1. PMID:24705649

  15. Structure and expression of the human L-myc gene reveal a complex pattern of alternative mRNA processing

    SciTech Connect

    Kaye, F.; Battey, J.; Nau, M.; Brooks, B.; Seifter, E.; De Greve, J.; Birrer, M.; Sausville, E.; Minna, J.

    1988-01-01

    The authors' analyzed in detail the structure of the L-myc gene isolated from human placental DNA and characterized its expression in several small-cell lung cancer cell lines. The gene is composed of three exons and two introns spanning 6.6 kilobases in human DNA. Several distinct mRNA species are produced in all small-cell lung cancer cell lines that express L-myc. These transcripts are generated from a single gene by alternative splicing of introns 1 and 2 and by use of alternative polyadenylation signals. In some mRNAs that is a long open reading frame with a predicted translated protein of 364 residues. Amino acid sequence comparison with c-myc and N-myc demonstrated multiple discrete regions with extensive homology. In contrast, other mRNA transcripts, generated by alternative processing, could encode a truncated protein with a novel carboxy-terminal end.

  16. The antiviral drug ribavirin does not mimic the 7-methylguanosine moiety of the mRNA cap structure in vitro.

    PubMed

    Westman, Belinda; Beeren, Lisa; Grudzien, Ewa; Stepinski, Janusz; Worch, Remigiusz; Zuberek, Joanna; Jemielity, Jacek; Stolarski, Ryszard; Darzynkiewicz, Edward; Rhoads, Robert E; Preiss, Thomas

    2005-10-01

    The eukaryotic initiation factor eIF4E binds the mRNA 5' cap structure and has a central role during translational initiation. eIF4E and the mechanisms to control its activity have oncogenic properties and thus have become targets for anticancer drug development. A recent study (Kentsis et al. 2004) presented evidence that the antiviral nucleoside ribavirin and its phosphorylated derivatives were structural mimics of the mRNA cap, high-affinity ligands for eIF4E, and potent repressors of eIF4E-mediated cell transformation and tumor growth. Based on these findings, we tested ribavirin, ribavirin triphosphate (RTP), and the dinucleotide RpppG for their ability to inhibit translation in vitro. Surprisingly, the ribavirin-based compounds did not affect translation at concentrations where canonical cap analogs efficiently block cap-dependent translation. Using a set of reporter mRNAs that are translated via either cap-dependent or viral internal ribosome entry sites (IRES)-dependent initiation, we found that these ribavirin-containing compounds did inhibit translation at high (millimolar) concentrations, but there was no correlation of this inhibition with an eIF4E requirement for translation. The addition of a ribavirin-containing cap to mRNA did not stimulate translation. Fluorescence titration experiments with eIF4E and the nuclear cap-binding complex CBC indicated affinities for RTP and RpppG that were two to four orders of magnitude lower than those of m(7)GTP and m(7)GpppG. We conclude that, at least with respect to translation, ribavirin does not act in vitro as a functional mimic of the mRNA cap.

  17. PDZ Binding Domains, Structural Disorder and Phosphorylation: A Menage-a-trois Tailing Dcp2 mRNA Decapping Enzymes.

    PubMed

    Gunawardana, Dilantha

    2016-01-01

    Diverse cellular activities are mediated through the interaction of protein domains and their binding partners. One such protein domain widely distributed in the higher metazoan world is the PDZ domain, which facilitates abundant protein-protein interactions. The PDZ domain-PDZ binding domain interaction has been implicated in several pathologies including Alzheimer's disease, Parkinson's disease and Down syndrome. PDZ domains bind to C-terminal peptides/proteins which have either of the following combinations: S/T-X-hydrophobic-COOH for type I, hydrophobic-Xhydrophobic- COOH for type II, and D/E-X-hydrophobic-COOH for type III, although hydrophobicity in the termini form the key characteristic of the PDZ-binding domains. We identified and characterized a Dcp2 type mRNA decapping enzyme from Arabidopsis thaliana, a protein containing a putative PDZ-binding domain using mutagenesis and protein biochemistry. Now we are using bioinformatics to study the Cterminal end of mRNA decapping enzymes from complex metazoans with the aim of (1) identifying putative PDZ-binding domains (2) Correlating structural disorder with PDZ binding domains and (3) Demonstrating the presence of phosphorylation sites in C-terminal extremities of Dcp2 type mRNA decapping enzymes. It is proposed here that the trinity of PDZbinding domains, structural disorder and phosphorylation-susceptible sites are a feature of the Dcp2 family of decapping enzymes and perhaps is a wider trick in protein evolution where scaffolding/tethering is a requirement for localization and function. It is critical though laboratory-based supporting evidence is sought to back-up this bioinformatics exploration into tail regions of mRNA decapping enzymes. PMID:27151193

  18. The antiviral drug ribavirin does not mimic the 7-methylguanosine moiety of the mRNA cap structure in vitro

    PubMed Central

    WESTMAN, BELINDA; BEEREN, LISA; GRUDZIEN, EWA; STEPINSKI, JANUSZ; WORCH, REMIGIUSZ; ZUBEREK, JOANNA; JEMIELITY, JACEK; STOLARSKI, RYSZARD; DARZYNKIEWICZ, EDWARD; RHOADS, ROBERT E.; PREISS, THOMAS

    2005-01-01

    The eukaryotic initiation factor eIF4E binds the mRNA 5′ cap structure and has a central role during translational initiation. eIF4E and the mechanisms to control its activity have oncogenic properties and thus have become targets for anticancer drug development. A recent study (Kentsis et al. 2004) presented evidence that the antiviral nucleoside ribavirin and its phosphorylated derivatives were structural mimics of the mRNA cap, high-affinity ligands for eIF4E, and potent repressors of eIF4E-mediated cell transformation and tumor growth. Based on these findings, we tested ribavirin, ribavirin triphosphate (RTP), and the dinucleotide RpppG for their ability to inhibit translation in vitro. Surprisingly, the ribavirin-based compounds did not affect translation at concentrations where canonical cap analogs efficiently block cap-dependent translation. Using a set of reporter mRNAs that are translated via either cap-dependent or viral internal ribosome entry sites (IRES)-dependent initiation, we found that these ribavirin-containing compounds did inhibit translation at high (millimolar) concentrations, but there was no correlation of this inhibition with an eIF4E requirement for translation. The addition of a ribavirin-containing cap to mRNA did not stimulate translation. Fluorescence titration experiments with eIF4E and the nuclear cap-binding complex CBC indicated affinities for RTP and RpppG that were two to four orders of magnitude lower than those of m7GTP and m7GpppG. We conclude that, at least with respect to translation, ribavirin does not act in vitro as a functional mimic of the mRNA cap. PMID:16131589

  19. Protein secondary structure prediction using logic-based machine learning.

    PubMed

    Muggleton, S; King, R D; Sternberg, M J

    1992-10-01

    Many attempts have been made to solve the problem of predicting protein secondary structure from the primary sequence but the best performance results are still disappointing. In this paper, the use of a machine learning algorithm which allows relational descriptions is shown to lead to improved performance. The Inductive Logic Programming computer program, Golem, was applied to learning secondary structure prediction rules for alpha/alpha domain type proteins. The input to the program consisted of 12 non-homologous proteins (1612 residues) of known structure, together with a background knowledge describing the chemical and physical properties of the residues. Golem learned a small set of rules that predict which residues are part of the alpha-helices--based on their positional relationships and chemical and physical properties. The rules were tested on four independent non-homologous proteins (416 residues) giving an accuracy of 81% (+/- 2%). This is an improvement, on identical data, over the previously reported result of 73% by King and Sternberg (1990, J. Mol. Biol., 216, 441-457) using the machine learning program PROMIS, and of 72% using the standard Garnier-Osguthorpe-Robson method. The best previously reported result in the literature for the alpha/alpha domain type is 76%, achieved using a neural net approach. Machine learning also has the advantage over neural network and statistical methods in producing more understandable results. PMID:1480619

  20. Secondary Structure of Rat and Human Amylin across Force Fields.

    PubMed

    Hoffmann, Kyle Quynn; McGovern, Michael; Chiu, Chi-Cheng; de Pablo, Juan J

    2015-01-01

    The aggregation of human amylin has been strongly implicated in the progression of Type II diabetes. This 37-residue peptide forms a variety of secondary structures, including random coils, α-helices, and β-hairpins. The balance between these structures depends on the chemical environment, making amylin an ideal candidate to examine inherent biases in force fields. Rat amylin differs from human amylin by only 6 residues; however, it does not form fibrils. Therefore it provides a useful complement to human amylin in studies of the key events along the aggregation pathway. In this work, the free energy of rat and human amylin was determined as a function of α-helix and β-hairpin content for the Gromos96 53a6, OPLS-AA/L, CHARMM22/CMAP, CHARMM22*, Amberff99sb*-ILDN, and Amberff03w force fields using advanced sampling techniques, specifically bias exchange metadynamics. This work represents a first systematic attempt to evaluate the conformations and the corresponding free energy of a large, clinically relevant disordered peptide in solution across force fields. The NMR chemical shifts of rIAPP were calculated for each of the force fields using their respective free energy maps, allowing us to quantitatively assess their predictions. We show that the predicted distribution of secondary structures is sensitive to the choice of force-field: Gromos53a6 is biased towards β-hairpins, while CHARMM22/CMAP predicts structures that are overly α-helical. OPLS-AA/L favors disordered structures. Amberff99sb*-ILDN, AmberFF03w and CHARMM22* provide the balance between secondary structures that is most consistent with available experimental data. In contrast to previous reports, our findings suggest that the equilibrium conformations of human and rat amylin are remarkably similar, but that subtle differences arise in transient alpha-helical and beta-strand containing structures that the human peptide can more readily adopt. We hypothesize that these transient states enable

  1. Secondary Structure of Rat and Human Amylin across Force Fields.

    PubMed

    Hoffmann, Kyle Quynn; McGovern, Michael; Chiu, Chi-Cheng; de Pablo, Juan J

    2015-01-01

    The aggregation of human amylin has been strongly implicated in the progression of Type II diabetes. This 37-residue peptide forms a variety of secondary structures, including random coils, α-helices, and β-hairpins. The balance between these structures depends on the chemical environment, making amylin an ideal candidate to examine inherent biases in force fields. Rat amylin differs from human amylin by only 6 residues; however, it does not form fibrils. Therefore it provides a useful complement to human amylin in studies of the key events along the aggregation pathway. In this work, the free energy of rat and human amylin was determined as a function of α-helix and β-hairpin content for the Gromos96 53a6, OPLS-AA/L, CHARMM22/CMAP, CHARMM22*, Amberff99sb*-ILDN, and Amberff03w force fields using advanced sampling techniques, specifically bias exchange metadynamics. This work represents a first systematic attempt to evaluate the conformations and the corresponding free energy of a large, clinically relevant disordered peptide in solution across force fields. The NMR chemical shifts of rIAPP were calculated for each of the force fields using their respective free energy maps, allowing us to quantitatively assess their predictions. We show that the predicted distribution of secondary structures is sensitive to the choice of force-field: Gromos53a6 is biased towards β-hairpins, while CHARMM22/CMAP predicts structures that are overly α-helical. OPLS-AA/L favors disordered structures. Amberff99sb*-ILDN, AmberFF03w and CHARMM22* provide the balance between secondary structures that is most consistent with available experimental data. In contrast to previous reports, our findings suggest that the equilibrium conformations of human and rat amylin are remarkably similar, but that subtle differences arise in transient alpha-helical and beta-strand containing structures that the human peptide can more readily adopt. We hypothesize that these transient states enable

  2. Secondary Structure of Rat and Human Amylin across Force Fields

    PubMed Central

    Hoffmann, Kyle Quynn; McGovern, Michael; Chiu, Chi-cheng; de Pablo, Juan J.

    2015-01-01

    The aggregation of human amylin has been strongly implicated in the progression of Type II diabetes. This 37-residue peptide forms a variety of secondary structures, including random coils, α-helices, and β-hairpins. The balance between these structures depends on the chemical environment, making amylin an ideal candidate to examine inherent biases in force fields. Rat amylin differs from human amylin by only 6 residues; however, it does not form fibrils. Therefore it provides a useful complement to human amylin in studies of the key events along the aggregation pathway. In this work, the free energy of rat and human amylin was determined as a function of α-helix and β-hairpin content for the Gromos96 53a6, OPLS-AA/L, CHARMM22/CMAP, CHARMM22*, Amberff99sb*-ILDN, and Amberff03w force fields using advanced sampling techniques, specifically bias exchange metadynamics. This work represents a first systematic attempt to evaluate the conformations and the corresponding free energy of a large, clinically relevant disordered peptide in solution across force fields. The NMR chemical shifts of rIAPP were calculated for each of the force fields using their respective free energy maps, allowing us to quantitatively assess their predictions. We show that the predicted distribution of secondary structures is sensitive to the choice of force-field: Gromos53a6 is biased towards β-hairpins, while CHARMM22/CMAP predicts structures that are overly α-helical. OPLS-AA/L favors disordered structures. Amberff99sb*-ILDN, AmberFF03w and CHARMM22* provide the balance between secondary structures that is most consistent with available experimental data. In contrast to previous reports, our findings suggest that the equilibrium conformations of human and rat amylin are remarkably similar, but that subtle differences arise in transient alpha-helical and beta-strand containing structures that the human peptide can more readily adopt. We hypothesize that these transient states enable

  3. Secondary structure of rat and human amylin across force fields

    DOE PAGESBeta

    Hoffmann, Kyle Quynn; McGovern, Michael; Chiu, Chi -cheng; de Pablo, Juan J.; Paci, Emanuele

    2015-07-29

    The aggregation of human amylin has been strongly implicated in the progression of Type II diabetes. This 37-residue peptide forms a variety of secondary structures, including random coils, α-helices, and β-hairpins. The balance between these structures depends on the chemical environment, making amylin an ideal candidate to examine inherent biases in force fields. Rat amylin differs from human amylin by only 6 residues; however, it does not form fibrils. Therefore it provides a useful complement to human amylin in studies of the key events along the aggregation pathway. In this work, the free energy of rat and human amylin wasmore » determined as a function of α-helix and β-hairpin content for the Gromos96 53a6, OPLS-AA/L, CHARMM22/CMAP, CHARMM22*, Amberff99sb*-ILDN, and Amberff03w force fields using advanced sampling techniques, specifically bias exchange metadynamics. This work represents a first systematic attempt to evaluate the conformations and the corresponding free energy of a large, clinically relevant disordered peptide in solution across force fields. The NMR chemical shifts of rIAPP were calculated for each of the force fields using their respective free energy maps, allowing us to quantitatively assess their predictions. We show that the predicted distribution of secondary structures is sensitive to the choice of force-field: Gromos53a6 is biased towards β-hairpins, while CHARMM22/CMAP predicts structures that are overly α-helical. OPLS-AA/L favors disordered structures. Amberff99sb*-ILDN, AmberFF03w and CHARMM22* provide the balance between secondary structures that is most consistent with available experimental data. In contrast to previous reports, our findings suggest that the equilibrium conformations of human and rat amylin are remarkably similar, but that subtle differences arise in transient alpha-helical and beta-strand containing structures that the human peptide can more readily adopt. We hypothesize that these transient states

  4. Secondary structure of rat and human amylin across force fields

    SciTech Connect

    Hoffmann, Kyle Quynn; McGovern, Michael; Chiu, Chi -cheng; de Pablo, Juan J.; Paci, Emanuele

    2015-07-29

    The aggregation of human amylin has been strongly implicated in the progression of Type II diabetes. This 37-residue peptide forms a variety of secondary structures, including random coils, α-helices, and β-hairpins. The balance between these structures depends on the chemical environment, making amylin an ideal candidate to examine inherent biases in force fields. Rat amylin differs from human amylin by only 6 residues; however, it does not form fibrils. Therefore it provides a useful complement to human amylin in studies of the key events along the aggregation pathway. In this work, the free energy of rat and human amylin was determined as a function of α-helix and β-hairpin content for the Gromos96 53a6, OPLS-AA/L, CHARMM22/CMAP, CHARMM22*, Amberff99sb*-ILDN, and Amberff03w force fields using advanced sampling techniques, specifically bias exchange metadynamics. This work represents a first systematic attempt to evaluate the conformations and the corresponding free energy of a large, clinically relevant disordered peptide in solution across force fields. The NMR chemical shifts of rIAPP were calculated for each of the force fields using their respective free energy maps, allowing us to quantitatively assess their predictions. We show that the predicted distribution of secondary structures is sensitive to the choice of force-field: Gromos53a6 is biased towards β-hairpins, while CHARMM22/CMAP predicts structures that are overly α-helical. OPLS-AA/L favors disordered structures. Amberff99sb*-ILDN, AmberFF03w and CHARMM22* provide the balance between secondary structures that is most consistent with available experimental data. In contrast to previous reports, our findings suggest that the equilibrium conformations of human and rat amylin are remarkably similar, but that subtle differences arise in transient alpha-helical and beta-strand containing structures that the human peptide can more readily adopt. We hypothesize that these transient states enable

  5. Function of RNA secondary structures in transcriptional attenuation of the Bacillus subtilis pyr operon.

    PubMed

    Lu, Y; Turner, R J; Switzer, R L

    1996-12-10

    The Bacillus subtilis pyr operon is regulated by exogenous pyrimidines by a transcriptional attenuation mechanism. Transcription in vitro from pyr DNA templates specifying attenuation regions yielded terminated and read-through transcripts of the expected lengths. Addition of the PyrR regulatory protein plus UMP led to greatly increased termination. Synthetic antisense deoxyoligonucleotides were used to probe possible secondary structures in the pyr mRNA that were proposed to play roles in controlling attenuation. Oligonucleotides predicted to disrupt terminator structures suppressed termination, whereas oligonucleotides predicted to disrupt the stem of antiterminator stem-loops strongly promoted termination at the usual termination site. Oligonucleotides that disrupt a previously unrecognized stem-loop structure, called the anti-antiterminator, the formation of which interferes with formation of the downstream antiterminator, suppressed termination. We propose that transcriptional attenuation of the pyr operon is governed by switching between alternative antiterminator versus anti-antiterminator plus terminator structures, and that PyrR acts by UMP-dependent binding to and stabilization of the anti-antiterminator.

  6. RNA Secondary Structure Prediction by Using Discrete Mathematics: An Interdisciplinary Research Experience for Undergraduate Students

    ERIC Educational Resources Information Center

    Ellington, Roni; Wachira, James; Nkwanta, Asamoah

    2010-01-01

    The focus of this Research Experience for Undergraduates (REU) project was on RNA secondary structure prediction by using a lattice walk approach. The lattice walk approach is a combinatorial and computational biology method used to enumerate possible secondary structures and predict RNA secondary structure from RNA sequences. The method uses…

  7. Mutational analysis of the 5' non-coding region of human immunodeficiency virus type 1: effects of secondary structure on translation.

    PubMed Central

    Parkin, N T; Cohen, E A; Darveau, A; Rosen, C; Haseltine, W; Sonenberg, N

    1988-01-01

    The first 111 nt from the 5' end of human immunodeficiency virus type 1 (HIV-1) mRNAs are shown to have a strong inhibitory effect on the translation of mRNA in in vitro translation extracts as well as in Xenopus oocytes. Mutations in the sequence of the 5' untranslated region (UTR) designed to disrupt predicted secondary structure of this region relieve the inhibition. Inhibition is restored by mutations that reconstruct the predicted secondary structure. The accessibility of the 5'-terminal cap structure was also found to be increased by some of these mutations. We conclude that secondary structure in the 5' UTR of HIV-1 mRNAs and resultant inaccessibility of the cap structure is responsible for the inhibition of translation. The implications of these findings for the understanding of the life cycle of HIV-1 are discussed. Images PMID:3181141

  8. A phase transition in energy-filtered RNA secondary structures.

    PubMed

    Han, Hillary S W; Reidys, Christian M

    2012-10-01

    In this article we study the effect of energy parameters on minimum free energy (mfe) RNA secondary structures. Employing a simplified combinatorial energy model that is only dependent on the diagram representation and is not sequence-specific, we prove the following dichotomy result. Mfe structures derived via the Turner energy parameters contain only finitely many complex irreducible substructures, and just minor parameter changes produce a class of mfe structures that contain a large number of small irreducibles. We localize the exact point at which the distribution of irreducibles experiences this phase transition from a discrete limit to a central limit distribution and, subsequently, put our result into the context of quantifying the effect of sparsification of the folding of these respective mfe structures. We show that the sparsification of realistic mfe structures leads to a constant time and space reduction, and that the sparsification of the folding of structures with modified parameters leads to a linear time and space reduction. We, furthermore, identify the limit distribution at the phase transition as a Rayleigh distribution.

  9. Coding of odor stimulus features among secondary olfactory structures.

    PubMed

    Xia, Christina Z; Adjei, Stacey; Wesson, Daniel W

    2015-07-01

    Sensory systems must represent stimuli in manners dependent upon a wealth of factors, including stimulus intensity and duration. One way the brain might handle these complex functions is to assign the tasks throughout distributed nodes, each contributing to information processing. We sought to explore this important aspect of sensory network function in the mammalian olfactory system, wherein the intensity and duration of odor exposure are critical contributors to odor perception. This is a quintessential model for exploring processing schemes given the distribution of odor information by olfactory bulb mitral and tufted cells into several anatomically distinct secondary processing stages, including the piriform cortex (PCX) and olfactory tubercle (OT), whose unique contributions to odor coding are unresolved. We explored the coding of PCX and OT neuron responses to odor intensity and duration. We found that both structures similarly partake in representing descending intensities of odors by reduced recruitment and modulation of neurons. Additionally, while neurons in the OT adapt to odor exposure, they display reduced capacity to adapt to either repeated presentations of odor or a single prolonged odor presentation compared with neurons in the PCX. These results provide insights into manners whereby secondary olfactory structures may, at least in some cases, uniquely represent stimulus features. PMID:26041832

  10. Coding of odor stimulus features among secondary olfactory structures

    PubMed Central

    Xia, Christina Z.; Adjei, Stacey

    2015-01-01

    Sensory systems must represent stimuli in manners dependent upon a wealth of factors, including stimulus intensity and duration. One way the brain might handle these complex functions is to assign the tasks throughout distributed nodes, each contributing to information processing. We sought to explore this important aspect of sensory network function in the mammalian olfactory system, wherein the intensity and duration of odor exposure are critical contributors to odor perception. This is a quintessential model for exploring processing schemes given the distribution of odor information by olfactory bulb mitral and tufted cells into several anatomically distinct secondary processing stages, including the piriform cortex (PCX) and olfactory tubercle (OT), whose unique contributions to odor coding are unresolved. We explored the coding of PCX and OT neuron responses to odor intensity and duration. We found that both structures similarly partake in representing descending intensities of odors by reduced recruitment and modulation of neurons. Additionally, while neurons in the OT adapt to odor exposure, they display reduced capacity to adapt to either repeated presentations of odor or a single prolonged odor presentation compared with neurons in the PCX. These results provide insights into manners whereby secondary olfactory structures may, at least in some cases, uniquely represent stimulus features. PMID:26041832

  11. Peptoid nanosheets exhibit a new secondary-structure motif

    NASA Astrophysics Data System (ADS)

    Mannige, Ranjan V.; Haxton, Thomas K.; Proulx, Caroline; Robertson, Ellen J.; Battigelli, Alessia; Butterfoss, Glenn L.; Zuckermann, Ronald N.; Whitelam, Stephen

    2015-10-01

    A promising route to the synthesis of protein-mimetic materials that are capable of complex functions, such as molecular recognition and catalysis, is provided by sequence-defined peptoid polymers--structural relatives of biologically occurring polypeptides. Peptoids, which are relatively non-toxic and resistant to degradation, can fold into defined structures through a combination of sequence-dependent interactions. However, the range of possible structures that are accessible to peptoids and other biological mimetics is unknown, and our ability to design protein-like architectures from these polymer classes is limited. Here we use molecular-dynamics simulations, together with scattering and microscopy data, to determine the atomic-resolution structure of the recently discovered peptoid nanosheet, an ordered supramolecular assembly that extends macroscopically in only two dimensions. Our simulations show that nanosheets are structurally and dynamically heterogeneous, can be formed only from peptoids of certain lengths, and are potentially porous to water and ions. Moreover, their formation is enabled by the peptoids' adoption of a secondary structure that is not seen in the natural world. This structure, a zigzag pattern that we call a Σ(`sigma')-strand, results from the ability of adjacent backbone monomers to adopt opposed rotational states, thereby allowing the backbone to remain linear and untwisted. Linear backbones tiled in a brick-like way form an extended two-dimensional nanostructure, the Σ-sheet. The binary rotational-state motif of the Σ-strand is not seen in regular protein structures, which are usually built from one type of rotational state. We also show that the concept of building regular structures from multiple rotational states can be generalized beyond the peptoid nanosheet system.

  12. RNAex: an RNA secondary structure prediction server enhanced by high-throughput structure-probing data.

    PubMed

    Wu, Yang; Qu, Rihao; Huang, Yiming; Shi, Binbin; Liu, Mengrong; Li, Yang; Lu, Zhi John

    2016-07-01

    Several high-throughput technologies have been developed to probe RNA base pairs and loops at the transcriptome level in multiple species. However, to obtain the final RNA secondary structure, extensive effort and considerable expertise is required to statistically process the probing data and combine them with free energy models. Therefore, we developed an RNA secondary structure prediction server that is enhanced by experimental data (RNAex). RNAex is a web interface that enables non-specialists to easily access cutting-edge structure-probing data and predict RNA secondary structures enhanced by in vivo and in vitro data. RNAex annotates the RNA editing, RNA modification and SNP sites on the predicted structures. It provides four structure-folding methods, restrained MaxExpect, SeqFold, RNAstructure (Fold) and RNAfold that can be selected by the user. The performance of these four folding methods has been verified by previous publications on known structures. We re-mapped the raw sequencing data of the probing experiments to the whole genome for each species. RNAex thus enables users to predict secondary structures for both known and novel RNA transcripts in human, mouse, yeast and Arabidopsis The RNAex web server is available at http://RNAex.ncrnalab.org/.

  13. RNAex: an RNA secondary structure prediction server enhanced by high-throughput structure-probing data

    PubMed Central

    Wu, Yang; Qu, Rihao; Huang, Yiming; Shi, Binbin; Liu, Mengrong; Li, Yang; Lu, Zhi John

    2016-01-01

    Several high-throughput technologies have been developed to probe RNA base pairs and loops at the transcriptome level in multiple species. However, to obtain the final RNA secondary structure, extensive effort and considerable expertise is required to statistically process the probing data and combine them with free energy models. Therefore, we developed an RNA secondary structure prediction server that is enhanced by experimental data (RNAex). RNAex is a web interface that enables non-specialists to easily access cutting-edge structure-probing data and predict RNA secondary structures enhanced by in vivo and in vitro data. RNAex annotates the RNA editing, RNA modification and SNP sites on the predicted structures. It provides four structure-folding methods, restrained MaxExpect, SeqFold, RNAstructure (Fold) and RNAfold that can be selected by the user. The performance of these four folding methods has been verified by previous publications on known structures. We re-mapped the raw sequencing data of the probing experiments to the whole genome for each species. RNAex thus enables users to predict secondary structures for both known and novel RNA transcripts in human, mouse, yeast and Arabidopsis. The RNAex web server is available at http://RNAex.ncrnalab.org/. PMID:27137891

  14. Computer-aided prediction of RNA secondary structures.

    PubMed Central

    Auron, P E; Rindone, W P; Vary, C P; Celentano, J J; Vournakis, J N

    1982-01-01

    A brief survey of computer algorithms that have been developed to generate predictions of the secondary structures of RNA molecules is presented. Two particular methods are described in some detail. The first utilizes a thermodynamic energy minimization algorithm that takes into account the likelihood that short-range folding tends to be favored over long-range interactions. The second utilizes an interactive computer graphic modelling algorithm that enables the user to consider thermodynamic criteria as well as structural data obtained by nuclease susceptibility, chemical reactivity and phylogenetic studies. Examples of structures for prokaryotic 16S and 23S ribosomal RNAs, several eukaryotic 5S ribosomal RNAs and rabbit beta-globin messenger RNA are presented as case studies in order to describe the two techniques. Anm argument is made for integrating the two approaches presented in this paper, enabling the user to generate proposed structures using thermodynamic criteria, allowing interactive refinement of these structures through the application of experimentally derived data. PMID:6174937

  15. Importance of RNA secondary structure information for yeast donor and acceptor splice site predictions by neural networks.

    PubMed

    Marashi, Sayed-Amir; Goodarzi, Hani; Sadeghi, Mehdi; Eslahchi, Changiz; Pezeshk, Hamid

    2006-02-01

    Previously, Patterson et al. showed that mRNA structure information aids splice site prediction in human genes [Patterson, D.J., Yasuhara, K., Ruzzo, W.L., 2002. Pre-mRNA secondary structure prediction aids splice site prediction. Pac. Symp. Biocomput. 7, 223-234]. Here, we have attempted to predict splice sites in selected genes of Saccharomyces cerevisiae using the information obtained from the secondary structures of corresponding mRNAs. From Ares database, 154 genes were selected and their structures were predicted by Mfold. We selected a 20-nucleotide window around each site, each containing 4 nucleotides in the exon region. Based on whether the nucleotide is in a stem or not, the conventional four-letter nucleotide alphabet was translated into an eight-letter alphabet. Two different three-layer-based perceptron neural networks were devised to predict the 5' and 3' splice sites. In case of 5' site determination, a network with 3 neurons at the hidden layer was chosen, while in case of 3' site 20 neurons acted more efficiently. Both neural nets were trained applying Levenberg-Marquardt backpropagation method, using half of the available genes as training inputs and the other half for testing and cross-validations. Sequences with GUs and AGs non-sites were used as negative controls. The correlation coefficients in the predictions of 5' and 3' splice sites using eight-letter alphabet were 98.0% and 69.6%, respectively, while these values were 89.3% and 57.1% when four-letter alphabet is applied. Our results suggest that considering the secondary structure of mRNA molecules positively affects both donor and acceptor site predictions by increasing the capacity of neural networks in learning the patterns.

  16. Mature MiRNAs Form Secondary Structure, which Suggests Their Function beyond RISC

    PubMed Central

    Belter, Agnieszka; Gudanis, Dorota; Rolle, Katarzyna; Piwecka, Monika; Gdaniec, Zofia; Naskręt-Barciszewska, Mirosława Z.; Barciszewski, Jan

    2014-01-01

    The generally accepted model of the miRNA-guided RNA down-regulation suggests that mature miRNA targets mRNA in a nucleotide sequence-specific manner. However, we have shown that the nucleotide sequence of miRNA is not the only determinant of miRNA specificity. Using specific nucleases, T1, V1 and S1 as well as NMR, UV/Vis and CD spectroscopies, we found that miR-21, miR-93 and miR-296 can adopt hairpin and/or homoduplex structures. The secondary structure of those miRNAs in solution is a function of RNA concentration and ionic conditions. Additionally, we have shown that a formation of miRNA hairpin is facilitated by cellular environment.Looking for functional consequences of this observation, we have perceived that structure of these miRNAs resemble RNA aptamers, short oligonucleotides forming a stable 3D structures with a high affinity and specificity for their targets. We compared structures of anti-tenascin C (anti-Tn-C) aptamers, which inhibit brain tumor glioblastoma multiforme (GBM, WHO IV) and selected miRNA. A strong overexpression of miR-21, miR-93 as well Tn-C in GBM may imply some connections between them. The structural similarity of these miRNA hairpins and anti-Tn-C aptamers indicates that miRNAs may function also beyond RISC and are even more sophisticated regulators, that it was previously expected. We think that the knowledge of the miRNA structure may give a new insight into miRNA-dependent gene regulation mechanism and be a step forward in the understanding their function and involvement in cancerogenesis. This may improve design process of anti-miRNA therapeutics. PMID:25423301

  17. Protein Secondary Structure Prediction Using Deep Convolutional Neural Fields.

    PubMed

    Wang, Sheng; Peng, Jian; Ma, Jianzhu; Xu, Jinbo

    2016-01-01

    Protein secondary structure (SS) prediction is important for studying protein structure and function. When only the sequence (profile) information is used as input feature, currently the best predictors can obtain ~80% Q3 accuracy, which has not been improved in the past decade. Here we present DeepCNF (Deep Convolutional Neural Fields) for protein SS prediction. DeepCNF is a Deep Learning extension of Conditional Neural Fields (CNF), which is an integration of Conditional Random Fields (CRF) and shallow neural networks. DeepCNF can model not only complex sequence-structure relationship by a deep hierarchical architecture, but also interdependency between adjacent SS labels, so it is much more powerful than CNF. Experimental results show that DeepCNF can obtain ~84% Q3 accuracy, ~85% SOV score, and ~72% Q8 accuracy, respectively, on the CASP and CAMEO test proteins, greatly outperforming currently popular predictors. As a general framework, DeepCNF can be used to predict other protein structure properties such as contact number, disorder regions, and solvent accessibility. PMID:26752681

  18. Protein Secondary Structure Prediction Using Deep Convolutional Neural Fields

    NASA Astrophysics Data System (ADS)

    Wang, Sheng; Peng, Jian; Ma, Jianzhu; Xu, Jinbo

    2016-01-01

    Protein secondary structure (SS) prediction is important for studying protein structure and function. When only the sequence (profile) information is used as input feature, currently the best predictors can obtain ~80% Q3 accuracy, which has not been improved in the past decade. Here we present DeepCNF (Deep Convolutional Neural Fields) for protein SS prediction. DeepCNF is a Deep Learning extension of Conditional Neural Fields (CNF), which is an integration of Conditional Random Fields (CRF) and shallow neural networks. DeepCNF can model not only complex sequence-structure relationship by a deep hierarchical architecture, but also interdependency between adjacent SS labels, so it is much more powerful than CNF. Experimental results show that DeepCNF can obtain ~84% Q3 accuracy, ~85% SOV score, and ~72% Q8 accuracy, respectively, on the CASP and CAMEO test proteins, greatly outperforming currently popular predictors. As a general framework, DeepCNF can be used to predict other protein structure properties such as contact number, disorder regions, and solvent accessibility.

  19. An RNA secondary structure prediction method based on minimum and suboptimal free energy structures.

    PubMed

    Fu, Haoyue; Yang, Lianping; Zhang, Xiangde

    2015-09-01

    The function of an RNA-molecule is mainly determined by its tertiary structures. And its secondary structure is an important determinant of its tertiary structure. The comparative methods usually give better results than the single-sequence methods. Based on minimum and suboptimal free energy structures, the paper presents a novel method for predicting conserved secondary structure of a group of related RNAs. In the method, the information from the known RNA structures is used as training data in a SVM (Support Vector Machine) classifier. Our method has been tested on the benchmark dataset given by Puton et al. The results show that the average sensitivity of our method is higher than that of other comparative methods such as CentroidAlifold, MXScrana, RNAalifold, and TurboFold. PMID:26100179

  20. Consequential secondary structure alterations and aggregation during prolonged casein glycation.

    PubMed

    Jindal, Supriya; Naeem, Aabgeena

    2013-05-01

    Non-enzymatic glycosylation (glycation) of casein is a process used not just to ameliorate the quality of dairy products but also to increase the shelf life of canned foods, including baby milk supplements. Incubation of κ-casein with reducing sugars for 15 days at physiological temperature showed the formation of a molten globule state at day 9 and 12 during fructation and glucation respectively. This state exhibits substantial secondary structure and maximum ANS binding. Later on, glycation resulted in the formation of aggregates at day 12 in presence of fructose and day 15 in presence of glucose. Aggregates possess extensive β-sheet structure as revealed by far-UV CD and FTIR. These aggregates showed altered tryptophan environment, decrease ANS binding relative to molten globule state and increase in Thioflavin T fluorescence. Aggregates were also accompanied by the accumulation of AGEs, indicative of structural damage to the protein and formation of potentially harmful species at the physiological level. Fructose was more reactive than glucose and thus caused early and significant changes in the protein. From our studies, we conclude that controlling the extent of the Maillard reaction in the food industry is essential to counter its negative effects and expand its safety spectrum. PMID:23408088

  1. Toward a better understanding of structural divergences in proteins using different secondary structure assignment methods

    NASA Astrophysics Data System (ADS)

    Rocha, L. F. O.

    2014-04-01

    Structural disagreements on the location and quantity of secondary structure segments comprise a current challenging problem leading to several limitations for theoretical and applied research. This paper presents 116 structural evaluations by steric and hydrophobic interactions in secondary structures within a specific template group; determines simple prediction rules that calculate 88 occurrence frequencies of large and hydrophobic residues into target intra- and inter-subgroups with structure disagreements; and utilizes 42 comparisons between the methods PROMOTIF, DSSP and STRIDE. In the stereochemical predictions inside the subgroups there are predominantly excellent and/or good success amounts with their expected values, and the disclosure of a triple molecular mechanism by residue volumetric and hydrophobic ingredients. The method comparisons show high compatibility scores between them, therefore validating their seemingly incompatible assignments. Thus, the nonconsensual ascriptions are better understood and appreciated. Furthermore, such results suggest a broad utility of our assignment method for other benchmark datasets and known methods.

  2. PSRna: Prediction of small RNA secondary structures based on reverse complementary folding method.

    PubMed

    Li, Jin; Xu, Chengzhen; Wang, Lei; Liang, Hong; Feng, Weixing; Cai, Zhongxi; Wang, Ying; Cong, Wang; Liu, Yunlong

    2016-08-01

    Prediction of RNA secondary structures is an important problem in computational biology and bioinformatics, since RNA secondary structures are fundamental for functional analysis of RNA molecules. However, small RNA secondary structures are scarce and few algorithms have been specifically designed for predicting the secondary structures of small RNAs. Here we propose an algorithm named "PSRna" for predicting small-RNA secondary structures using reverse complementary folding and characteristic hairpin loops of small RNAs. Unlike traditional algorithms that usually generate multi-branch loops and 5[Formula: see text] end self-folding, PSRna first estimated the maximum number of base pairs of RNA secondary structures based on the dynamic programming algorithm and a path matrix is constructed at the same time. Second, the backtracking paths are extracted from the path matrix based on backtracking algorithm, and each backtracking path represents a secondary structure. To improve accuracy, the predicted RNA secondary structures are filtered based on their free energy, where only the secondary structure with the minimum free energy was identified as the candidate secondary structure. Our experiments on real data show that the proposed algorithm is superior to two popular methods, RNAfold and RNAstructure, in terms of sensitivity, specificity and Matthews correlation coefficient (MCC). PMID:27045556

  3. PSRna: Prediction of small RNA secondary structures based on reverse complementary folding method.

    PubMed

    Li, Jin; Xu, Chengzhen; Wang, Lei; Liang, Hong; Feng, Weixing; Cai, Zhongxi; Wang, Ying; Cong, Wang; Liu, Yunlong

    2016-08-01

    Prediction of RNA secondary structures is an important problem in computational biology and bioinformatics, since RNA secondary structures are fundamental for functional analysis of RNA molecules. However, small RNA secondary structures are scarce and few algorithms have been specifically designed for predicting the secondary structures of small RNAs. Here we propose an algorithm named "PSRna" for predicting small-RNA secondary structures using reverse complementary folding and characteristic hairpin loops of small RNAs. Unlike traditional algorithms that usually generate multi-branch loops and 5[Formula: see text] end self-folding, PSRna first estimated the maximum number of base pairs of RNA secondary structures based on the dynamic programming algorithm and a path matrix is constructed at the same time. Second, the backtracking paths are extracted from the path matrix based on backtracking algorithm, and each backtracking path represents a secondary structure. To improve accuracy, the predicted RNA secondary structures are filtered based on their free energy, where only the secondary structure with the minimum free energy was identified as the candidate secondary structure. Our experiments on real data show that the proposed algorithm is superior to two popular methods, RNAfold and RNAstructure, in terms of sensitivity, specificity and Matthews correlation coefficient (MCC).

  4. Secondary use of structured patient data: interim results of a systematic review.

    PubMed

    Vuokko, Riikka; Mäkelä-Bengs, Päivi; Hyppönen, Hannele; Doupi, Persephone

    2015-01-01

    In addition to patient care, EHR data are increasingly in demand for secondary purposes, e.g. administration, research and enterprise resource planning. We conducted a systematic literature review and subsequent analysis of 85 articles focusing on the secondary use of structured patient records. We grounded the analysis on how patient records have been structured, how these structures have been evaluated and what are the main results achieved from the secondary use viewpoint. We conclude that secondary use requires complete and interoperable patient records, which in turn depend on better alignment of primary and secondary users' needs and benefits. PMID:25991152

  5. Structure of 13Be probed via secondary-beam reactions

    NASA Astrophysics Data System (ADS)

    Randisi, G.; Leprince, A.; Al Falou, H.; Orr, N. A.; Marqués, F. M.; Achouri, N. L.; Angélique, J.-C.; Ashwood, N.; Bastin, B.; Bloxham, T.; Brown, B. A.; Catford, W. N.; Curtis, N.; Delaunay, F.; Freer, M.; de Góes Brennand, E.; Haigh, P.; Hanappe, F.; Harlin, C.; Laurent, B.; Lecouey, J.-L.; Ninane, A.; Patterson, N.; Price, D.; Stuttgé, L.; Thomas, J. S.

    2014-03-01

    The low-lying level structure of the unbound neutron-rich nucleus 13Be has been investigated via breakup on a carbon target of secondary beams of 14,15B at 35 MeV/nucleon. The coincident detection of the beam velocity 12Be fragments and neutrons permitted the invariant mass of the 12Be+n and 12Be+n+n systems to be reconstructed. In the case of the breakup of 15B, a very narrow structure at threshold was observed in the 12Be+n channel. Analysis of the 12Be+n+n events demonstrated that this resulted from the sequential decay of the unbound 14Be(2+) state rather than a strongly interacting s-wave virtual state in 13Be, as had been surmised in stable beam fragmentation studies. Single-proton removal from 14B was found to populate a broad low-lying structure some 0.7 MeV above the neutron-decay threshold, in addition to a less prominent feature at around 2.4 MeV. Based on the selectivity of the reaction and a comparison with (0-3)ℏω shell-model calculations, the low-lying structure is concluded to arise from closely spaced Jπ=1/2+ and 5/2+ resonances (Er=0.40±0.03 and 0.85-0.11+0.15 MeV), while the broad higher-lying feature is a second 5/2+ level (Er=2.35±0.14 MeV). Taken in conjunction with earlier studies, the results suggest that the lowest 1/2+ and 1/2- levels lie relatively close together below 1 MeV.

  6. Expected distance between terminal nucleotides of RNA secondary structures.

    PubMed

    Clote, Peter; Ponty, Yann; Steyaert, Jean-Marc

    2012-09-01

    In "The ends of a large RNA molecule are necessarily close", Yoffe et al. (Nucleic Acids Res 39(1):292-299, 2011) used the programs RNAfold [resp. RNAsubopt] from Vienna RNA Package to calculate the distance between 5' and 3' ends of the minimum free energy secondary structure [resp. thermal equilibrium structures] of viral and random RNA sequences. Here, the 5'-3' distance is defined to be the length of the shortest path from 5' node to 3' node in the undirected graph, whose edge set consists of edges {i, i + 1} corresponding to covalent backbone bonds and of edges {i, j} corresponding to canonical base pairs. From repeated simulations and using a heuristic theoretical argument, Yoffe et al. conclude that the 5'-3' distance is less than a fixed constant, independent of RNA sequence length. In this paper, we provide a rigorous, mathematical framework to study the expected distance from 5' to 3' ends of an RNA sequence. We present recurrence relations that precisely define the expected distance from 5' to 3' ends of an RNA sequence, both for the Turner nearest neighbor energy model, as well as for a simple homopolymer model first defined by Stein and Waterman. We implement dynamic programming algorithms to compute (rather than approximate by repeated application of Vienna RNA Package) the expected distance between 5' and 3' ends of a given RNA sequence, with respect to the Turner energy model. Using methods of analytical combinatorics, that depend on complex analysis, we prove that the asymptotic expected 5'-3' distance of length n homopolymers is approximately equal to the constant 5.47211, while the asymptotic distance is 6.771096 if hairpins have a minimum of 3 unpaired bases and the probability that any two positions can form a base pair is 1/4. Finally, we analyze the 5'-3' distance for secondary structures from the STRAND database, and conclude that the 5'-3' distance is correlated with RNA sequence length.

  7. Expected distance between terminal nucleotides of RNA secondary structures.

    PubMed

    Clote, Peter; Ponty, Yann; Steyaert, Jean-Marc

    2012-09-01

    In "The ends of a large RNA molecule are necessarily close", Yoffe et al. (Nucleic Acids Res 39(1):292-299, 2011) used the programs RNAfold [resp. RNAsubopt] from Vienna RNA Package to calculate the distance between 5' and 3' ends of the minimum free energy secondary structure [resp. thermal equilibrium structures] of viral and random RNA sequences. Here, the 5'-3' distance is defined to be the length of the shortest path from 5' node to 3' node in the undirected graph, whose edge set consists of edges {i, i + 1} corresponding to covalent backbone bonds and of edges {i, j} corresponding to canonical base pairs. From repeated simulations and using a heuristic theoretical argument, Yoffe et al. conclude that the 5'-3' distance is less than a fixed constant, independent of RNA sequence length. In this paper, we provide a rigorous, mathematical framework to study the expected distance from 5' to 3' ends of an RNA sequence. We present recurrence relations that precisely define the expected distance from 5' to 3' ends of an RNA sequence, both for the Turner nearest neighbor energy model, as well as for a simple homopolymer model first defined by Stein and Waterman. We implement dynamic programming algorithms to compute (rather than approximate by repeated application of Vienna RNA Package) the expected distance between 5' and 3' ends of a given RNA sequence, with respect to the Turner energy model. Using methods of analytical combinatorics, that depend on complex analysis, we prove that the asymptotic expected 5'-3' distance of length n homopolymers is approximately equal to the constant 5.47211, while the asymptotic distance is 6.771096 if hairpins have a minimum of 3 unpaired bases and the probability that any two positions can form a base pair is 1/4. Finally, we analyze the 5'-3' distance for secondary structures from the STRAND database, and conclude that the 5'-3' distance is correlated with RNA sequence length. PMID:21984358

  8. Control of cerium oxidation state through metal complex secondary structures

    DOE PAGESBeta

    Levin, Jessica R.; Dorfner, Walter L.; Carroll, Patrick J.; Schelter, Eric J.

    2015-08-11

    A series of alkali metal cerium diphenylhydrazido complexes, Mx(py)y[Ce(PhNNPh)4], M = Li, Na, and K, x = 4 (Li and Na) or 5 (K), and y = 4 (Li), 8 (Na), or 7 (K), were synthesized to probe how a secondary coordination sphere would modulate electronic structures at a cerium cation. The resulting electronic structures of the heterobimetallic cerium diphenylhydrazido complexes were found to be strongly dependent on the identity of the alkali metal cations. When M = Li+ or Na+, the cerium(III) starting material was oxidized with concomitant reduction of 1,2-diphenylhydrazine to aniline. Reduction of 1,2-diphenylhydrazine was not observedmore » when M = K+, and the complex remained in the cerium(III) oxidation state. Oxidation of the cerium(III) diphenylhydrazido complex to the Ce(IV) diphenylhydrazido one was achieved through a simple cation exchange reaction of the alkali metals. As a result, UV-Vis spectroscopy, FTIR spectroscopy, electrochemistry, magnetic susceptibility, and DFT studies were used to probe the oxidation state and the electronic changes that occurred at the metal centre.« less

  9. Changes in secondary structure of gluten proteins due to emulsifiers

    NASA Astrophysics Data System (ADS)

    Gómez, Analía V.; Ferrer, Evelina G.; Añón, María C.; Puppo, María C.

    2013-02-01

    Changes in the secondary structure of gluten proteins due to emulsifiers were analyzed by Raman Spectroscopy. The protein folding induced by 0.25% SSL (Sodium Stearoyl Lactylate) (GS0.25, Gluten + 0.25% SSL) included an increase in α-helix conformation and a decrease in β-sheet, turns and random coil. The same behavior, although in a less degree, was observed for 0.5% gluten-DATEM (Diacetyl Tartaric Acid Esters of Monoglycerides) system. The low burial of Tryptophan residues to a more hydrophobic environment and the low percentage area of the C-H stretching band for GS0.25 (Gluten + 0.25% SSL), could be related to the increased in α-helix conformation. This behavior was also confirmed by changes in stretching vibrational modes of disulfide bridges (S-S) and the low exposure of Tyrosine residues. High levels of SSL (0.5% and 1.0%) and DATEM (1.0%) led to more disordered protein structures, with different gluten networks. SSL (1.0%) formed a more disordered and opened gluten matrix than DATEM, the last one being laminar and homogeneous.

  10. Control of cerium oxidation state through metal complex secondary structures

    SciTech Connect

    Levin, Jessica R.; Dorfner, Walter L.; Carroll, Patrick J.; Schelter, Eric J.

    2015-08-11

    A series of alkali metal cerium diphenylhydrazido complexes, Mx(py)y[Ce(PhNNPh)4], M = Li, Na, and K, x = 4 (Li and Na) or 5 (K), and y = 4 (Li), 8 (Na), or 7 (K), were synthesized to probe how a secondary coordination sphere would modulate electronic structures at a cerium cation. The resulting electronic structures of the heterobimetallic cerium diphenylhydrazido complexes were found to be strongly dependent on the identity of the alkali metal cations. When M = Li+ or Na+, the cerium(III) starting material was oxidized with concomitant reduction of 1,2-diphenylhydrazine to aniline. Reduction of 1,2-diphenylhydrazine was not observed when M = K+, and the complex remained in the cerium(III) oxidation state. Oxidation of the cerium(III) diphenylhydrazido complex to the Ce(IV) diphenylhydrazido one was achieved through a simple cation exchange reaction of the alkali metals. As a result, UV-Vis spectroscopy, FTIR spectroscopy, electrochemistry, magnetic susceptibility, and DFT studies were used to probe the oxidation state and the electronic changes that occurred at the metal centre.

  11. Gene structure, chromosomal location, and basis for alternative mRNA splicing of the human VCAM1 gene

    SciTech Connect

    Cybulsky, M.I.; Fries, J.W.U.; Williams, A.J.; Sultan, P.; Gimbrone, M.A. Jr.; Collins, T. ); Eddy, R.; Byers, M.; Shows, T. )

    1991-09-01

    Vascular cell adhesion molecule 1 (VCAM-1) is a cell surface glycoprotein adhesive for certain blood leukocytes and tumor cells, which is expressed by activated endothelium in a variety of pathologic conditions including atherosclerosis. Genomic clones encoding the VCAM1 gene were isolated and the organization of the gene was determined. The gene, which is present in a single copy in the human genome, contains 9 exons spanning {approx}25 kilobases of DNA. Exons 2-8 contain C2 or H-type immunoglobulin domains. At least two different VCAM-1 precursors can be generated from the human gene as a result of alternative mRNA splicing events, which include or exclude exon 5. A consensus TATAA element is located upstream of the transcriptional start site. The VCAM1 promoter contains consensus binding sites for NF-{kappa}B, the GATA family of transcription factors, as well as an AP1 site. The VCAM1 gene was assigned to the 1p31-32 region of chromosome 1 based on the analysis of human-mouse hybrid cell lines and in situ hybridization. Structural analysis of the human VCAM1 gene provides the basis for alternative mRNA splicing and an initial approach to elucidating the regulation of VCAM-1 expression.

  12. S6:S18 ribosomal protein complex interacts with a structural motif present in its own mRNA

    PubMed Central

    Matelska, Dorota; Purta, Elzbieta; Panek, Sylwia; Boniecki, Michal J.; Bujnicki, Janusz M.; Dunin-Horkawicz, Stanislaw

    2013-01-01

    Prokaryotic ribosomal protein genes are typically grouped within highly conserved operons. In many cases, one or more of the encoded proteins not only bind to a specific site in the ribosomal RNA, but also to a motif localized within their own mRNA, and thereby regulate expression of the operon. In this study, we computationally predicted an RNA motif present in many bacterial phyla within the 5′ untranslated region of operons encoding ribosomal proteins S6 and S18. We demonstrated that the S6:S18 complex binds to this motif, which we hereafter refer to as the S6:S18 complex-binding motif (S6S18CBM). This motif is a conserved CCG sequence presented in a bulge flanked by a stem and a hairpin structure. A similar structure containing a CCG trinucleotide forms the S6:S18 complex binding site in 16S ribosomal RNA. We have constructed a 3D structural model of a S6:S18 complex with S6S18CBM, which suggests that the CCG trinucleotide in a specific structural context may be specifically recognized by the S18 protein. This prediction was supported by site-directed mutagenesis of both RNA and protein components. These results provide a molecular basis for understanding protein-RNA recognition and suggest that the S6S18CBM is involved in an auto-regulatory mechanism. PMID:23980204

  13. Template-directed synthesis of a small molecule-antisense conjugate targeting an mRNA structure

    PubMed Central

    Liu, Yang; Rodriguez, Lilia; Wolfe, Michael S.

    2014-01-01

    The targeting of structural features in mRNA with specificity remains a great chemical challenge. A hairpin structure near exon 10 in the pre-mRNA encoding the tau protein controls its splicing, and dementia-causing mutations that disrupt this structure increase exon 10 splicing. We previously reported the discovery of small molecules, mitoxantrone (MTX) and analogs, which bind to the tau RNA hairpin structure and the design of bipartite antisense oligonucleotides (ASOs) that simultaneously bind to the discontinuous sequences that flank this hairpin. Herein we report the synthesis of a bipartite ASO conjugated to MTX using the tau RNA hairpin and flanking sequences as a template. A set of six MTX analogs, each containing a linker-azide, and a set of ten bipartite ASOs, each containing a branched linker-alkyne, were synthesized and tested in combinatorial fashion for their ability to conjugate in the presence or absence of template RNA. A single template-dependent MTX–ASO conjugate was identified from among the 60 reaction mixtures, demonstrating that the MTX and ASO precursors could simultaneously bind the RNA template and allow proper positioning of azide and alkyne for 1,3-cycloaddition. While the MTX–ASO conjugate proved too cytotoxic for cell-based assays, the conjugate inhibited tau exon 10 splicing under cell-free conditions more effectively than MTX or bipartite ASO alone. PMID:24691171

  14. Structure of the Saccharomyces cerevisiae Cet1-Ceg1 mRNA Capping Apparatus

    SciTech Connect

    Gu, Meigang; Rajashankar, Kanagalaghatta R.; Lima, Christopher D.

    2010-05-04

    The 5{prime} guanine-N7 cap is the first cotranscriptional modification of messenger RNA. In Saccharomyces cerevisiae, the first two steps in capping are catalyzed by the RNA triphosphatase Cet1 and RNA guanylyltransferase Ceg1, which form a complex that is directly recruited to phosphorylated RNA polymerase II (RNAP IIo), primarily via contacts between RNAP IIo and Ceg1. A 3.0 {angstrom} crystal structure of Cet1-Ceg1 revealed a 176 kDa heterotetrameric complex composed of one Cet1 homodimer that associates with two Ceg1 molecules via interactions between the Ceg1 oligonucleotide binding domain and an extended Cet1 WAQKW amino acid motif. The WAQKW motif is followed by a flexible linker that would allow Ceg1 to achieve conformational changes required for capping while maintaining interactions with both Cet1 and RNAP IIo. The impact of mutations as assessed through genetic analysis in S. cerevisiae is consonant with contacts observed in the Cet1-Ceg1 structure.

  15. Energy-based RNA consensus secondary structure prediction in multiple sequence alignments.

    PubMed

    Washietl, Stefan; Bernhart, Stephan H; Kellis, Manolis

    2014-01-01

    Many biologically important RNA structures are conserved in evolution leading to characteristic mutational patterns. RNAalifold is a widely used program to predict consensus secondary structures in multiple alignments by combining evolutionary information with traditional energy-based RNA folding algorithms. Here we describe the theory and applications of the RNAalifold algorithm. Consensus secondary structure prediction not only leads to significantly more accurate structure models, but it also allows to study structural conservation of functional RNAs. PMID:24639158

  16. Energy-based RNA consensus secondary structure prediction in multiple sequence alignments.

    PubMed

    Washietl, Stefan; Bernhart, Stephan H; Kellis, Manolis

    2014-01-01

    Many biologically important RNA structures are conserved in evolution leading to characteristic mutational patterns. RNAalifold is a widely used program to predict consensus secondary structures in multiple alignments by combining evolutionary information with traditional energy-based RNA folding algorithms. Here we describe the theory and applications of the RNAalifold algorithm. Consensus secondary structure prediction not only leads to significantly more accurate structure models, but it also allows to study structural conservation of functional RNAs.

  17. Interaction of Bacillus subtilis Polynucleotide Phosphorylase and RNase Y: STRUCTURAL MAPPING AND EFFECT ON mRNA TURNOVER.

    PubMed

    Salvo, Elizabeth; Alabi, Shanique; Liu, Bo; Schlessinger, Avner; Bechhofer, David H

    2016-03-25

    Polynucleotide phosphorylase (PNPase), a 3'-to-5' phosphorolytic exoribonuclease, is thought to be the primary enzyme responsible for turnover ofBacillus subtilismRNA. The role of PNPase inB. subtilismRNA decay has been analyzed previously by comparison of mRNA profiles in a wild-type strainversusa strain that is deleted forpnpA, the gene encoding PNPase. Recent studies have provided evidence for a degradosome-like complex inB. subtilisthat is built around the major decay-initiating endonuclease, RNase Y, and there is ample evidence for a strong interaction between PNPase and RNase Y. The role of the PNPase-RNase Y interaction in the exonucleolytic function of PNPase needs to be clarified. We sought to construct aB. subtilisstrain containing a catalytically active PNPase that could not interact with RNase Y. Mapping studies of the PNPase-RNase Y interaction were guided by a homology model ofB. subtilisPNPase based on the known structure of theEscherichia coliPNPase in complex with an RNase E peptide. Mutations inB. subtilisresidues predicted to be involved in RNase Y binding showed a loss of PNPase-RNase Y interaction. Two mRNAs whose decay is dependent on RNase Y and PNPase were examined in strains containing full-length PNPase that was either catalytically active but unable to interact with RNase Y, or catalytically inactive but able to interact with RNase Y. At least for these two mRNAs, disruption of the PNPase-RNase Y interaction did not appear to affect mRNA turnover.

  18. Rtools: a web server for various secondary structural analyses on single RNA sequences.

    PubMed

    Hamada, Michiaki; Ono, Yukiteru; Kiryu, Hisanori; Sato, Kengo; Kato, Yuki; Fukunaga, Tsukasa; Mori, Ryota; Asai, Kiyoshi

    2016-07-01

    The secondary structures, as well as the nucleotide sequences, are the important features of RNA molecules to characterize their functions. According to the thermodynamic model, however, the probability of any secondary structure is very small. As a consequence, any tool to predict the secondary structures of RNAs has limited accuracy. On the other hand, there are a few tools to compensate the imperfect predictions by calculating and visualizing the secondary structural information from RNA sequences. It is desirable to obtain the rich information from those tools through a friendly interface. We implemented a web server of the tools to predict secondary structures and to calculate various structural features based on the energy models of secondary structures. By just giving an RNA sequence to the web server, the user can get the different types of solutions of the secondary structures, the marginal probabilities such as base-paring probabilities, loop probabilities and accessibilities of the local bases, the energy changes by arbitrary base mutations as well as the measures for validations of the predicted secondary structures. The web server is available at http://rtools.cbrc.jp, which integrates software tools, CentroidFold, CentroidHomfold, IPKnot, CapR, Raccess, Rchange and RintD. PMID:27131356

  19. Rtools: a web server for various secondary structural analyses on single RNA sequences

    PubMed Central

    Hamada, Michiaki; Ono, Yukiteru; Kiryu, Hisanori; Sato, Kengo; Kato, Yuki; Fukunaga, Tsukasa; Mori, Ryota; Asai, Kiyoshi

    2016-01-01

    The secondary structures, as well as the nucleotide sequences, are the important features of RNA molecules to characterize their functions. According to the thermodynamic model, however, the probability of any secondary structure is very small. As a consequence, any tool to predict the secondary structures of RNAs has limited accuracy. On the other hand, there are a few tools to compensate the imperfect predictions by calculating and visualizing the secondary structural information from RNA sequences. It is desirable to obtain the rich information from those tools through a friendly interface. We implemented a web server of the tools to predict secondary structures and to calculate various structural features based on the energy models of secondary structures. By just giving an RNA sequence to the web server, the user can get the different types of solutions of the secondary structures, the marginal probabilities such as base-paring probabilities, loop probabilities and accessibilities of the local bases, the energy changes by arbitrary base mutations as well as the measures for validations of the predicted secondary structures. The web server is available at http://rtools.cbrc.jp, which integrates software tools, CentroidFold, CentroidHomfold, IPKnot, CapR, Raccess, Rchange and RintD. PMID:27131356

  20. Comparative analysis of secondary structure of insect mitochondrial small subunit ribosomal RNA using maximum weighted matching.

    PubMed

    Page, R D

    2000-10-15

    Comparative analysis is the preferred method of inferring RNA secondary structure, but its use requires considerable expertise and manual effort. As the importance of secondary structure for accurate sequence alignment and phylogenetic analysis becomes increasingly realised, the need for secondary structure models for diverse taxonomic groups becomes more pressing. The number of available structures bears little relation to the relative diversity or importance of the different taxonomic groups. Insects, for example, comprise the largest group of animals and yet are very poorly represented in secondary structure databases. This paper explores the utility of maximum weighted matching (MWM) to help automate the process of comparative analysis by inferring secondary structure for insect mitochondrial small subunit (12S) rRNA sequences. By combining information on correlated changes in substitutions and helix dot plots, MWM can rapidly generate plausible models of secondary structure. These models can be further refined using standard comparative techniques. This paper presents a secondary structure model for insect 12S rRNA based on an alignment of 225 insect sequences and an alignment for 16 exemplar insect sequences. This alignment is used as a template for a web server that automatically generates secondary structures for insect sequences.

  1. mRNA expression profiling of laser microbeam microdissected cells from slender embryonic structures.

    PubMed

    Scheidl, Stefan J; Nilsson, Sven; Kalén, Mattias; Hellström, Mats; Takemoto, Minoru; Håkansson, Joakim; Lindahl, Per

    2002-03-01

    Microarray hybridization has rapidly evolved as an important tool for genomic studies and studies of gene regulation at the transcriptome level. Expression profiles from homogenous samples such as yeast and mammalian cell cultures are currently extending our understanding of biology, whereas analyses of multicellular organisms are more difficult because of tissue complexity. The combination of laser microdissection, RNA amplification, and microarray hybridization has the potential to provide expression profiles from selected populations of cells in vivo. In this article, we present and evaluate an experimental procedure for global gene expression analysis of slender embryonic structures using laser microbeam microdissection and laser pressure catapulting. As a proof of principle, expression profiles from 1000 cells in the mouse embryonic (E9.5) dorsal aorta were generated and compared with profiles for captured mesenchymal cells located one cell diameter further away from the aortic lumen. A number of genes were overexpressed in the aorta, including 11 previously known markers for blood vessels. Among the blood vessel markers were endoglin, tie-2, PDGFB, and integrin-beta1, that are important regulators of blood vessel formation. This demonstrates that microarray analysis of laser microbeam micro-dissected cells is sufficiently sensitive for identifying genes with regulative functions.

  2. Sucrose prevents protein fibrillation through compaction of the tertiary structure but hardly affects the secondary structure.

    PubMed

    Estrela, Nídia; Franquelim, Henri G; Lopes, Carlos; Tavares, Evandro; Macedo, Joana A; Christiansen, Gunna; Otzen, Daniel E; Melo, Eduardo P

    2015-11-01

    Amyloid fibers, implicated in a wide range of diseases, are formed when proteins misfold and stick together in long rope-like structures. As a natural mechanism, osmolytes can be used to modulate protein aggregation pathways with no interference with other cellular functions. The osmolyte sucrose delays fibrillation of the ribosomal protein S6 leading to softer and less shaped-defined fibrils. The molecular mechanism used by sucrose to delay S6 fibrillation was studied based on the two-state unfolding kinetics of the secondary and tertiary structures. It was concluded that the delay in S6 fibrillation results from stabilization and compaction of the slightly expanded tertiary native structure formed under fibrillation conditions. Interestingly, this compaction extends to almost all S6 tertiary structure but hardly affects its secondary structure. The part of the S6 tertiary structure that suffered more compaction by sucrose is known to be the first part to unfold, indicating that the native S6 has entered the unfolding pathway under fibrillation conditions.

  3. Secondary Impacts on Structures on the Lunar Surface

    NASA Technical Reports Server (NTRS)

    Christiansen, Eric; Walker, James D.; Grosch, Donald J.

    2010-01-01

    The Altair Lunar Lander is being designed for the planned return to the Moon by 2020. Since it is hoped that lander components will be re-used by later missions, studies are underway to examine the exposure threat to the lander sitting on the Lunar surface for extended periods. These threats involve both direct strikes of meteoroids on the vehicle as well as strikes from Lunar regolith and rock thrown by nearby meteorite strikes. Currently, the lander design is comprised of up to 10 different types of pressure vessels. These vessels included the manned habitation module, fuel, cryogenic fuel and gas storage containers, and instrument bays. These pressure vessels have various wall designs, including various aluminum alloys, honeycomb, and carbon-fiber composite materials. For some of the vessels, shielding is being considered. This program involved the test and analysis of six pressure vessel designs, one of which included a Whipple bumper shield. In addition to the pressure vessel walls, all the pressure vessels are wrapped in multi-layer insulation (MLI). Two variants were tested without the MLI to better understand the role of the MLI in the impact performance. The tests of performed were to examine the secondary impacts on these structures as they rested on the Lunar surface. If a hypervelocity meteor were to strike the surface nearby, it would throw regolith and rock debris into the structure at a much lower velocity. Also, when the manned module departs for the return to Earth, its rocket engines throw up debris that can impact the remaining lander components and cause damage. Glass spheres were used as a stimulant for the regolith material. Impact tests were performed with a gas gun to find the V50 of various sized spheres striking the pressure vessels. The impacts were then modeled and a fast-running approximate model for the V50 data was developed. This model was for performing risk analysis to assist in the vessel design and in the identification of ideal

  4. Self-Renewing Secondary Schools: The Relationship between Structural and Cultural Change.

    ERIC Educational Resources Information Center

    Hannay, Lynne M.; Ross, John A.

    This paper explores the deep-reform efforts of 9 secondary schools over a 3-year period. The reforms occurred in an Ontario, Canada, school district that empowered their secondary schools to develop site-specific organizational structures that deviated from the traditional subject, departmental structure. The sample for the study included all…

  5. CSI 3.0: a web server for identifying secondary and super-secondary structure in proteins using NMR chemical shifts.

    PubMed

    Hafsa, Noor E; Arndt, David; Wishart, David S

    2015-07-01

    The Chemical Shift Index or CSI 3.0 (http://csi3.wishartlab.com) is a web server designed to accurately identify the location of secondary and super-secondary structures in protein chains using only nuclear magnetic resonance (NMR) backbone chemical shifts and their corresponding protein sequence data. Unlike earlier versions of CSI, which only identified three types of secondary structure (helix, β-strand and coil), CSI 3.0 now identifies total of 11 types of secondary and super-secondary structures, including helices, β-strands, coil regions, five common β-turns (type I, II, I', II' and VIII), β hairpins as well as interior and edge β-strands. CSI 3.0 accepts experimental NMR chemical shift data in multiple formats (NMR Star 2.1, NMR Star 3.1 and SHIFTY) and generates colorful CSI plots (bar graphs) and secondary/super-secondary structure assignments. The output can be readily used as constraints for structure determination and refinement or the images may be used for presentations and publications. CSI 3.0 uses a pipeline of several well-tested, previously published programs to identify the secondary and super-secondary structures in protein chains. Comparisons with secondary and super-secondary structure assignments made via standard coordinate analysis programs such as DSSP, STRIDE and VADAR on high-resolution protein structures solved by X-ray and NMR show >90% agreement between those made with CSI 3.0. PMID:25979265

  6. CSI 3.0: a web server for identifying secondary and super-secondary structure in proteins using NMR chemical shifts.

    PubMed

    Hafsa, Noor E; Arndt, David; Wishart, David S

    2015-07-01

    The Chemical Shift Index or CSI 3.0 (http://csi3.wishartlab.com) is a web server designed to accurately identify the location of secondary and super-secondary structures in protein chains using only nuclear magnetic resonance (NMR) backbone chemical shifts and their corresponding protein sequence data. Unlike earlier versions of CSI, which only identified three types of secondary structure (helix, β-strand and coil), CSI 3.0 now identifies total of 11 types of secondary and super-secondary structures, including helices, β-strands, coil regions, five common β-turns (type I, II, I', II' and VIII), β hairpins as well as interior and edge β-strands. CSI 3.0 accepts experimental NMR chemical shift data in multiple formats (NMR Star 2.1, NMR Star 3.1 and SHIFTY) and generates colorful CSI plots (bar graphs) and secondary/super-secondary structure assignments. The output can be readily used as constraints for structure determination and refinement or the images may be used for presentations and publications. CSI 3.0 uses a pipeline of several well-tested, previously published programs to identify the secondary and super-secondary structures in protein chains. Comparisons with secondary and super-secondary structure assignments made via standard coordinate analysis programs such as DSSP, STRIDE and VADAR on high-resolution protein structures solved by X-ray and NMR show >90% agreement between those made with CSI 3.0.

  7. Characterization of a Trifunctional Mimivirus mRNA Capping Enzyme and Crystal Structure of the RNA Triphosphatase Domain

    SciTech Connect

    Benarroch,D.; Smith, P.; Shuman, S.

    2008-01-01

    The RNA triphosphatase (RTPase) components of the mRNA capping apparatus are a bellwether of eukaryal taxonomy. Fungal and protozoal RTPases belong to the triphosphate tunnel metalloenzyme (TTM) family, exemplified by yeast Cet1. Several large DNA viruses encode metal-dependent RTPases unrelated to the cysteinyl-phosphatase RTPases of their metazoan host organisms. The origins of DNA virus RTPases are unclear because they are structurally uncharacterized. Mimivirus, a giant virus of amoeba, resembles poxviruses in having a trifunctional capping enzyme composed of a metal-dependent RTPase module fused to guanylyltransferase (GTase) and guanine-N7 methyltransferase domains. The crystal structure of mimivirus RTPase reveals a minimized tunnel fold and an active site strikingly similar to that of Cet1. Unlike homodimeric fungal RTPases, mimivirus RTPase is a monomer. The mimivirus TTM-type RTPase-GTase fusion resembles the capping enzymes of amoebae, providing evidence that the ancestral large DNA virus acquired its capping enzyme from a unicellular host.

  8. ExSer: A standalone tool to mine protein data bank (PDB) for secondary structural elements

    PubMed Central

    Vignesh, Dhandapani; Daniel, Paul; Raja, Natarajan; Balasubramanian, Ponnusamy; Arul, Loganathan

    2010-01-01

    Detailed structural analysis of protein necessitates investigation at primary, secondary and tertiary levels, respectively. Insight into protein secondary structures pave way for understanding the type of secondary structural elements involved (α-helices, β-strands etc.), the amino acid sequence that encode the secondary structural elements, number of residues, length and, percentage composition of the respective elements in the protein. Here we present a standalone tool entitled “ExSer” which facilitate an automated extraction of the amino acid sequence that encode for the secondary structural regions of a protein from the protein data bank (PDB) file. Availability ExSer is freely downloadable from http://code.google.com/p/tool-exser/ PMID:20975886

  9. Evaluation of the information content of RNA structure mapping data for secondary structure prediction.

    PubMed

    Quarrier, Scott; Martin, Joshua S; Davis-Neulander, Lauren; Beauregard, Arthur; Laederach, Alain

    2010-06-01

    Structure mapping experiments (using probes such as dimethyl sulfate [DMS], kethoxal, and T1 and V1 RNases) are used to determine the secondary structures of RNA molecules. The process is iterative, combining the results of several probes with constrained minimum free-energy calculations to produce a model of the structure. We aim to evaluate whether particular probes provide more structural information, and specifically, how noise in the data affects the predictions. Our approach involves generating "decoy" RNA structures (using the sFold Boltzmann sampling procedure) and evaluating whether we are able to identify the correct structure from this ensemble of structures. We show that with perfect information, we are always able to identify the optimal structure for five RNAs of known structure. We then collected orthogonal structure mapping data (DMS and RNase T1 digest) under several solution conditions using our high-throughput capillary automated footprinting analysis (CAFA) technique on two group I introns of known structure. Analysis of these data reveals the error rates in the data under optimal (low salt) and suboptimal solution conditions (high MgCl(2)). We show that despite these errors, our computational approach is less sensitive to experimental noise than traditional constraint-based structure prediction algorithms. Finally, we propose a novel approach for visualizing the interaction of chemical and enzymatic mapping data with RNA structure. We project the data onto the first two dimensions of a multidimensional scaling of the sFold-generated decoy structures. We are able to directly visualize the structural information content of structure mapping data and reconcile multiple data sets.

  10. Evaluation of the information content of RNA structure mapping data for secondary structure prediction.

    PubMed

    Quarrier, Scott; Martin, Joshua S; Davis-Neulander, Lauren; Beauregard, Arthur; Laederach, Alain

    2010-06-01

    Structure mapping experiments (using probes such as dimethyl sulfate [DMS], kethoxal, and T1 and V1 RNases) are used to determine the secondary structures of RNA molecules. The process is iterative, combining the results of several probes with constrained minimum free-energy calculations to produce a model of the structure. We aim to evaluate whether particular probes provide more structural information, and specifically, how noise in the data affects the predictions. Our approach involves generating "decoy" RNA structures (using the sFold Boltzmann sampling procedure) and evaluating whether we are able to identify the correct structure from this ensemble of structures. We show that with perfect information, we are always able to identify the optimal structure for five RNAs of known structure. We then collected orthogonal structure mapping data (DMS and RNase T1 digest) under several solution conditions using our high-throughput capillary automated footprinting analysis (CAFA) technique on two group I introns of known structure. Analysis of these data reveals the error rates in the data under optimal (low salt) and suboptimal solution conditions (high MgCl(2)). We show that despite these errors, our computational approach is less sensitive to experimental noise than traditional constraint-based structure prediction algorithms. Finally, we propose a novel approach for visualizing the interaction of chemical and enzymatic mapping data with RNA structure. We project the data onto the first two dimensions of a multidimensional scaling of the sFold-generated decoy structures. We are able to directly visualize the structural information content of structure mapping data and reconcile multiple data sets. PMID:20413617

  11. Visualizing the global secondary structure of a viral RNA genome with cryo-electron microscopy.

    PubMed

    Garmann, Rees F; Gopal, Ajaykumar; Athavale, Shreyas S; Knobler, Charles M; Gelbart, William M; Harvey, Stephen C

    2015-05-01

    The lifecycle, and therefore the virulence, of single-stranded (ss)-RNA viruses is regulated not only by their particular protein gene products, but also by the secondary and tertiary structure of their genomes. The secondary structure of the entire genomic RNA of satellite tobacco mosaic virus (STMV) was recently determined by selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE). The SHAPE analysis suggested a single highly extended secondary structure with much less branching than occurs in the ensemble of structures predicted by purely thermodynamic algorithms. Here we examine the solution-equilibrated STMV genome by direct visualization with cryo-electron microscopy (cryo-EM), using an RNA of similar length transcribed from the yeast genome as a control. The cryo-EM data reveal an ensemble of branching patterns that are collectively consistent with the SHAPE-derived secondary structure model. Thus, our results both elucidate the statistical nature of the secondary structure of large ss-RNAs and give visual support for modern RNA structure determination methods. Additionally, this work introduces cryo-EM as a means to distinguish between competing secondary structure models if the models differ significantly in terms of the number and/or length of branches. Furthermore, with the latest advances in cryo-EM technology, we suggest the possibility of developing methods that incorporate restraints from cryo-EM into the next generation of algorithms for the determination of RNA secondary and tertiary structures.

  12. Pfold: RNA secondary structure prediction using stochastic context-free grammars.

    PubMed

    Knudsen, Bjarne; Hein, Jotun

    2003-07-01

    RNA secondary structures are important in many biological processes and efficient structure prediction can give vital directions for experimental investigations. Many available programs for RNA secondary structure prediction only use a single sequence at a time. This may be sufficient in some applications, but often it is possible to obtain related RNA sequences with conserved secondary structure. These should be included in structural analyses to give improved results. This work presents a practical way of predicting RNA secondary structure that is especially useful when related sequences can be obtained. The method improves a previous algorithm based on an explicit evolutionary model and a probabilistic model of structures. Predictions can be done on a web server at http://www.daimi.au.dk/~compbio/pfold.

  13. Formation of circular polyribosomes on eukaryotic mRNA without cap-structure and poly(A)-tail: a cryo electron tomography study.

    PubMed

    Afonina, Zhanna A; Myasnikov, Alexander G; Shirokov, Vladimir A; Klaholz, Bruno P; Spirin, Alexander S

    2014-08-01

    The polyribosomes newly formed on recombinant GFP-encoding mRNAs in a wheat germ cell-free translation system were analyzed using cryo-electron tomography, with sub-tomogram averaging of polysomal ribosomes and reconstruction of 3D structures of individual polyribosomes. The achieved level of resolution in the reconstructed polyribosomes allowed deducing the mRNA path by connecting adjacent exit and entry sites at the ribosomes inside each polyribosome. In this way, the circularity of a significant fraction (about 50%) of translating polyribosomes was proved in the case of the capped poly(A)-tailed mRNA, in agreement with the existing paradigm of the circularization via interaction of cap-bound initiation factor eIF4F with poly(A)-binding protein. However, translation of the capped mRNA construct without poly(A) tail, but with unspecific 3'-UTR derived from non-coding plasmid sequence, also led to the formation of circular polyribosomes in similar proportion (40%). Moreover, the polyribosomes formed on the uncapped non-polyadenylated mRNA with non-synergistic 5'- and 3'-UTRs proved to be circular as well, and appeared in the same proportion as in the previous cases. Thus, the formation of circular polyribosomes was found to be virtually independent of the presence of cap structure and poly(A) tail in mRNA, in contrast to the longstanding paradigm in the field.

  14. Efficient detection of secondary structure folded nucleic acids related to Alzheimer's disease based on junction probes.

    PubMed

    Li, Juan; Qi, Xiu-Juan; Du, Yan-Yan; Fu, Hua-E; Chen, Guo-Nan; Yang, Huang-Hao

    2012-01-01

    Single stranded DNA often forms stable secondary structures under physiological conditions. These DNA secondary structures play important physiological roles. However, the analysis of such secondary structure folded DNA is often complicated because of its high thermodynamic stability and slow hybridization kinetics. In this article, we demonstrate that Y-shaped junction probes could be used for rapid and highly efficient detection of secondary structure folded DNA. Our approach contained a molecular beacon (MB) probe and an assistant probe. In the absence of target, the MB probe failed to hybridize with the assistant probe. Whereas, the MB probe and the assistant probe could cooperatively unwind the secondary structure folded DNA target to form a ternary Y-shaped junction structure. In this condition, the MB probe was also opened, resulting in separating the fluorophores from the quenching moiety and emitting the fluorescence signal. This approach allowed for the highly sensitive detection of secondary structure folded DNA target, such as a tau specific DNA fragment related to Alzheimer's disease in this case. Additionally, this approach showed strong SNPs identifying capability. Furthermore, it was noteworthy that this newly proposed approach was capable of detecting secondary structure folded DNA target in cell lysate samples.

  15. Deciphering the shape and deformation of secondary structures through local conformation analysis

    PubMed Central

    2011-01-01

    Background Protein deformation has been extensively analysed through global methods based on RMSD, torsion angles and Principal Components Analysis calculations. Here we use a local approach, able to distinguish among the different backbone conformations within loops, α-helices and β-strands, to address the question of secondary structures' shape variation within proteins and deformation at interface upon complexation. Results Using a structural alphabet, we translated the 3 D structures of large sets of protein-protein complexes into sequences of structural letters. The shape of the secondary structures can be assessed by the structural letters that modeled them in the structural sequences. The distribution analysis of the structural letters in the three protein compartments (surface, core and interface) reveals that secondary structures tend to adopt preferential conformations that differ among the compartments. The local description of secondary structures highlights that curved conformations are preferred on the surface while straight ones are preferred in the core. Interfaces display a mixture of local conformations either preferred in core or surface. The analysis of the structural letters transition occurring between protein-bound and unbound conformations shows that the deformation of secondary structure is tightly linked to the compartment preference of the local conformations. Conclusion The conformation of secondary structures can be further analysed and detailed thanks to a structural alphabet which allows a better description of protein surface, core and interface in terms of secondary structures' shape and deformation. Induced-fit modification tendencies described here should be valuable information to identify and characterize regions under strong structural constraints for functional reasons. PMID:21284872

  16. Structural analysis of human 2'-O-ribose methyltransferases involved in mRNA cap structure formation

    NASA Astrophysics Data System (ADS)

    Smietanski, Miroslaw; Werner, Maria; Purta, Elzbieta; Kaminska, Katarzyna H.; Stepinski, Janusz; Darzynkiewicz, Edward; Nowotny, Marcin; Bujnicki, Janusz M.

    2014-01-01

    The 5' cap of human messenger RNA contains 2'-O-methylation of the first and often second transcribed nucleotide that is important for its processing, translation and stability. Human enzymes that methylate these nucleotides, termed CMTr1 and CMTr2, respectively, have recently been identified. However, the structures of these enzymes and their mechanisms of action remain unknown. In the present study, we solve the crystal structures of the active CMTr1 catalytic domain in complex with a methyl group donor and a capped oligoribonucleotide, thereby revealing the mechanism of specific recognition of capped RNA. This mechanism differs significantly from viral enzymes, thus providing a framework for their specific targeting. Based on the crystal structure of CMTr1, a comparative model of the CMTr2 catalytic domain is generated. This model, together with mutational analysis, leads to the identification of residues involved in RNA and methyl group donor binding.

  17. Structural analysis of human 2′-O-ribose methyltransferases involved in mRNA cap structure formation

    PubMed Central

    Smietanski, Miroslaw; Werner, Maria; Purta, Elzbieta; Kaminska, Katarzyna H.; Stepinski, Janusz; Darzynkiewicz, Edward; Nowotny, Marcin; Bujnicki, Janusz M.

    2014-01-01

    The 5′ cap of human messenger RNA contains 2′-O-methylation of the first and often second transcribed nucleotide that is important for its processing, translation and stability. Human enzymes that methylate these nucleotides, termed CMTr1 and CMTr2, respectively, have recently been identified. However, the structures of these enzymes and their mechanisms of action remain unknown. In the present study, we solve the crystal structures of the active CMTr1 catalytic domain in complex with a methyl group donor and a capped oligoribonucleotide, thereby revealing the mechanism of specific recognition of capped RNA. This mechanism differs significantly from viral enzymes, thus providing a framework for their specific targeting. Based on the crystal structure of CMTr1, a comparative model of the CMTr2 catalytic domain is generated. This model, together with mutational analysis, leads to the identification of residues involved in RNA and methyl group donor binding. PMID:24402442

  18. Quantifying the energetic interplay of RNA tertiary and secondary structure interactions.

    PubMed Central

    Silverman, S K; Zheng, M; Wu, M; Tinoco, I; Cech, T R

    1999-01-01

    To understand the RNA-folding problem, we must know the extent to which RNA structure formation is hierarchical (tertiary folding of preformed secondary structure). Recently, nuclear magnetic resonance (NMR) spectroscopy was used to show that Mg2+-dependent tertiary interactions force secondary structure rearrangement in the 56-nt tP5abc RNA, a truncated subdomain of the Tetrahymena group I intron. Here we combine mutagenesis with folding computations, nondenaturing gel electrophoresis, high-resolution NMR spectroscopy, and chemical-modification experiments to probe further the energetic interplay of tertiary and secondary interactions in tP5abc. Point mutations predicted to destabilize the secondary structure of folded tP5abc greatly disrupt its Mg2+-dependent folding, as monitored by nondenaturing gels. Imino proton assignments and sequential NOE walks of the two-dimensional NMR spectrum of one of the tP5abc mutants confirm the predicted secondary structure, which does not change in the presence of Mg2+. In contrast to these data on tP5abc, the same point mutations in the context of the P4-P6 domain (of which P5abc is a subdomain) shift the Mg2+ dependence of P4-P6 folding only moderately, and dimethyl sulfate (DMS) modification experiments demonstrate that Mg2+ does cause secondary structure rearrangement of the P4-P6 mutants' P5abc subdomains. Our data provide experimental support for two simple conclusions: (1) Even single point mutations at bases involved only in secondary structure can be enough to tip the balance between RNA tertiary and secondary interactions. (2) Domain context must be considered in evaluating the relative importance of tertiary and secondary contributions. This tertiary/secondary interplay is likely relevant to the folding of many large RNA and to bimolecular snRNA-snRNA and snRNA-intron RNA interactions. PMID:10606276

  19. Secondary structure models for the internal transcribed spacer (ITS) region 1 from symbiotic dinoflagellates.

    PubMed

    Thornhill, Daniel J; Lord, Jenna B

    2010-07-01

    Ribosomal genes and their spacers have been extensively utilized to examine the biodiversity and phylogenetics of protists. Among these, the internal transcribed spacer regions 1 and 2 (ITS1 and ITS2) are known to form secondary structures that are critically important for proper processing of the pre-rRNA into mature ribosomes. Although the secondary structure of ITS2 has been widely investigated, considerably less is known about ITS1 and its secondary structure. Here, secondary structures of the ITS1 were modeled for 46 ITS "types" from Symbiodinium, a diverse dinoflagellate genus that forms symbioses with many protists and metazoans, using comparative phylogenetic and minimum free energy approaches. The predicted ITS1 secondary structures for each Symbiodinium "type" were highly stable (DeltaG=-46.40 to -85.30 kcal mol(-1) at 37 degrees C) and consisted of an open loop with five helices separated by single-stranded regions. Several structural characteristics were conserved within monophyletic sub-groups, providing additional support for the predicted structures and the relationships within this genus. Finally, the structures were applied to identify potential pseudogenes from five Symbiodinium ITS1 datasets. Consequently, ITS1 secondary structures are useful in understanding the biology and phylogenetics, as well as recognizing and excluding questionable sequences from datasets, of protists such as Symbiodinium.

  20. Determination of Secondary School Students' Cognitive Structure, and Misconception in Ecological Concepts through Word Association Test

    ERIC Educational Resources Information Center

    Yücel, Elif Özata; Özkan, Mulis

    2015-01-01

    In this study, we determined cognitive structures and misconceptions about basic ecological concepts by using "word association" tests on secondary school students, age between 12-14 years. Eighty-nine students participated in this study. Before WAT was generated, basic ecological concepts that take place in the secondary science…

  1. Non-B DNA Secondary Structures and Their Resolution by RecQ Helicases

    PubMed Central

    Sharma, Sudha

    2011-01-01

    In addition to the canonical B-form structure first described by Watson and Crick, DNA can adopt a number of alternative structures. These non-B-form DNA secondary structures form spontaneously on tracts of repeat sequences that are abundant in genomes. In addition, structured forms of DNA with intrastrand pairing may arise on single-stranded DNA produced transiently during various cellular processes. Such secondary structures have a range of biological functions but also induce genetic instability. Increasing evidence suggests that genomic instabilities induced by non-B DNA secondary structures result in predisposition to diseases. Secondary DNA structures also represent a new class of molecular targets for DNA-interactive compounds that might be useful for targeting telomeres and transcriptional control. The equilibrium between the duplex DNA and formation of multistranded non-B-form structures is partly dependent upon the helicases that unwind (resolve) these alternate DNA structures. With special focus on tetraplex, triplex, and cruciform, this paper summarizes the incidence of non-B DNA structures and their association with genomic instability and emphasizes the roles of RecQ-like DNA helicases in genome maintenance by resolution of DNA secondary structures. In future, RecQ helicases are anticipated to be additional molecular targets for cancer chemotherapeutics. PMID:21977309

  2. Students' understanding of primary and secondary protein structure: drawing secondary protein structure reveals student understanding better than simple recognition of structures.

    PubMed

    Harle, Marissa; Towns, Marcy H

    2013-01-01

    The interdisciplinary nature of biochemistry courses requires students to use both chemistry and biology knowledge to understand biochemical concepts. Research that has focused on external representations in biochemistry has uncovered student difficulties in comprehending and interpreting external representations in addition to a fragmented understanding of fundamental biochemistry concepts. This project focuses on students' understanding of primary and secondary protein structure and drawings (representations) of hydrogen-bonding in alpha helices and beta sheets. Analysis demonstrated that students can recognize and identify primary protein structure concepts when given a polypeptide. However, when asked to draw alpha helices and beta sheets and explain the role of hydrogen bonding their drawings students exhibited a fragmented understanding that lacked coherence. Faculty are encouraged to have students draw molecular level representations to make their mental models more explicit, complete, and coherent. This is in contrast to recognition and identification tasks, which do not adequately probe mental models and molecular level understanding.

  3. Putative secondary structures of unusually long strepsipteran SSU rRNAs and its phylogenetic implications.

    PubMed

    Choe, C P; Hwang, U W; Kim, W

    1999-04-30

    We constructed the putative secondary structures of the small subunit rRNAs (SSU rRNA) from three strepsipteran insects. The primary sequences of the strepsipteran SSU rRNAs are unusually long due to unique and long insertions. In spite of these insertions, the basic shapes of their secondary structures are well maintained as shown in those of other eukaryotes, because these insertions appear mainly in the variable regions. The secondary structures for the V1, V3, V5, V8, and V9 regions are well conserved, even though the primary structures of V1, V5, and V8 regions are quite variable. However, the predicted secondary structures for the V2, V4, and V7 regions are quite different from those of other insects. In the V4 and V7 regions, helices specific to the Strepsiptera exist. These helices have not been reported in other organisms so far. Similarly, four eukaryotic specific helices (E8-1, E10-2, E23-4 and E45-1) not reported in insects exist in the V2, V4, and V8 regions. These helices are formed by the inserted sequences. The secondary structures of the expanded segments of the strepsipteran SSU rRNA were applied to infer the phylogenetic position of Strepsiptera, one of the most enigmatic problems in insect phylogeny. Only the secondary structure of the V7 region showed the weak Strepsiptera/Diptera sister-group relationship. PMID:10340475

  4. Nuclear rRNA transcript processing versus internal transcribed spacer secondary structure.

    PubMed

    Coleman, Annette W

    2015-03-01

    rRNA is one of the few universal features of life, making it uniquely suited to assess phylogenetic relationships. The processing of the initial polycistronic rRNA transcript is also a conserved process, involving numerous cleavage events and the generation of secondary structures. The secondary structure of the internal transcribed spacer (ITS) regions of nuclear rRNA transcripts are well known for a wide variety of eukaryotes and have been used to aid in the alignment of these sequences for phylogenetic comparisons. By contrast, study of the processing of the initial rRNA transcripts has been largely limited to yeast, mice, rats, and humans. Here I examine the known cleavage sites in the two ITS regions and their positions relative to the secondary structure. A better understanding of the conservation of secondary structures and cleavage sites within the ITS regions will improve evolutionary inferences based on these sequences.

  5. Sheath structure transition controlled by secondary electron emission

    NASA Astrophysics Data System (ADS)

    Schweigert, I. V.; Langendorf, S. J.; Walker, M. L. R.; Keidar, M.

    2015-04-01

    In particle-in-cell Monte Carlo collision (PIC MCC) simulations and in an experiment we study sheath formation over an emissive floating Al2O3 plate in a direct current discharge plasma at argon gas pressure 10-4 Torr. The discharge glow is maintained by the beam electrons emitted from a negatively biased hot cathode. We observe three types of sheaths near the floating emissive plate and the transition between them is driven by changing the negative bias. The Debye sheath appears at lower voltages, when secondary electron emission is negligible. With increasing applied voltage, secondary electron emission switches on and a first transition to a new sheath type, beam electron emission (BEE), takes place. For the first time we find this specific regime of sheath operation near the floating emissive surface. In this regime, the potential drop over the plate sheath is about four times larger than the temperature of plasma electrons. The virtual cathode appears near the emissive plate and its modification helps to maintain the BEE regime within some voltage range. Further increase of the applied voltage U initiates the second smooth transition to the plasma electron emission sheath regime and the ratio Δφs/Te tends to unity with increasing U. The oscillatory behavior of the emissive sheath is analyzed in PIC MCC simulations. A plasmoid of slow electrons is formed near the plate and transported to the bulk plasma periodically with a frequency of about 25 kHz.

  6. Structure of the no-go mRNA decay complex Dom34-Hbs1 bound to a stalled 80S ribosome.

    PubMed

    Becker, Thomas; Armache, Jean-Paul; Jarasch, Alexander; Anger, Andreas M; Villa, Elizabeth; Sieber, Heidemarie; Motaal, Basma Abdel; Mielke, Thorsten; Berninghausen, Otto; Beckmann, Roland

    2011-06-01

    No-go decay (NGD) is a mRNA quality-control mechanism in eukaryotic cells that leads to degradation of mRNAs stalled during translational elongation. The key factors triggering NGD are Dom34 and Hbs1. We used cryo-EM to visualize NGD intermediates resulting from binding of the Dom34-Hbs1 complex to stalled ribosomes. At subnanometer resolution, all domains of Dom34 and Hbs1 were identified, allowing the docking of crystal structures and homology models. Moreover, the close structural similarity of Dom34 and Hbs1 to eukaryotic release factors (eRFs) enabled us to propose a model for the ribosome-bound eRF1-eRF3 complex. Collectively, our data provide structural insights into how stalled mRNA is recognized on the ribosome and how the eRF complex can simultaneously recognize stop codons and catalyze peptide release.

  7. Structural basis for the recognition of cellular mRNA export factor REF by herpes viral proteins HSV-1 ICP27 and HVS ORF57.

    PubMed

    Tunnicliffe, Richard B; Hautbergue, Guillaume M; Kalra, Priti; Jackson, Brian R; Whitehouse, Adrian; Wilson, Stuart A; Golovanov, Alexander P

    2011-01-06

    The herpesvirus proteins HSV-1 ICP27 and HVS ORF57 promote viral mRNA export by utilizing the cellular mRNA export machinery. This function is triggered by binding to proteins of the transcription-export (TREX) complex, in particular to REF/Aly which directs viral mRNA to the TAP/NFX1 pathway and, subsequently, to the nuclear pore for export to the cytoplasm. Here we have determined the structure of the REF-ICP27 interaction interface at atomic-resolution and provided a detailed comparison of the binding interfaces between ICP27, ORF57 and REF using solution-state NMR. Despite the absence of any obvious sequence similarity, both viral proteins bind on the same site of the folded RRM domain of REF, via short but specific recognition sites. The regions of ICP27 and ORF57 involved in binding by REF have been mapped as residues 104-112 and 103-120, respectively. We have identified the pattern of residues critical for REF/Aly recognition, common to both ICP27 and ORF57. The importance of the key amino acid residues within these binding sites was confirmed by site-directed mutagenesis. The functional significance of the ORF57-REF/Aly interaction was also probed using an ex vivo cytoplasmic viral mRNA accumulation assay and this revealed that mutants that reduce the protein-protein interaction dramatically decrease the ability of ORF57 to mediate the nuclear export of intronless viral mRNA. Together these data precisely map amino acid residues responsible for the direct interactions between viral adaptors and cellular REF/Aly and provide the first molecular details of how herpes viruses access the cellular mRNA export pathway.

  8. Structural Basis for the Recognition of Cellular mRNA Export Factor REF by Herpes Viral Proteins HSV-1 ICP27 and HVS ORF57

    PubMed Central

    Tunnicliffe, Richard B.; Hautbergue, Guillaume M.; Kalra, Priti; Jackson, Brian R.; Whitehouse, Adrian; Wilson, Stuart A.; Golovanov, Alexander P.

    2011-01-01

    The herpesvirus proteins HSV-1 ICP27 and HVS ORF57 promote viral mRNA export by utilizing the cellular mRNA export machinery. This function is triggered by binding to proteins of the transcription-export (TREX) complex, in particular to REF/Aly which directs viral mRNA to the TAP/NFX1 pathway and, subsequently, to the nuclear pore for export to the cytoplasm. Here we have determined the structure of the REF-ICP27 interaction interface at atomic-resolution and provided a detailed comparison of the binding interfaces between ICP27, ORF57 and REF using solution-state NMR. Despite the absence of any obvious sequence similarity, both viral proteins bind on the same site of the folded RRM domain of REF, via short but specific recognition sites. The regions of ICP27 and ORF57 involved in binding by REF have been mapped as residues 104–112 and 103–120, respectively. We have identified the pattern of residues critical for REF/Aly recognition, common to both ICP27 and ORF57. The importance of the key amino acid residues within these binding sites was confirmed by site-directed mutagenesis. The functional significance of the ORF57-REF/Aly interaction was also probed using an ex vivo cytoplasmic viral mRNA accumulation assay and this revealed that mutants that reduce the protein-protein interaction dramatically decrease the ability of ORF57 to mediate the nuclear export of intronless viral mRNA. Together these data precisely map amino acid residues responsible for the direct interactions between viral adaptors and cellular REF/Aly and provide the first molecular details of how herpes viruses access the cellular mRNA export pathway. PMID:21253573

  9. Knowledge base and neural network approach for protein secondary structure prediction.

    PubMed

    Patel, Maulika S; Mazumdar, Himanshu S

    2014-11-21

    Protein structure prediction is of great relevance given the abundant genomic and proteomic data generated by the genome sequencing projects. Protein secondary structure prediction is addressed as a sub task in determining the protein tertiary structure and function. In this paper, a novel algorithm, KB-PROSSP-NN, which is a combination of knowledge base and modeling of the exceptions in the knowledge base using neural networks for protein secondary structure prediction (PSSP), is proposed. The knowledge base is derived from a proteomic sequence-structure database and consists of the statistics of association between the 5-residue words and corresponding secondary structure. The predicted results obtained using knowledge base are refined with a Backpropogation neural network algorithm. Neural net models the exceptions of the knowledge base. The Q3 accuracy of 90% and 82% is achieved on the RS126 and CB396 test sets respectively which suggest improvement over existing state of art methods.

  10. RNACluster: An integrated tool for RNA secondary structure comparison and clustering.

    PubMed

    Liu, Qi; Olman, V; Liu, Huiqing; Ye, Xiuzi; Qiu, Shilun; Xu, Ying

    2008-07-15

    RNA structure comparison is a fundamental problem in structural biology, structural chemistry, and bioinformatics. It can be used for analysis of RNA energy landscapes, conformational switches, and facilitating RNA structure prediction. The purpose of our integrated tool RNACluster is twofold: to provide a platform for computing and comparison of different distances between RNA secondary structures, and to perform cluster identification to derive useful information of RNA structure ensembles, using a minimum spanning tree (MST) based clustering algorithm. RNACluster employs a cluster identification approach based on a MST representation of the RNA ensemble data and currently supports six distance measures between RNA secondary structures. RNACluster provides a user-friendly graphical interface to allow a user to compare different structural distances, analyze the structure ensembles, and visualize predicted structural clusters. PMID:18271070

  11. RNACluster: An integrated tool for RNA secondary structure comparison and clustering.

    PubMed

    Liu, Qi; Olman, V; Liu, Huiqing; Ye, Xiuzi; Qiu, Shilun; Xu, Ying

    2008-07-15

    RNA structure comparison is a fundamental problem in structural biology, structural chemistry, and bioinformatics. It can be used for analysis of RNA energy landscapes, conformational switches, and facilitating RNA structure prediction. The purpose of our integrated tool RNACluster is twofold: to provide a platform for computing and comparison of different distances between RNA secondary structures, and to perform cluster identification to derive useful information of RNA structure ensembles, using a minimum spanning tree (MST) based clustering algorithm. RNACluster employs a cluster identification approach based on a MST representation of the RNA ensemble data and currently supports six distance measures between RNA secondary structures. RNACluster provides a user-friendly graphical interface to allow a user to compare different structural distances, analyze the structure ensembles, and visualize predicted structural clusters.

  12. The role of a metastable RNA secondary structure in hepatitis delta virus genotype III RNA editing

    PubMed Central

    Linnstaedt, Sarah D.; Kasprzak, Wojciech K.; Shapiro, Bruce A.; Casey, John L.

    2006-01-01

    RNA editing plays a critical role in the life cycle of hepatitis delta virus (HDV). The host editing enzyme ADAR1 recognizes specific RNA secondary structure features around the amber/W site in the HDV antigenome and deaminates the amber/W adenosine. A previous report suggested that a branched secondary structure is necessary for editing in HDV genotype III. This branched structure, which is distinct from the characteristic unbranched rod structure required for HDV replication, was only partially characterized, and knowledge concerning its formation and stability was limited. Here, we examine the secondary structures, conformational dynamics, and amber/W site editing of HDV genotype III RNA using a miniaturized HDV genotype III RNA in vitro. Computational analysis of this RNA using the MPGAfold algorithm indicated that the RNA has a tendency to form both metastable and stable unbranched secondary structures. Moreover, native polyacrylamide gel electrophoresis demonstrated that this RNA forms both branched and unbranched rod structures when transcribed in vitro. As predicted, the branched structure is a metastable structure that converts readily to the unbranched rod structure. Only branched RNA was edited at the amber/W site by ADAR1 in vitro. The structural heterogeneity of HDV genotype III RNA is significant because not only are both conformations of the RNA functionally important for viral replication, but the ratio of the two forms could modulate editing by determining the amount of substrate RNA available for modification. PMID:16790843

  13. Efficient sampling of RNA secondary structures from the Boltzmann ensemble of low-energy: the boustrophedon method.

    PubMed

    Ponty, Yann

    2008-01-01

    We adapt here a surprising technique, the boustrophedon method, to speed up the sampling of RNA secondary structures from the Boltzmann low-energy ensemble. This technique is simple and its implementation straight-forward, as it only requires a permutation in the order of some operations already performed in the stochastic traceback stage of these algorithms. It nevertheless greatly improves their worst-case complexity from O(n2)] to O(n log (n)), for n the size of the original sequence. Moreover the average-case complexity of the generation is shown to be improved from O(n square root n) to (O n log ((n)) in an Boltzmann-weighted homopolymer model based on the Nussinov-Jacobson free-energy model. These results are extended to the more realistic Turner free-energy model through experiments performed on both structured (Drosophilia melanogaster mRNA 5S) and hybrid (Staphylococcus aureus RNAIII) RNA sequences, using a boustrophedon modified version of the popular software UnaFold. This improvement allows for the sampling of greater and more significant sets of structures in a given time.

  14. Secondary structures of proteins from the 30S subunit of the Escherichia coli ribosome.

    PubMed

    Dzionara, M; Robinson, S M; Wittmann-Liebold, B

    1977-08-01

    The secondary structures of the proteins S4, S6, S8, S9, S12, S13, S15, S16, S18, S20 and S21 from the subunit of the E. coli ribosome were predicted according to four different methods. From the resultant diagrams indicating regions of helix, turn, extended structure and random coil, average values for the respective secondary structures could be calculated for each protein. Using the known relative distances for residues in the helical, turn and sheet or allowed random conformations, estimates are made of the maximum possible lengths of the proteins in order to correlate these with results obtained from antibody binding studies to the 30S subunit as determined by electron microscopy. The influence of amino acid changes on the predicted secondary structures of proteins from a few selected mutants was studied. The altered residues tend to be structurally conservative or to induce only minimal local changes.

  15. Structural evolution of gold nanorods during controlled secondary growth.

    PubMed

    Keul, Heidrun A; Möller, Martin; Bockstaller, Michael R

    2007-09-25

    Single-crystalline gold nanorods synthesized by the Ag(I)-mediated seeded-growth method (see: El-Sayed, M. A.; Nikoobakht, B. Chem. Mater. 2003, 15, 1957) were used as seeds for the preferential overgrowth of gold on particular crystallographic facets by systematic variation of the conditions during overgrowth. The results support previous reports about the relevance of the cationic surfactant cetyltrimethylammonium bromide (CTAB) and Ag(I) in stabilizing anisotropic particle shapes and demonstrate that the regulation of the amount of ascorbic acid facilitates the preferential overgrowth of {111} crystal facets to form Xi-type particle shapes. Interestingly, secondary overgrowth is found to inevitably result in a loss of particle shape anisotropy. A mechanism based on surface reconstruction is proposed to rationalize the "shape-reversal" that is generally observed in the nanorod growth process, that is, the initial increase and subsequent decrease of particle anisotropy with increasing reaction time. High-resolution electron microscopy analysis of gold nanorods reveals clear evidence for (1 x 2) missing row surface reconstruction of high energetic {110} facets that form during the initial phase during particle growth. PMID:17713936

  16. Probing the glycosidic linkage: secondary structures in the gas phase

    NASA Astrophysics Data System (ADS)

    Simons, John P.; Cristina Stanca-Kaposta, E.; Cocinero, Emilio J.; Liu, B.; Davis, Benjamin G.; Gamblin, David P.; Kroemer, Romano T.

    2008-10-01

    The functional importance of carbohydrates in biological processes, particularly those involving specific molecular recognition, is immense. Characterizing the three-dimensional (3D) structures of carbohydrates and glycoproteins, and their interactions with other molecules, not least the ubiquitous solvent, water, is a key starting point for understanding these processes. The combination of laser-based electronic and vibrational spectroscopy of mass-selected carbohydrate molecules and their hydrated complexes, conducted under molecular beam conditions, with ab initio computation is providing a uniquely powerful means of characterizing 3D carbohydrate conformations; the structures of their hydrated complexes, the hydrogen-bonded networks they support (or which support them); and the factors that determine their conformational and structural preferences.

  17. Fragile X Mental Retardation Protein Interactions with a G quadruplex structure in the 3′-Untranslated Region of NR2B mRNA

    PubMed Central

    Stefanovic, Snezana; DeMarco, Brett A.; Underwood, Ayana; Williams, Kathryn R.; Bassell, Gary J.; Mihailescu, Mihaela Rita

    2015-01-01

    Fragile X syndrome, the most common cause of inherited intellectual disability, is caused by a trinucleotide CGG expansion in the 5′-untranslated region of the FMR1 gene, which leads to the loss of expression of the fragile X mental retardation protein (FMRP). FMRP, an RNA-binding protein that regulates the translation of specific mRNAs, has been shown to bind a subset of its mRNA targets by recognizing G quadruplex structures. It has been suggested that FMRP controls the local protein synthesis of several protein components of the Post Synaptic Density (PSD) in response to specific cellular needs. We have previously shown that the interactions between FMRP and mRNAs of the PSD scaffold proteins PSD-95 and Shank1 are mediated via stable G-quadruplex structures formed within the 3′-untranslated regions of these mRNAs. In this study we used biophysical methods to show that a comparable G quadruplex structure forms in the 3′-untranslated region of the glutamate receptor subunit NR2B mRNA encoding for a subunit of N-methyl-D-aspartate (NMDA) receptors that is recognized specifically by FMRP, suggesting a common theme for FMRP recognition of its dendritic mRNA targets. PMID:26412477

  18. Structural Characterization of the Chaetomium thermophilum TREX-2 Complex and its Interaction with the mRNA Nuclear Export Factor Mex67:Mtr2

    PubMed Central

    Dimitrova, Lyudmila; Valkov, Eugene; Aibara, Shintaro; Flemming, Dirk; McLaughlin, Stephen H.; Hurt, Ed; Stewart, Murray

    2015-01-01

    Summary The TREX-2 complex integrates mRNA nuclear export into the gene expression pathway and is based on a Sac3 scaffold to which Thp1, Sem1, Sus1, and Cdc31 bind. TREX-2 also binds the mRNA nuclear export factor, Mex67:Mtr2, through the Sac3 N-terminal region (Sac3N). Here, we characterize Chaetomium thermophilum TREX-2, show that the in vitro reconstituted complex has an annular structure, and define the structural basis for interactions between Sac3, Sus1, Cdc31, and Mex67:Mtr2. Crystal structures show that the binding of C. thermophilum Sac3N to the Mex67 NTF2-like domain (Mex67NTF2L) is mediated primarily through phenylalanine residues present in a series of repeating sequence motifs that resemble those seen in many nucleoporins, and Mlp1 also binds Mex67:Mtr2 using a similar motif. Deletion of Sac3N generated growth and mRNA export defects in Saccharomyces cerevisiae, and we propose TREX-2 and Mlp1 function to facilitate export by concentrating mature messenger ribonucleoparticles at the nuclear pore entrance. PMID:26051714

  19. Porous carbonaceous electrode structure and method for secondary electrochemical cell

    DOEpatents

    Kaun, Thomas D.

    1977-03-08

    Positive and negative electrodes are provided as rigid, porous carbonaceous matrices with particulate active material fixedly embedded. Active material such as metal chalcogenides, solid alloys of alkali metal or alkaline earth metals along with other metals and their oxides in particulate form are blended with a thermosetting resin and a solid volatile to form a paste mixture. Various electrically conductive powders or current collector structures can be blended or embedded into the paste mixture which can be molded to the desired electrode shape. The molded paste is heated to a temperature at which the volatile transforms into vapor to impart porosity as the resin begins to cure into a rigid solid structure.

  20. Free energy minimization to predict RNA secondary structures and computational RNA design.

    PubMed

    Churkin, Alexander; Weinbrand, Lina; Barash, Danny

    2015-01-01

    Determining the RNA secondary structure from sequence data by computational predictions is a long-standing problem. Its solution has been approached in two distinctive ways. If a multiple sequence alignment of a collection of homologous sequences is available, the comparative method uses phylogeny to determine conserved base pairs that are more likely to form as a result of billions of years of evolution than by chance. In the case of single sequences, recursive algorithms that compute free energy structures by using empirically derived energy parameters have been developed. This latter approach of RNA folding prediction by energy minimization is widely used to predict RNA secondary structure from sequence. For a significant number of RNA molecules, the secondary structure of the RNA molecule is indicative of its function and its computational prediction by minimizing its free energy is important for its functional analysis. A general method for free energy minimization to predict RNA secondary structures is dynamic programming, although other optimization methods have been developed as well along with empirically derived energy parameters. In this chapter, we introduce and illustrate by examples the approach of free energy minimization to predict RNA secondary structures.

  1. GTfold: Enabling parallel RNA secondary structure prediction on multi-core desktops

    PubMed Central

    2012-01-01

    Background Accurate and efficient RNA secondary structure prediction remains an important open problem in computational molecular biology. Historically, advances in computing technology have enabled faster and more accurate RNA secondary structure predictions. Previous parallelized prediction programs achieved significant improvements in runtime, but their implementations were not portable from niche high-performance computers or easily accessible to most RNA researchers. With the increasing prevalence of multi-core desktop machines, a new parallel prediction program is needed to take full advantage of today’s computing technology. Findings We present here the first implementation of RNA secondary structure prediction by thermodynamic optimization for modern multi-core computers. We show that GTfold predicts secondary structure in less time than UNAfold and RNAfold, without sacrificing accuracy, on machines with four or more cores. Conclusions GTfold supports advances in RNA structural biology by reducing the timescales for secondary structure prediction. The difference will be particularly valuable to researchers working with lengthy RNA sequences, such as RNA viral genomes. PMID:22747589

  2. Evaluation of the Information Content in Infrared Spectra for Protein Secondary Structure Determination

    PubMed Central

    Goormaghtigh, Erik; Ruysschaert, Jean-Marie; Raussens, Vincent

    2006-01-01

    Fourier-transform infrared spectroscopy is a method of choice for the experimental determination of protein secondary structure. Numerous approaches have been developed during the past 15 years. A critical parameter that has not been taken into account systematically is the selection of the wavenumbers used for building the mathematical models used for structure prediction. The high quality of the current Fourier-transform infrared spectrometers makes the absorbance at every single wavenumber a valid and almost noiseless type of information. We address here the question of the amount of independent information present in the infrared spectra of proteins for the prediction of the different secondary structure contents. It appears that, at most, the absorbance at three distinct frequencies of the spectra contain all the nonredundant information that can be related to one secondary structure content. The ascending stepwise method proposed here identifies the relevance of each wavenumber of the infrared spectrum for the prediction of a given secondary structure and yields a particularly simple method for computing the secondary structure content. Using the 50-protein database built beforehand to contain as little fold redundancy as possible, the standard error of prediction in cross-validation is 5.5% for the α-helix, 6.6% for the β-sheet, and 3.4% for the β-turn. PMID:16428280

  3. Argumentation in Secondary School Students' Structured and Unstructured Chat Discussions

    ERIC Educational Resources Information Center

    Salminen, Timo; Marttunen, Miika; Laurinen, Leena

    2012-01-01

    Joint construction of new knowledge demands that persons can express their statements in a convincing way and explore other people's arguments constructively. For this reason, more knowledge on different means to support collaborative argumentation is needed. This study clarifies whether structured interaction supports students' critical and…

  4. Multiple Structurally Distinct ERα mRNA Variants in Zebrafish are Differentially Expressed by Tissue Type, Stage of Development and Estrogen Exposure

    PubMed Central

    Cotter, Kellie A.; Yershov, Anya; Novillo, Apolonia; Callard, Gloria V.

    2013-01-01

    It is well established that estrogen-like environmental chemicals interact with the ligand-binding site of estrogen receptors (ER) to disrupt transcriptional control of estrogen responsive targets. Here we investigate the possibility that estrogens also impact splicing decisions on estrogen responsive genes, such as that encoding ERα itself. Targeted PCR cloning was applied to identify six ERα mRNA variants in zebrafish. Sequencing revealed alternate use of transcription and translation start sites, multiple exon deletions, intron retention and alternate polyadenylation. As determined by quantitative (q)PCR, N-terminal mRNA variants predicting long (ERαL) and short (ERαS) isoforms were differentially expressed by tissue-type, sex, stage of development and estrogen exposure. Whereas ERαL mRNA was diffusely distributed in liver, brain, heart, eye, and gonads, ERαS mRNA was preferentially expressed in liver (female > male) and ovary. Neither ERαL nor ERαS transcripts varied significantly during development, but 17β-estradiol selectively increased accumulation of ERαS mRNA (~170-fold by 120 hpf), an effect mimicked by bisphenol-A and diethylstilbestrol. Significantly, a C-truncated variant (ERαS-Cx) lacking most of the ligand binding and AF-2 domains was transcribed exclusively from the short isoform promoter and was similar to ERαS in its tissue-, stage- and estrogen inducible expression. These results support the idea that promoter choice and alternative splicing of the esr1 gene of zebrafish are part of the autoregulatory mechanism by which estrogen modulates subsequent ERα expression, and further suggest that environmental estrogens could exert some of their toxic effects by altering the relative abundance of structurally and functionally distinct ERα isoforms. PMID:24090614

  5. Exploring accessibility of structural elements of the mammalian 40S ribosomal mRNA entry channel at various steps of translation initiation.

    PubMed

    Sharifulin, Dmitri E; Bartuli, Yulia S; Meschaninova, Maria I; Ven'yaminova, Aliya G; Graifer, Dmitri M; Karpova, Galina G

    2016-10-01

    In this work, we studied how the accessibility of structural elements of the mammalian 40S ribosomal mRNA entry channel, ribosomal protein (rp) uS3 and helix (h) 16 of the 18S rRNA, changes upon the translation initiation. In particular, we examined the accessibility of rp uS3 for binding of unstructured RNAs and of riboses in h16 towards attack with benzoyl cyanide (BzCN) in complexes assembled in rabbit reticulocyte lysate utilizing synthetic oligoribonucleotides as well as full-length and truncated up to the initiation AUG codon hepatitis C virus IRES as model mRNAs. With both mRNA types, the rp uS3 peptide recognizing single-stranded RNAs was shown to become shielded only in those 48S preinitiation complexes (PICs) that contained eIF3j bound to 40S subunit in the area between the decoding site and the mRNA entry channel. Chemical probing with BzCN revealed that h16 in the 48S PICs containing eIF3j or scanning factor DHX29 is strongly shielded; the effect was observed with all the mRNAs used, and h16 remained protected as well in 80S post-initiation complexes lacking these factors. Altogether, the obtained results allowed us to suggest that eIF3j bound at the 48S PICs makes the rp uS3 inaccessible for binding of RNAs and this factor subunit is responsible for the decrease of h16 conformational flexibility; the latter is manifested as reduced accessibility of h16 to BzCN. Thus, our findings provide new insights into how eIF3j is implicated in ensuring the proper conformation of the mRNA entry channel, thereby facilitating mRNA loading. PMID:27346718

  6. Parameter-Free Hydrogen-Bond Definition to Classify Protein Secondary Structure.

    PubMed

    Haghighi, Hasti; Higham, Jonathan; Henchman, Richard H

    2016-08-25

    DSSP is the most commonly used method to assign protein secondary structure. It is based on a hydrogen-bond definition with an energy cutoff. To assess whether hydrogen bonds defined in a parameter-free way may give more generality while preserving accuracy, we examine a series of hydrogen-bond definitions to assign secondary structure for a series of proteins. Assignment by the strongest-acceptor bifurcated definition with provision for unassigned donor hydrogens, termed the SABLE method, is found to match DSSP with 95% agreement. The small disagreement mainly occurs for helices, turns, and bends. While there is no absolute way to assign protein secondary structure, avoiding molecule-specific cutoff parameters should be advantageous in generalizing structure-assignment methods to any hydrogen-bonded system. PMID:27067825

  7. Charge‐Induced Unzipping of Isolated Proteins to a Defined Secondary Structure

    PubMed Central

    González Flórez, Ana Isabel; Mucha, Eike; Ahn, Doo‐Sik; Gewinner, Sandy; Schöllkopf, Wieland; Pagel, Kevin

    2016-01-01

    Abstract Here we present a combined experimental and theoretical study on the secondary structure of isolated proteins as a function of charge state. In infrared spectra of the proteins ubiquitin and cytochrome c, amide I (C=O stretch) and amide II (N–H bend) bands can be found at positions that are typical for condensed‐phase proteins. For high charge states a new band appears, substantially red‐shifted from the amide II band observed at lower charge states. The observations are interpreted in terms of Coulomb‐driven transitions in secondary structures from mostly helical to extended C5‐type hydrogen‐bonded structures. Support for this interpretation comes from simple energy considerations as well as from quantum chemical calculations on model peptides. This transition in secondary structure is most likely universal for isolated proteins that occur in mass spectrometric experiments. PMID:26847383

  8. Residue-residue contacts: application to analysis of secondary structure interactions.

    PubMed

    Potapov, Vladimir; Edelman, Marvin; Sobolev, Vladimir

    2013-01-01

    Protein structures and their complexes are formed and stabilized by interactions, both inside and outside of the protein. Analysis of such interactions helps in understanding different levels of structures (secondary, super-secondary, and oligomeric states). It can also assist molecular biologists in understanding structural consequences of modifying proteins and/or ligands. In this chapter, our definition of atom-atom and residue-residue contacts is described and applied to analysis of protein-protein interactions in dimeric β-sandwich proteins.

  9. Use of secondary structural information and C alpha-C alpha distance restraints to model protein structures with MODELLER.

    PubMed

    Reddy, Boojala V B; Kaznessis, Yiannis N

    2007-08-01

    Protein secondary structure predictions and amino acid long range contact map predictions from primary sequence of proteins have been explored to aid in modelling protein tertiary structures. In order to evaluate the usefulness of secondary structure and 3D-residue contact prediction methods to model protein structures we have used the known Q3 (alpha-helix,beta-strands and irregular turns/loops) secondary structure information, along with residue-residue contact information as restraints for MODELLER. We present here results of our modelling studies on 30 best resolved single domain protein structures of varied lengths. The results shows that it is very difficult to obtain useful models even with 100% accurate secondary structure predictions and accurate residue contact predictions for up to 30% of residues in a sequence. The best models that we obtained for proteins of lengths 37, 70, 118, 136 and 193 amino acid residues are of RMSDs 4.17, 5.27, 9.12, 7.89 and 9.69,respectively. The results show that one can obtain better models for the proteins which have high percent of alpha-helix content. This analysis further shows that MODELLER restrain optimization program can be useful only if we have truly homologous structure(s) as a template where it derives numerous restraints, almost identical to the templates used. This analysis also clearly indicates that even if we satisfy several true residue-residue contact distances, up to 30%of their sequence length with fully known secondary structural information, we end up predicting model structures much distant from their corresponding native structures.

  10. Macromolecular ab initio phasing enforcing secondary and tertiary structure

    PubMed Central

    Millán, Claudia; Sammito, Massimo; Usón, Isabel

    2015-01-01

    Ab initio phasing of macromolecular structures, from the native intensities alone with no experimental phase information or previous particular structural knowledge, has been the object of a long quest, limited by two main barriers: structure size and resolution of the data. Current approaches to extend the scope of ab initio phasing include use of the Patterson function, density modification and data extrapolation. The authors’ approach relies on the combination of locating model fragments such as polyalanine α-helices with the program PHASER and density modification with the program SHELXE. Given the difficulties in discriminating correct small substructures, many putative groups of fragments have to be tested in parallel; thus calculations are performed in a grid or supercomputer. The method has been named after the Italian painter Arcimboldo, who used to compose portraits out of fruit and vegetables. With ARCIMBOLDO, most collections of fragments remain a ‘still-life’, but some are correct enough for density modification and main-chain tracing to reveal the protein’s true portrait. Beyond α-helices, other fragments can be exploited in an analogous way: libraries of helices with modelled side chains, β-strands, predictable fragments such as DNA-binding folds or fragments selected from distant homologues up to libraries of small local folds that are used to enforce nonspecific tertiary structure; thus restoring the ab initio nature of the method. Using these methods, a number of unknown macromolecules with a few thousand atoms and resolutions around 2 Å have been solved. In the 2014 release, use of the program has been simplified. The software mediates the use of massive computing to automate the grid access required in difficult cases but may also run on a single multicore workstation (http://chango.ibmb.csic.es/ARCIMBOLDO_LITE) to solve straightforward cases. PMID:25610631

  11. Macromolecular ab initio phasing enforcing secondary and tertiary structure.

    PubMed

    Millán, Claudia; Sammito, Massimo; Usón, Isabel

    2015-01-01

    Ab initio phasing of macromolecular structures, from the native intensities alone with no experimental phase information or previous particular structural knowledge, has been the object of a long quest, limited by two main barriers: structure size and resolution of the data. Current approaches to extend the scope of ab initio phasing include use of the Patterson function, density modification and data extrapolation. The authors' approach relies on the combination of locating model fragments such as polyalanine α-helices with the program PHASER and density modification with the program SHELXE. Given the difficulties in discriminating correct small substructures, many putative groups of fragments have to be tested in parallel; thus calculations are performed in a grid or supercomputer. The method has been named after the Italian painter Arcimboldo, who used to compose portraits out of fruit and vegetables. With ARCIMBOLDO, most collections of fragments remain a 'still-life', but some are correct enough for density modification and main-chain tracing to reveal the protein's true portrait. Beyond α-helices, other fragments can be exploited in an analogous way: libraries of helices with modelled side chains, β-strands, predictable fragments such as DNA-binding folds or fragments selected from distant homologues up to libraries of small local folds that are used to enforce nonspecific tertiary structure; thus restoring the ab initio nature of the method. Using these methods, a number of unknown macromolecules with a few thousand atoms and resolutions around 2 Å have been solved. In the 2014 release, use of the program has been simplified. The software mediates the use of massive computing to automate the grid access required in difficult cases but may also run on a single multicore workstation (http://chango.ibmb.csic.es/ARCIMBOLDO_LITE) to solve straightforward cases.

  12. Macromolecular ab initio phasing enforcing secondary and tertiary structure.

    PubMed

    Millán, Claudia; Sammito, Massimo; Usón, Isabel

    2015-01-01

    Ab initio phasing of macromolecular structures, from the native intensities alone with no experimental phase information or previous particular structural knowledge, has been the object of a long quest, limited by two main barriers: structure size and resolution of the data. Current approaches to extend the scope of ab initio phasing include use of the Patterson function, density modification and data extrapolation. The authors' approach relies on the combination of locating model fragments such as polyalanine α-helices with the program PHASER and density modification with the program SHELXE. Given the difficulties in discriminating correct small substructures, many putative groups of fragments have to be tested in parallel; thus calculations are performed in a grid or supercomputer. The method has been named after the Italian painter Arcimboldo, who used to compose portraits out of fruit and vegetables. With ARCIMBOLDO, most collections of fragments remain a 'still-life', but some are correct enough for density modification and main-chain tracing to reveal the protein's true portrait. Beyond α-helices, other fragments can be exploited in an analogous way: libraries of helices with modelled side chains, β-strands, predictable fragments such as DNA-binding folds or fragments selected from distant homologues up to libraries of small local folds that are used to enforce nonspecific tertiary structure; thus restoring the ab initio nature of the method. Using these methods, a number of unknown macromolecules with a few thousand atoms and resolutions around 2 Å have been solved. In the 2014 release, use of the program has been simplified. The software mediates the use of massive computing to automate the grid access required in difficult cases but may also run on a single multicore workstation (http://chango.ibmb.csic.es/ARCIMBOLDO_LITE) to solve straightforward cases. PMID:25610631

  13. Interconnection of Salt-induced Hydrophobic Compaction and Secondary Structure Formation Depends on Solution Conditions

    PubMed Central

    Haldar, Shubhasis; Chattopadhyay, Krishnananda

    2012-01-01

    What happens in the early stage of protein folding remains an interesting unsolved problem. Rapid kinetics measurements with cytochrome c using submillisecond continuous flow mixing devices suggest simultaneous formation of a compact collapsed state and secondary structure. These data seem to indicate that collapse formation is guided by specific short and long range interactions (heteropolymer collapse). A contrasting interpretation also has been proposed, which suggests that the collapse formation is rapid, nonspecific, and a trivial solvent related compaction, which could as well be observed by a homopolymer (homopolymer collapse). We address this controversy using fluorescence correlation spectroscopy (FCS), which enables us to monitor the salt-induced compaction accompanying collapse formation and the associated time constant directly at single molecule resolution. In addition, we follow the formation of secondary structure using far UV CD. The data presented here suggest that both these models (homopolymer and heteropolymer) could be applicable depending on the solution conditions. For example, the formation of secondary structure and compact state is not simultaneous in aqueous buffer. In aqueous buffer, formation of the compact state occurs through a two-state co-operative transition following heteropolymer formalism, whereas secondary structure formation takes place gradually. In contrast, in the presence of urea, a compaction of the protein radius occurs gradually over an extended range of salt concentration following homopolymer formalism. The salt-induced compaction and the formation of secondary structure take place simultaneously in the presence of urea. PMID:22303014

  14. The Role of Structural Elements of the 5'-Terminal Region of p53 mRNA in Translation under Stress Conditions Assayed by the Antisense Oligonucleotide Approach

    PubMed Central

    Swiatkowska, Agata; Zydowicz, Paulina; Gorska, Agnieszka; Suchacka, Julia; Dutkiewicz, Mariola; Ciesiołka, Jerzy

    2015-01-01

    The p53 protein is one of the major factors responsible for cell cycle regulation and stress response. In the 5’-terminal region of p53 mRNA, an IRES element has been found which takes part in the translational regulation of p53 expression. Two characteristic hairpin motifs are present in this mRNA region: G56-C169, with the first AUG codon, and U180-A218, which interacts with the Hdm2 protein (human homolog of mouse double minute 2 protein). 2′-OMe modified antisense oligomers hybridizing to the 5'-terminal region of p53 mRNA were applied to assess the role of these structural elements in translation initiation under conditions of cellular stress. Structural changes in the RNA target occurring upon oligomers’ binding were monitored by the Pb2+-induced cleavage method. The impact of antisense oligomers on the synthesis of two proteins, the full-length p53 and its isoform Δ40p53, was analysed in HT-29, MCF-7 and HepG2 cells, under normal conditions and under stress, as well as in vitro conditions. The results revealed that the hairpin U180-A218 and adjacent single-stranded region A219-A228 were predominantly responsible for high efficacy of IRES-mediated translation in the presence of stress factors. These motifs play a role of cis-acting elements which are able to modulate IRES activity, likely via interactions with protein factors. PMID:26513723

  15. Structuring Free-text Microbiology Culture Reports For Secondary Use

    PubMed Central

    Yim, Wen-wai; Evans, Heather L.; Yetisgen, Meliha

    2015-01-01

    Microbiology lab culture reports are a frequently used diagnostic tool for clinical providers. However, their incorporation into clinical surveillance applications and evidence-based medicine can be severely hindered by the free-text nature of these reports. In this work, we (1) created a microbiology culture template to structure free-text microbiology reports, (2) generated an annotated microbiology report corpus, and (3) built a microbiology information extraction system. Specifically, we combined rule-based, hybrid, and statistical techniques to extract microbiology entities and fill templates for structuring data. System performances were favorable, with entity f1-score 0.889 and relation f1-score 0.795. We plan to incorporate these extractions as features for our ongoing ventilator-associated pneumonia surveillance project, though this tool can be used as an upstream process in other applications. Our newly created corpus includes 1442 unique gram stain and culture microbiology reports generated from a cohort of 715 patients at the University of Washington Medical Facilities. PMID:26306288

  16. Fast and accurate determination of sites along the FUT2 in vitro transcript that are accessible to antisense oligonucleotides by application of secondary structure predictions and RNase H in combination with MALDI-TOF mass spectrometry

    PubMed Central

    Gabler, Angelika; Krebs, Stefan; Seichter, Doris; Förster, Martin

    2003-01-01

    Alteration of gene expression by use of antisense oligonucleotides has considerable potential for therapeutic purposes and scientific studies. Although applied for almost 25 years, this technique is still associated with difficulties in finding antisense-effective regions along the target mRNA. This is mainly due to strong secondary structures preventing binding of antisense oligonucleotides and RNase H, playing a major role in antisense-mediated degradation of the mRNA. These difficulties make empirical testing of a large number of sequences complementary to various sites in the target mRNA a very lengthy and troublesome procedure. To overcome this problem, more recent strategies to find efficient antisense sites are based on secondary structure prediction and RNase H-dependent mechanisms. We were the first who directly combined these two strategies; antisense oligonucleotides complementary to predicted unpaired target mRNA regions were designed and hybridized to the corresponding RNAs. Incubation with RNase H led to cleavage of the RNA at the respective hybridization sites. Analysis of the RNA fragments by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, which has not been used in this context before, allowed exact determination of the cleavage site. Thus the technique described here is very promising when searching for effective antisense sites. PMID:12888531

  17. Parity-Violation Energy of Biomolecules - IV: Protein Secondary Structure

    NASA Astrophysics Data System (ADS)

    Faglioni, Francesco; Cuesta, Inmaculada García

    2011-06-01

    The parity-violation energy difference between enantiomeric forms of the same amino acid sequence, from the amyloid β-peptide involved in Alzheimer's desease, in both α-helix and β-sheet configurations, is investigated with ab-initio techniques. To this end, we develop an extension of the N2 computational scheme that selectively includes neighboring amino acids to preserve the relevant H-bonds. In agreement with previous speculations, it is found that the helical α structure is associated with larger parity-violation energy differences than the corresponding β form. Implications for the evolution of biological homochirality are discussed as well as the relative importance of various effects in determining the parity-violation energy.

  18. Regulation of tyrosine hydroxylase transcription by hnRNP K and DNA secondary structure

    PubMed Central

    Banerjee, Kasturi; Wang, Meng; Cai, Elizabeth; Fujiwara, Nana; Baker, Harriet; Cave, John W.

    2014-01-01

    Regulation of tyrosine hydroxylase gene (Th) transcription is critical for specifying and maintaining the dopaminergic neuronal phenotype. Here we define a molecular regulatory mechanism for Th transcription conserved in tetrapod vertebrates. We show that heterogeneous nuclear ribonucleoprotein (hnRNP) K is a transactivator of Th transcription. It binds to previously unreported and evolutionarily conserved G:C-rich regions in the Th proximal promoter. hnRNP K directly binds C-rich single DNA strands within these conserved regions and also associates with double-stranded sequences when proteins, such as CREB, are bound to an adjacent cis-regulatory element. The single DNA strands within the conserved G:C-rich regions adopt either G-quadruplex or i-motif secondary structures. We also show that small molecule-mediated stabilization of these secondary structures represses Th promoter activity. These data suggest that these secondary structures are targets for pharmacological modulation of the dopaminergic phenotype. PMID:25493445

  19. The FT-IR spectrometric analysis of the changes of polyphenol oxidase II secondary structure

    NASA Astrophysics Data System (ADS)

    Shi, Chunhua; Dai, Ya; Liu, Qingliang; Xie, Yongshu; Xu, Xiaolong

    2003-01-01

    Polyphenol oxidase II is a novel protein purified from tobacco, which acts as a key role in plant defense system. From the analysis of FT-IR spectrums, Fourier self-deconvolution (FSD) spectrums and second-derivative spectrums of PPO II at different pH and peroxide PPO II adduct, the secondary structure fractions are analyzed. PPO II at low pH (pH=3.0) and peroxide PPO II adduct almost keep the same secondary structure of native PPO II. The percentages of β-turn and random coil increase rapidly and the percentages of α-helix and anti-parallel β-sheet decrease rapidly at high pH (pH=10.0) comparing with that of native PPO II. All these conclusions are proved by the secondary structure calculations of circular dichroism spectrums in different states.

  20. Relationship between chain collapse and secondary structure formation in a partially folded protein.

    PubMed

    Nakagawa, Kanako; Yamada, Yoshiteru; Matsumura, Yoshitaka; Tsukamoto, Seiichi; Yamamoto-Ohtomo, Mio; Ohtomo, Hideaki; Okabe, Takahiro; Fujiwara, Kazuo; Ikeguchi, Masamichi

    2014-06-01

    Chain collapse and secondary structure formation are frequently observed during the early stages of protein folding. Is the chain collapse brought about by interactions between secondary structure units or is it due to polymer behavior in a poor solvent (coil-globule transition)? To answer this question, we measured small-angle X-ray scattering for a series of β-lactoglobulin mutants under conditions in which they assume a partially folded state analogous to the folding intermediates. Mutants that were designed to disrupt the secondary structure units showed the gyration radii similar to that of the wild type protein, indicating that chain collapse is due to coil-globule transitions. PMID:25100622

  1. RNA secondary structure prediction based on SHAPE data in helix regions.

    PubMed

    Lotfi, Mohadeseh; Zare-Mirakabad, Fatemeh; Montaseri, Soheila

    2015-09-01

    RNA molecules play important and fundamental roles in biological processes. Frequently, the functional form of single-stranded RNA molecules requires a specific tertiary structure. Classically, RNA structure determination has mostly been accomplished by X-Ray crystallography or Nuclear Magnetic Resonance approaches. These experimental methods are time consuming and expensive. In the past two decades, some computational methods and algorithms have been developed for RNA secondary structure prediction. In these algorithms, minimum free energy is known as the best criterion. However, the results of algorithms show that minimum free energy is not a sufficient criterion to predict RNA secondary structure. These algorithms need some additional knowledge about the structure, which has to be added in the methods. Recently, the information obtained from some experimental data, called SHAPE, can greatly improve the consistency between the native and predicted RNA secondary structure. In this paper, we investigate the influence of SHAPE data on four types of RNA substructures, helices, loops, base pairs from the start and end of helices and two base pairs from the start and end of helices. The results show that SHAPE data in helix regions can improve the prediction. We represent a new method to apply SHAPE data in helix regions for finding RNA secondary structure. Finally, we compare the results of the method on a set of RNAs to predict minimum free energy structure based on considering all SHAPE data and only SHAPE data in helix regions as pseudo free energy and without SHAPE data (without any pseudo free energy). The results show that RNA secondary structure prediction based on considering only SHAPE data in helix regions is more successful than not considering SHAPE data and it provides competitive results in comparison with considering all SHAPE data.

  2. Dynamics of beta and proliferating cell nuclear antigen sliding clamps in traversing DNA secondary structure.

    PubMed

    Yao, N; Hurwitz, J; O'Donnell, M

    2000-01-14

    Chromosomal replicases of cellular organisms utilize a ring shaped protein that encircles DNA as a mobile tether for high processivity in DNA synthesis. These "sliding clamps" have sufficiently large linear diameters to encircle duplex DNA and are perhaps even large enough to slide over certain DNA secondary structural elements. This report examines the Escherichia coli beta and human proliferating cell nuclear antigen clamps for their ability to slide over various DNA secondary structures. The results show that these clamps are capable of traversing a 13-nucleotide ssDNA loop, a 4-base pair stem-loop, a 4-nucleotide 5' tail, and a 15-mer bubble within the duplex. However, upon increasing the size of these structures (20-nucleotide loop, 12-base pair stem-loop, 28-nucleotide 5' tail, and 20-nucleotide bubble) the sliding motion of the beta and proliferating cell nuclear antigen over these elements is halted. Studies of the E. coli replicase, DNA polymerase III holoenzyme, in chain elongation with the beta clamp demonstrate that upon encounter with an oligonucleotide annealed in its path, it traverses the duplex and resumes synthesis on the 3' terminus of the oligonucleotide. This sliding and resumption of synthesis occurs even when the oligonucleotide contains a secondary structure element, provided the beta clamp can traverse the structure. However, upon encounter with a downstream oligonucleotide containing a large internal secondary structure, the holoenzyme clears the obstacle by strand displacing the oligonucleotide from the template. Implications of these protein dynamics to DNA transactions are discussed. PMID:10625694

  3. A set of nearest neighbor parameters for predicting the enthalpy change of RNA secondary structure formation

    PubMed Central

    Lu, Zhi John; Turner, Douglas H.; Mathews, David H.

    2006-01-01

    A complete set of nearest neighbor parameters to predict the enthalpy change of RNA secondary structure formation was derived. These parameters can be used with available free energy nearest neighbor parameters to extend the secondary structure prediction of RNA sequences to temperatures other than 37°C. The parameters were tested by predicting the secondary structures of sequences with known secondary structure that are from organisms with known optimal growth temperatures. Compared with the previous set of enthalpy nearest neighbor parameters, the sensitivity of base pair prediction improved from 65.2 to 68.9% at optimal growth temperatures ranging from 10 to 60°C. Base pair probabilities were predicted with a partition function and the positive predictive value of structure prediction is 90.4% when considering the base pairs in the lowest free energy structure with pairing probability of 0.99 or above. Moreover, a strong correlation is found between the predicted melting temperatures of RNA sequences and the optimal growth temperatures of the host organism. This indicates that organisms that live at higher temperatures have evolved RNA sequences with higher melting temperatures. PMID:16982646

  4. RNAmutants: a web server to explore the mutational landscape of RNA secondary structures.

    PubMed

    Waldispühl, Jerome; Devadas, Srinivas; Berger, Bonnie; Clote, Peter

    2009-07-01

    The history and mechanism of molecular evolution in DNA have been greatly elucidated by contributions from genetics, probability theory and bioinformatics--indeed, mathematical developments such as Kimura's neutral theory, Kingman's coalescent theory and efficient software such as BLAST, ClustalW, Phylip, etc., provide the foundation for modern population genetics. In contrast to DNA, the function of most noncoding RNA depends on tertiary structure, experimentally known to be largely determined by secondary structure, for which dynamic programming can efficiently compute the minimum free energy secondary structure. For this reason, understanding the effect of pointwise mutations in RNA secondary structure could reveal fundamental properties of structural RNA molecules and improve our understanding of molecular evolution of RNA. The web server RNAmutants provides several efficient tools to compute the ensemble of low-energy secondary structures for all k-mutants of a given RNA sequence, where k is bounded by a user-specified upper bound. As we have previously shown, these tools can be used to predict putative deleterious mutations and to analyze regulatory sequences from the hepatitis C and human immunodeficiency genomes. Web server is available at http://bioinformatics.bc.edu/clotelab/RNAmutants/, and downloadable binaries at http://rnamutants.csail.mit.edu/.

  5. RNAmutants: a web server to explore the mutational landscape of RNA secondary structures.

    PubMed

    Waldispühl, Jerome; Devadas, Srinivas; Berger, Bonnie; Clote, Peter

    2009-07-01

    The history and mechanism of molecular evolution in DNA have been greatly elucidated by contributions from genetics, probability theory and bioinformatics--indeed, mathematical developments such as Kimura's neutral theory, Kingman's coalescent theory and efficient software such as BLAST, ClustalW, Phylip, etc., provide the foundation for modern population genetics. In contrast to DNA, the function of most noncoding RNA depends on tertiary structure, experimentally known to be largely determined by secondary structure, for which dynamic programming can efficiently compute the minimum free energy secondary structure. For this reason, understanding the effect of pointwise mutations in RNA secondary structure could reveal fundamental properties of structural RNA molecules and improve our understanding of molecular evolution of RNA. The web server RNAmutants provides several efficient tools to compute the ensemble of low-energy secondary structures for all k-mutants of a given RNA sequence, where k is bounded by a user-specified upper bound. As we have previously shown, these tools can be used to predict putative deleterious mutations and to analyze regulatory sequences from the hepatitis C and human immunodeficiency genomes. Web server is available at http://bioinformatics.bc.edu/clotelab/RNAmutants/, and downloadable binaries at http://rnamutants.csail.mit.edu/. PMID:19531740

  6. Fabrication of experimental three-meter space telescope primary and secondary mirror support structure

    NASA Technical Reports Server (NTRS)

    Mishler, H. W.

    1974-01-01

    The fabrication of prototype titanium alloy primary and secondary mirror support structures for a proposed experimental three-meter space telescope is discussed. The structure was fabricated entirely of Ti-6Al-4V tubing and plate. Fabrication included the development of procedures including welding, forming, and machining. Most of the structures was fabricated by gas-shielding tungsten-arc (GTA) welding with several major components fabricated by high frequency resistance (HFR) welding.

  7. New charge-bearing amino acid residues that promote β-sheet secondary structure.

    PubMed

    Maynard, Stacy J; Almeida, Aaron M; Yoshimi, Yasuharu; Gellman, Samuel H

    2014-11-26

    Proteinogenic amino acid residues that promote β-sheet secondary structure are hydrophobic (e.g., Ile or Val) or only moderately polar (e.g., Thr). The design of peptides intended to display β-sheet secondary structure in water typically requires one set of residues to ensure conformational stability and an orthogonal set, with charged side chains, to ensure aqueous solubility and discourage self-association. Here we describe new amino acids that manifest substantial β-sheet propensity, by virtue of β-branching, and also bear an ionizable group in the side chain. PMID:25393077

  8. Artificial Intelligence in Prediction of Secondary Protein Structure Using CB513 Database

    PubMed Central

    Avdagic, Zikrija; Purisevic, Elvir; Omanovic, Samir; Coralic, Zlatan

    2009-01-01

    In this paper we describe CB513 a non-redundant dataset, suitable for development of algorithms for prediction of secondary protein structure. A program was made in Borland Delphi for transforming data from our dataset to make it suitable for learning of neural network for prediction of secondary protein structure implemented in MATLAB Neural-Network Toolbox. Learning (training and testing) of neural network is researched with different sizes of windows, different number of neurons in the hidden layer and different number of training epochs, while using dataset CB513. PMID:21347158

  9. FASTR: A novel data format for concomitant representation of RNA sequence and secondary structure information.

    PubMed

    Bose, Tungadri; Dutta, Anirban; Mh, Mohammed; Gandhi, Hemang; Mande, Sharmila S

    2015-09-01

    Given the importance of RNA secondary structures in defining their biological role, it would be convenient for researchers seeking RNA data if both sequence and structural information pertaining to RNA molecules are made available together. Current nucleotide data repositories archive only RNA sequence data. Furthermore, storage formats which can frugally represent RNA sequence as well as structure data in a single file, are currently unavailable. This article proposes a novel storage format, 'FASTR', for concomitant representation of RNA sequence and structure. The storage efficiency of the proposed FASTR format has been evaluated using RNA data from various microorganisms. Results indicate that the size of FASTR formatted files (containing both RNA sequence as well as structure information) are equivalent to that of FASTA-format files, which contain only RNA sequence information. RNA secondary structure is typically represented using a combination of a string of nucleotide characters along with the corresponding dot-bracket notation indicating structural attributes. 'FASTR' - the novel storage format proposed in the present study enables a frugal representation of both RNA sequence and structural information in the form of a single string. In spite of having a relatively smaller storage footprint, the resultant 'fastr' string(s) retain all sequence as well as secondary structural information that could be stored using a dot-bracket notation. An implementation of the 'FASTR' methodology is available for download at http://metagenomics.atc.tcs.com/compression/fastr.

  10. Secondary Structural Change Can Occur Diffusely and Not Modularly during Protein Folding and Unfolding Reactions.

    PubMed

    Malhotra, Pooja; Udgaonkar, Jayant B

    2016-05-11

    A major goal of protein folding studies is to understand the structural basis of the coupling between stabilizing interactions, which leads to cooperative conformational change. The goal is challenging because of the difficulty in simultaneously measuring global cooperativity by determining population distributions of the conformations present, and the structures of these conformations. Here, hydrogen exchange (HX) into the small protein monellin was carried out under conditions where structure-opening is rate limiting for most backbone amide sites. Detection by mass spectrometry allowed characterization of not only segment-specific structure-opening rates but also the cooperativity of unfolding of the different secondary structural segments of the protein. The segment-specific pattern of HX reveals that the backbone hydrogen-bonding network disassembles in a structurally diffuse, asynchronous manner. A comparison of the site-specific transient opening rates of secondary and tertiary structure in the protein provides a structural rationale for the observation that unfolding is hierarchical and describable by exponential kinetics, despite being diffuse. Since unfolding was studied in native conditions, the sequence of events during folding in the same conditions will be the reverse of the sequence of events observed during unfolding. Hence, the formation of secondary structural units during folding would also occur in a non-cooperative, diffuse, and asynchronous manner. PMID:27093885

  11. Web-Beagle: a web server for the alignment of RNA secondary structures

    PubMed Central

    Mattei, Eugenio; Pietrosanto, Marco; Ferrè, Fabrizio; Helmer-Citterich, Manuela

    2015-01-01

    Web-Beagle (http://beagle.bio.uniroma2.it) is a web server for the pairwise global or local alignment of RNA secondary structures. The server exploits a new encoding for RNA secondary structure and a substitution matrix of RNA structural elements to perform RNA structural alignments. The web server allows the user to compute up to 10 000 alignments in a single run, taking as input sets of RNA sequences and structures or primary sequences alone. In the latter case, the server computes the secondary structure prediction for the RNAs on-the-fly using RNAfold (free energy minimization). The user can also compare a set of input RNAs to one of five pre-compiled RNA datasets including lncRNAs and 3′ UTRs. All types of comparison produce in output the pairwise alignments along with structural similarity and statistical significance measures for each resulting alignment. A graphical color-coded representation of the alignments allows the user to easily identify structural similarities between RNAs. Web-Beagle can be used for finding structurally related regions in two or more RNAs, for the identification of homologous regions or for functional annotation. Benchmark tests show that Web-Beagle has lower computational complexity, running time and better performances than other available methods. PMID:25977293

  12. The vitellogenin of the bumblebee, Bombus hypocrita: studies on structural analysis of the cDNA and expression of the mRNA.

    PubMed

    Li, Jilian; Huang, Jiaxing; Cai, Wanzhi; Zhao, Zhangwu; Peng, Wenjun; Wu, Jie

    2010-02-01

    In this present study, the cDNA of Bombus hypocrita vitellogenin (Vg) was cloned and sequenced. It is composed of 5,478 bp and contains an ORF of 1,772 amino acids within a putative signal peptide of 16 residues. The deduced amino acid sequence shows significant similarity with Bombus ignitus (95%) and Apis mellifera (52%) and a high number of conserved motifs. Close to the C terminus there is a GL/ICG motif followed by nine cysteines, and a DGXR motif is located 18 residues upstream from the GL/ICG motif. Moreover, we predicted the 3D structure of B. hypocrita Vg. Furthermore, the Vg mRNA of B. hypocrita was spatio-temporally analyzed in different castes (such as queen, worker and drone) from pupae to adult. The Vg mRNA was found in the white-eyed pupal (Pw) stage in queens, and the expression increased during the entire pupal development and attained its peak in the dark brown pupal stage. It also had a high expression in the adult fat body. In workers, the Vg expression was detected in the Pw stage, and its levels increased with age with the highest in 15 days. Afterward, it decreased progressively. Vg mRNA was also observed in drones, with a higher level of expression shown in only freshly molted adult drones.

  13. Structure of the coding region and mRNA variants of the apyrase gene from pea (Pisum sativum)

    NASA Technical Reports Server (NTRS)

    Shibata, K.; Abe, S.; Davies, E.

    2001-01-01

    Partial amino acid sequences of a 49 kDa apyrase (ATP diphosphohydrolase, EC 3.6.1.5) from the cytoskeletal fraction of etiolated pea stems were used to derive oligonucleotide DNA primers to generate a cDNA fragment of pea apyrase mRNA by RT-PCR and these primers were used to screen a pea stem cDNA library. Two almost identical cDNAs differing in just 6 nucleotides within the coding regions were found, and these cDNA sequences were used to clone genomic fragments by PCR. Two nearly identical gene fragments containing 8 exons and 7 introns were obtained. One of them (H-type) encoded the mRNA sequence described by Hsieh et al. (1996) (DDBJ/EMBL/GenBank Z32743), while the other (S-type) differed by the same 6 nucleotides as the mRNAs, suggesting that these genes may be alleles. The six nucleotide differences between these two alleles were found solely in the first exon, and these mutation sites had two types of consensus sequences. These mRNAs were found with varying lengths of 3' untranslated regions (3'-UTR). There are some similarities between the 3'-UTR of these mRNAs and those of actin and actin binding proteins in plants. The putative roles of the 3'-UTR and alternative polyadenylation sites are discussed in relation to their possible role in targeting the mRNAs to different subcellular compartments.

  14. Secondary structure models of the 3′ untranslated regions of diverse R2 RNAs

    PubMed Central

    RUSCHAK, AMY M.; MATHEWS, DAVID H.; BIBILLO, ARKADIUSZ; SPINELLI, SHERRY L.; CHILDS, JESSICA L.; EICKBUSH, THOMAS H.; TURNER, DOUGLAS H.

    2004-01-01

    The RNA structure of the 3′ untranslated region (UTR) of the R2 retrotransposable element is recognized by the R2-encoded reverse transcriptase in a reaction called target primed reverse transcription (TPRT). To provide insight into structure–function relationships important for TPRT, we have created alignments that reveal the secondary structure for 22 Drosophila and five silkmoth 3′ UTR R2 sequences. In addition, free energy minimization has been used to predict the secondary structure for the 3′ UTR R2 RNA of Forficula auricularia. The predicted structures for Bombyx mori and F. auricularia are consistent with chemical modification data obtained with β-ethoxy-α-ketobutyraldehyde (kethoxal), dimethyl sulfate, and 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluene sulfonate. The structures appear to have common helices that are likely important for function. PMID:15146081

  15. Computer-aided nucleic acid secondary structure modeling incorporating enzymatic digestion data.

    PubMed Central

    Quigley, G J; Gehrke, L; Roth, D A; Auron, P E

    1984-01-01

    We present a computer-aided method for determining nucleic acid secondary structure. The method utilizes a program which has the capability to filter matrix diagonal data on the basis of diagonal length, stabilization energy, and chemical and enzymatic data. The program also allows the user to assign selected regions of the structure as uniquely single-stranded or paired, and to filter out "trade-off" structures on the basis of such pairing. In order to demonstrate the utility of the program we present a preliminary secondary structure for the 3' end of alfalfa mosaic virus RNA 4 (AMV-4 RNA). This structure is based on an analysis which includes the use of in vitro partial enzymatic digestion of the RNA. Images PMID:6320093

  16. New insights from cluster analysis methods for RNA secondary structure prediction.

    PubMed

    Rogers, Emily; Heitsch, Christine

    2016-05-01

    A widening gap exists between the best practices for RNA secondary structure prediction developed by computational researchers and the methods used in practice by experimentalists. Minimum free energy predictions, although broadly used, are outperformed by methods which sample from the Boltzmann distribution and data mine the results. In particular, moving beyond the single structure prediction paradigm yields substantial gains in accuracy. Furthermore, the largest improvements in accuracy and precision come from viewing secondary structures not at the base pair level but at lower granularity/higher abstraction. This suggests that random errors affecting precision and systematic ones affecting accuracy are both reduced by this 'fuzzier' view of secondary structures. Thus experimentalists who are willing to adopt a more rigorous, multilayered approach to secondary structure prediction by iterating through these levels of granularity will be much better able to capture fundamental aspects of RNA base pairing. WIREs RNA 2016, 7:278-294. doi: 10.1002/wrna.1334 For further resources related to this article, please visit the WIREs website.

  17. The four ingredients of single-sequence RNA secondary structure prediction. A unifying perspective

    PubMed Central

    Rivas, Elena

    2013-01-01

    Any method for RNA secondary structure prediction is determined by four ingredients. The architecture is the choice of features implemented by the model (such as stacked basepairs, loop length distributions, etc.). The architecture determines the number of parameters in the model. The scoring scheme is the nature of those parameters (whether thermodynamic, probabilistic, or weights). The parameterization stands for the specific values assigned to the parameters. These three ingredients are referred to as “the model.” The fourth ingredient is the folding algorithms used to predict plausible secondary structures given the model and the sequence of a structural RNA. Here, I make several unifying observations drawn from looking at more than 40 years of methods for RNA secondary structure prediction in the light of this classification. As a final observation, there seems to be a performance ceiling that affects all methods with complex architectures, a ceiling that impacts all scoring schemes with remarkable similarity. This suggests that modeling RNA secondary structure by using intrinsic sequence-based plausible “foldability” will require the incorporation of other forms of information in order to constrain the folding space and to improve prediction accuracy. This could give an advantage to probabilistic scoring systems since a probabilistic framework is a natural platform to incorporate different sources of information into one single inference problem. PMID:23695796

  18. Quantitative Correlation between the protein primary sequences and secondary structures in spider dragline silks.

    PubMed

    Jenkins, Janelle E; Creager, Melinda S; Lewis, Randolph V; Holland, Gregory P; Yarger, Jeffery L

    2010-01-11

    Synthetic spider silk holds great potential for use in various applications spanning medical uses to ultra lightweight armor; however, producing synthetic fibers with mechanical properties comparable to natural spider silk has eluded the scientific community. Natural dragline spider silks are commonly made from proteins that contain highly repetitive amino acid motifs, adopting an array of secondary structures. Before further advances can be made in the production of synthetic fibers based on spider silk proteins, it is imperative to know the percentage of each amino acid in the protein that forms a specific secondary structure. Linking these percentages to the primary amino acid sequence of the protein will establish a structural foundation for synthetic silk. In this study, nuclear magnetic resonance (NMR) techniques are used to quantify the percentage of Ala, Gly, and Ser that form both beta-sheet and helical secondary structures. The fraction of these three amino acids and their secondary structure are quantitatively correlated to the primary amino acid sequence for the proteins that comprise major and minor ampullate silk from the Nephila clavipes spider providing a blueprint for synthetic spider silks. PMID:20000730

  19. The four ingredients of single-sequence RNA secondary structure prediction. A unifying perspective.

    PubMed

    Rivas, Elena

    2013-07-01

    Any method for RNA secondary structure prediction is determined by four ingredients. The architecture is the choice of features implemented by the model (such as stacked basepairs, loop length distributions, etc.). The architecture determines the number of parameters in the model. The scoring scheme is the nature of those parameters (whether thermodynamic, probabilistic, or weights). The parameterization stands for the specific values assigned to the parameters. These three ingredients are referred to as "the model." The fourth ingredient is the folding algorithms used to predict plausible secondary structures given the model and the sequence of a structural RNA. Here, I make several unifying observations drawn from looking at more than 40 years of methods for RNA secondary structure prediction in the light of this classification. As a final observation, there seems to be a performance ceiling that affects all methods with complex architectures, a ceiling that impacts all scoring schemes with remarkable similarity. This suggests that modeling RNA secondary structure by using intrinsic sequence-based plausible "foldability" will require the incorporation of other forms of information in order to constrain the folding space and to improve prediction accuracy. This could give an advantage to probabilistic scoring systems since a probabilistic framework is a natural platform to incorporate different sources of information into one single inference problem.

  20. Beyond the known functions of the CCR4-NOT complex in gene expression regulatory mechanisms: New structural insights to unravel CCR4-NOT mRNA processing machinery.

    PubMed

    Ukleja, Marta; Valpuesta, José María; Dziembowski, Andrzej; Cuellar, Jorge

    2016-10-01

    Large protein assemblies are usually the effectors of major cellular processes. The intricate cell homeostasis network is divided into numerous interconnected pathways, each controlled by a set of protein machines. One of these master regulators is the CCR4-NOT complex, which ultimately controls protein expression levels. This multisubunit complex assembles around a scaffold platform, which enables a wide variety of well-studied functions from mRNA synthesis to transcript decay, as well as other tasks still being identified. Solving the structure of the entire CCR4-NOT complex will help to define the distribution of its functions. The recently published three-dimensional reconstruction of the complex, in combination with the known crystal structures of some of the components, has begun to address this. Methodological improvements in structural biology, especially in cryoelectron microscopy, encourage further structural and protein-protein interaction studies, which will advance our comprehension of the gene expression machinery.

  1. Flow structure in submarine meandering channels, a continuous discussion on secondary flow

    NASA Astrophysics Data System (ADS)

    Abad, J. D.; Parker, G.; Sequeiros, O.; Spinewine, B.; Garcia, M. H.; Pirmez, C.

    2011-12-01

    The understanding of the flow structure in deep-sea turbidity currents is important for the formation of submarine meandering channels. Similarly to the case of subaerial channels, several types of secondary flows include turbulence-, curvature- and bed morphodynamic-driven flow structures that modulate sediment transport and channel bed morphodynamics. This study focuses on [1] a review of long-time research effort (Abad et al., 2011) that tackles the description of the secondary flow associated with a subaqueous bottom current (saline) in a high-curvature meandering channel and [2] ongoing numerical simulations of similar settings as the experiments to describe the entire flow structure. In the case of subaerial channels, the classical Rozovskiian paradigm is often invoked which indicates that the near-bottom secondary flow in a bend is directed inward. It has recently been suggested based on experimental and theoretical considerations, however, that this pattern is reversed (near-bottom secondary flow is directed outward) in the case of submarine meandering channels. Experimental results presented here, on the other hand, indicate near-bottom secondary flows that have the same direction as observed in a river (normal secondary flow). The implication is an apparent contradiction between experimental results. This study combines theory, experiments, reconstructions of field flows and ongoing simulations to resolve this apparent contradiction based on the flow densimetric Froude number. Three ranges of densimetric Froude number are found, such that a) in an upper regime, secondary flow is reversed, b) in a middle regime, it is normal and c) in a lower regime, it is reversed. These results are applied to field scale channel-forming turbidity currents in the Amazon submarine canyon-fan system (Amazon Channel) and the Monterey canyon and a saline underflow in the Black Sea flowing from the Bosphorus. Our analysis indicates that secondary flow should be normal

  2. Reactivity of molybdovanadophosphoric acids: Influence of the presence of vanadium in the primary and secondary structure

    SciTech Connect

    Casarini, D.; Centi, G.; Lena, V.; Tvaruzkova, Z. ); Jiru, P. )

    1993-10-01

    The catalytic behavior in butadiene and n-butane oxidation of molybdovanadophosphoric acids with vanadium localized inside the primary (oxoanion) and/or the secondary structure is reported. The samples are characterized by infrared, [sup 31]P-NMR, [sup 51]V-NMR, and UV-visible diffuse reflectance spectroscopies in order to obtain information on the nature and localization of vanadium in the samples before reaction and the possible changes occurring during the course of the catalytic reaction. In particular, it is shown that vanadium localized initially in the secondary structure can exchange with the molybdenum atoms of the oxoanion during the catalytic reaction. Introduction of vanadium in the molybdophosphoric acid structure enhances the selective formation of maleic anhydride from the butadiene when vanadium is present both inside the oxoanion or localized in the secondary structure (before the catalytic tests), but the maximum in catalytic performance is found for different amounts of vanadium, depending on where the vanadium is localized initially. However, when present in the secondary structure, vanadium also has a negative influence on the activity of the heteropoly acid. On the contrary, in n-butane oxidation, the presence of vanadium enhances the rate of alkane activation due to the different rate-determining step. The presence of V ions also affects the maximum selectivity and yield to maleic anhydride from butane. V ions in the secondary structure are more selective at low conversion, while V ions inside the oxoanion are more selective at higher conversions and thus allow better maximum yields to maleic anhydride. 40 refs., 11 figs., 2 tabs.

  3. Bioinformatics approaches for structural and functional analysis of proteins in secondary metabolism in Withania somnifera.

    PubMed

    Sanchita; Singh, Swati; Sharma, Ashok

    2014-11-01

    Withania somnifera (Ashwagandha) is an affluent storehouse of large number of pharmacologically active secondary metabolites known as withanolides. These secondary metabolites are produced by withanolide biosynthetic pathway. Very less information is available on structural and functional aspects of enzymes involved in withanolides biosynthetic pathways of Withiana somnifera. We therefore performed a bioinformatics analysis to look at functional and structural properties of these important enzymes. The pathway enzymes taken for this study were 3-Hydroxy-3-methylglutaryl coenzyme A reductase, 1-Deoxy-D-xylulose-5-phosphate synthase, 1-Deoxy-D-xylulose-5-phosphate reductase, farnesyl pyrophosphate synthase, squalene synthase, squalene epoxidase, and cycloartenol synthase. The prediction of secondary structure was performed for basic structural information. Three-dimensional structures for these enzymes were predicted. The physico-chemical properties such as pI, AI, GRAVY and instability index were also studied. The current information will provide a platform to know the structural attributes responsible for the function of these protein until experimental structures become available.

  4. RNA secondary structure modeling at consistent high accuracy using differential SHAPE.

    PubMed

    Rice, Greggory M; Leonard, Christopher W; Weeks, Kevin M

    2014-06-01

    RNA secondary structure modeling is a challenging problem, and recent successes have raised the standards for accuracy, consistency, and tractability. Large increases in accuracy have been achieved by including data on reactivity toward chemical probes: Incorporation of 1M7 SHAPE reactivity data into an mfold-class algorithm results in median accuracies for base pair prediction that exceed 90%. However, a few RNA structures are modeled with significantly lower accuracy. Here, we show that incorporating differential reactivities from the NMIA and 1M6 reagents--which detect noncanonical and tertiary interactions--into prediction algorithms results in highly accurate secondary structure models for RNAs that were previously shown to be difficult to model. For these RNAs, 93% of accepted canonical base pairs were recovered in SHAPE-directed models. Discrepancies between accepted and modeled structures were small and appear to reflect genuine structural differences. Three-reagent SHAPE-directed modeling scales concisely to structurally complex RNAs to resolve the in-solution secondary structure analysis problem for many classes of RNA.

  5. Classroom Structure and Teacher Efficacy in Serving Students with Disabilities: Differences in Elementary and Secondary Teachers

    ERIC Educational Resources Information Center

    Shippen, Margaret E.; Flores, Margaret M.; Crites, Steven A.; Patterson, DaShaunda; Ramsey, Michelle L.; Houchins, David E.; Jolivette, Kristine

    2011-01-01

    The purpose of this study was to investigate the differential classroom structure and efficacy reported by general and special educators at the elementary and secondary level. General and special educators (n = 774, return rate of 37%) from a large school district in the southeast US participated in the study. The participants completed a modified…

  6. Understanding of Relation Structures of Graphical Models by Lower Secondary Students

    ERIC Educational Resources Information Center

    van Buuren, Onne; Heck, André; Ellermeijer, Ton

    2016-01-01

    A learning path has been developed on system dynamical graphical modelling, integrated into the Dutch lower secondary physics curriculum. As part of the developmental research for this learning path, students' understanding of the relation structures shown in the diagrams of graphical system dynamics based models has been investigated. One of our…

  7. Teachers Working in Collaborative Structures: A Case Study of a Secondary School in the USA

    ERIC Educational Resources Information Center

    Cameron, David Hagen

    2005-01-01

    This article reports on a study that explores collaborative structures of shared decision-making in an urban secondary school in the USA. The data in the study came from unstructured interviews with 20 teachers, the principal, the assistant principal, a counsellor and 10 students. The interviews took place over a three-week period in June of 2001…

  8. The Turn of the Screw: An Exercise in Protein Secondary Structure

    ERIC Educational Resources Information Center

    Pikaart, Michael

    2011-01-01

    An exercise using simple paper strips to illustrate protein helical and sheet secondary structures is presented. Drawing on the rich historical context of the use of physical models in protein biochemistry by early practitioners, in particular Linus Pauling, the purpose of this activity is to cultivate in students a hands-on, intuitive sense of…

  9. Secondary School Students' Understanding of Mathematical Induction: Structural Characteristics and the Process of Proof Construction

    ERIC Educational Resources Information Center

    Palla, Marina; Potari, Despina; Spyrou, Panagiotis

    2012-01-01

    In this study, we investigate the meaning students attribute to the structure of mathematical induction (MI) and the process of proof construction using mathematical induction in the context of a geometric recursion problem. Two hundred and thirteen 17-year-old students of an upper secondary school in Greece participated in the study. Students'…

  10. Assessing the impact of secondary structure and solvent accessibility on protein evolution.

    PubMed Central

    Goldman, N; Thorne, J L; Jones, D T

    1998-01-01

    Empirically derived models of amino acid replacement are employed to study the association between various physical features of proteins and evolution. The strengths of these associations are statistically evaluated by applying the models of protein evolution to 11 diverse sets of protein sequences. Parametric bootstrap tests indicate that the solvent accessibility status of a site has a particularly strong association with the process of amino acid replacement that it experiences. Significant association between secondary structure environment and the amino acid replacement process is also observed. Careful description of the length distribution of secondary structure elements and of the organization of secondary structure and solvent accessibility along a protein did not always significantly improve the fit of the evolutionary models to the data sets that were analyzed. As indicated by the strength of the association of both solvent accessibility and secondary structure with amino acid replacement, the process of protein evolution-both above and below the species level-will not be well understood until the physical constraints that affect protein evolution are identified and characterized. PMID:9584116

  11. [Conserved motifs in the primary and secondary ITS1 structures in bryophytes].

    PubMed

    Milyutina, I A; Ignatov, M S

    2015-01-01

    A study of the ITS1 nucleotide sequences of 1000 moss species of 62 families, 11 liverwort species from five orders, and one hornwort Anthoceros agrestis identified five highly conserved motifs (CM1-CM5), which are presumably involved in pre-rRNA processing. Although the ITS1 sequences substantially differ in length and the extent of divergence, the conserved motifs are found in all of them. ITS1 secondary structures were constructed for 76 mosses, and main regularities at conserved motif positioning were observed. The positions of processing sites in the ITS1 secondary structure of the yeast Saccharomyces cerevisiae were found to be similar to the positions of the conserved motifs in the ITS1 secondary structures of mosses and liverworts. In addition, a potential hairpin formation in the putative secondary structure of a pre-rRNA fragment was considered for the region between ITS1 CM4-CM5 and a highly conserved region between hairpins 49 and 50 (H49 and H50) of the 18S rRNA.

  12. [Conserved motifs in the primary and secondary ITS1 structures in bryophytes].

    PubMed

    Milyutina, I A; Ignatov, M S

    2015-01-01

    A study of the ITS1 nucleotide sequences of 1000 moss species of 62 families, 11 liverwort species from five orders, and one hornwort Anthoceros agrestis identified five highly conserved motifs (CM1-CM5), which are presumably involved in pre-rRNA processing. Although the ITS1 sequences substantially differ in length and the extent of divergence, the conserved motifs are found in all of them. ITS1 secondary structures were constructed for 76 mosses, and main regularities at conserved motif positioning were observed. The positions of processing sites in the ITS1 secondary structure of the yeast Saccharomyces cerevisiae were found to be similar to the positions of the conserved motifs in the ITS1 secondary structures of mosses and liverworts. In addition, a potential hairpin formation in the putative secondary structure of a pre-rRNA fragment was considered for the region between ITS1 CM4-CM5 and a highly conserved region between hairpins 49 and 50 (H49 and H50) of the 18S rRNA. PMID:26107892

  13. Topology and Secondary Structure of the N-terminal Domain of Diacylglycerol Kinase

    SciTech Connect

    Oxenoid, Kirill; Soennichsen, Frank D.; Sanders, Charles R.

    2002-09-28

    Prokaryotic diacylglycerol kinase (DAGK) functions as a homotrimer of 13 kDa subunits, each of which has three transmembrane segments. This enzyme is conditionally essential to some bacteria and serves as a model system for studies of membrane protein biocatalysis, stability, folding, and misfolding. In this work, the detailed topology and secondary structure of DAGKs N-terminus up through the loop

  14. Amide I'-II' 2D IR spectroscopy provides enhanced protein secondary structural sensitivity.

    PubMed

    Deflores, Lauren P; Ganim, Ziad; Nicodemus, Rebecca A; Tokmakoff, Andrei

    2009-03-11

    We demonstrate how multimode 2D IR spectroscopy of the protein amide I' and II' vibrations can be used to distinguish protein secondary structure. Polarization-dependent amide I'-II' 2D IR experiments on poly-l-lysine in the beta-sheet, alpha-helix, and random coil conformations show that a combination of amide I' and II' diagonal and cross peaks can effectively distinguish between secondary structural content, where amide I' infrared spectroscopy alone cannot. The enhanced sensitivity arises from frequency and amplitude correlations between amide II' and amide I' spectra that reflect the symmetry of secondary structures. 2D IR surfaces are used to parametrize an excitonic model for the amide I'-II' manifold suitable to predict protein amide I'-II' spectra. This model reveals that the dominant vibrational interaction contributing to this sensitivity is a combination of negative amide II'-II' through-bond coupling and amide I'-II' coupling within the peptide unit. The empirically determined amide II'-II' couplings do not significantly vary with secondary structure: -8.5 cm(-1) for the beta sheet, -8.7 cm(-1) for the alpha helix, and -5 cm(-1) for the coil.

  15. Secondary flow structure in a model curved artery: 3D morphology and circulation budget analysis

    NASA Astrophysics Data System (ADS)

    Bulusu, Kartik V.; Plesniak, Michael W.

    2015-11-01

    In this study, we examined the rate of change of circulation within control regions encompassing the large-scale vortical structures associated with secondary flows, i.e. deformed Dean-, Lyne- and Wall-type (D-L-W) vortices at planar cross-sections in a 180° curved artery model (curvature ratio, 1/7). Magnetic resonance velocimetry (MRV) and particle image velocimetry (PIV) experiments were performed independently, under the same physiological inflow conditions (Womersley number, 4.2) and using Newtonian blood-analog fluids. The MRV-technique performed at Stanford University produced phase-averaged, three-dimensional velocity fields. Secondary flow field comparisons of MRV-data to PIV-data at various cross-sectional planes and inflow phases were made. A wavelet-decomposition-based approach was implemented to characterize various secondary flow morphologies. We hypothesize that the persistence and decay of arterial secondary flow vortices is intrinsically related to the influence of the out-of-plane flow, tilting, in-plane convection and diffusion-related factors within the control regions. Evaluation of these factors will elucidate secondary flow structures in arterial hemodynamics. Supported by the National Science Foundation under Grant Number CBET-0828903, and GW Center for Biomimetics and Bioinspired Engineering (COBRE). The MRV data were acquired at Stanford University in collaboration with Christopher Elkins and John Eaton.

  16. Secondary structure of the 3' untranslated region of flaviviruses: similarities and differences.

    PubMed

    Proutski, V; Gould, E A; Holmes, E C

    1997-03-15

    Genetic algorithm-based RNA secondary structure prediction was used in combination with comparative sequence analysis to construct models of folding for the distal part of the 3'-untranslated region of flaviviruses belonging to four serological groups. Elements of RNA secondary structure that are preserved among all the flaviviruses studied were revealed, despite the high degree of sequence divergence between them. At the same time, structural elements were observed that distinguish members of different serological groups and, in particular, a region of remarkable structural divergence between the tick-borne and mosquito-borne flaviviruses was found. Application of the genetic algorithm also revealed that the 3'-terminus of flaviviral genomic RNA may take on alternative conformations, which are not observed in the 3'-terminus of complementary minus strand RNA. These alternative folding patterns may have roles in the regulation of transcription and translation initiation and in the switch between them.

  17. Shape and secondary structure prediction for ncRNAs including pseudoknots based on linear SVM

    PubMed Central

    2013-01-01

    Background Accurate secondary structure prediction provides important information to undefirstafinding the tertiary structures and thus the functions of ncRNAs. However, the accuracy of the native structure derivation of ncRNAs is still not satisfactory, especially on sequences containing pseudoknots. It is recently shown that using the abstract shapes, which retain adjacency and nesting of structural features but disregard the length details of helix and loop regions, can improve the performance of structure prediction. In this work, we use SVM-based feature selection to derive the consensus abstract shape of homologous ncRNAs and apply the predicted shape to structure prediction including pseudoknots. Results Our approach was applied to predict shapes and secondary structures on hundreds of ncRNA data sets with and without psuedoknots. The experimental results show that we can achieve 18% higher accuracy in shape prediction than the state-of-the-art consensus shape prediction tools. Using predicted shapes in structure prediction allows us to achieve approximate 29% higher sensitivity and 10% higher positive predictive value than other pseudoknot prediction tools. Conclusions Extensive analysis of RNA properties based on SVM allows us to identify important properties of sequences and structures related to their shapes. The combination of mass data analysis and SVM-based feature selection makes our approach a promising method for shape and structure prediction. The implemented tools, Knot Shape and Knot Structure are open source software and can be downloaded at: http://www.cse.msu.edu/~achawana/KnotShape. PMID:23369147

  18. RNA secondary structures in a polymer-zeta model how foldings should be shaped for sparsification to establish a linear speedup.

    PubMed

    Jin, Emma Yu; Nebel, Markus E

    2016-02-01

    Various tools used to predict the secondary structure for a given RNA sequence are based on dynamic programming used to compute a conformation of minimum free energy. For structures without pseudoknots, a worst-case runtime proportional to n3, with n being the length of the sequence, results since a table of dimension n2 has to be filled in while a single entry gives rise to a linear computational effort. However, it was recently observed that reformulating the corresponding dynamic programming recursion together with the bookkeeping of potential folding alternatives (a technique called sparsification) may reduce the runtime to n2 on average, assuming that nucleotides of distance d form a hydrogen bond (i..e., are paired) with probability b/d(c) for some constants b > 0, c > 1. The latter is called the polymer-zeta model and plays a crucial role in speeding up the above mentioned algorithm. In this paper we discuss the application of the polymer-zeta property for the analysis of sparsification, showing that it must be applied conditionally on first and last positions to pair. Afterwards, we will investigate the combinatorics of RNA secondary structures assuming that the corresponding conditional probabilities behave according to a polymer-zeta probability model. We show that even if some of the structural parameters exhibit an almost realistic behavior on average, the expected shape of a folding in that model must be assumed to highly differ from those observed in nature. More precisely, we prove our polymer-zeta model to be appropriate for mRNA molecules but to fail in connection with almost every other family of RNA. Those findings explain the huge speedup of the dynamic programming algorithm observed empirically by Wexler et al. when applying sparsification in connection with mRNA data.

  19. RNA secondary structures in a polymer-zeta model how foldings should be shaped for sparsification to establish a linear speedup.

    PubMed

    Jin, Emma Yu; Nebel, Markus E

    2016-02-01

    Various tools used to predict the secondary structure for a given RNA sequence are based on dynamic programming used to compute a conformation of minimum free energy. For structures without pseudoknots, a worst-case runtime proportional to n3, with n being the length of the sequence, results since a table of dimension n2 has to be filled in while a single entry gives rise to a linear computational effort. However, it was recently observed that reformulating the corresponding dynamic programming recursion together with the bookkeeping of potential folding alternatives (a technique called sparsification) may reduce the runtime to n2 on average, assuming that nucleotides of distance d form a hydrogen bond (i..e., are paired) with probability b/d(c) for some constants b > 0, c > 1. The latter is called the polymer-zeta model and plays a crucial role in speeding up the above mentioned algorithm. In this paper we discuss the application of the polymer-zeta property for the analysis of sparsification, showing that it must be applied conditionally on first and last positions to pair. Afterwards, we will investigate the combinatorics of RNA secondary structures assuming that the corresponding conditional probabilities behave according to a polymer-zeta probability model. We show that even if some of the structural parameters exhibit an almost realistic behavior on average, the expected shape of a folding in that model must be assumed to highly differ from those observed in nature. More precisely, we prove our polymer-zeta model to be appropriate for mRNA molecules but to fail in connection with almost every other family of RNA. Those findings explain the huge speedup of the dynamic programming algorithm observed empirically by Wexler et al. when applying sparsification in connection with mRNA data. PMID:26001743

  20. Single-molecule reconstruction of oligonucleotide secondary structure by atomic force microscopy.

    PubMed

    Pyne, Alice; Thompson, Ruth; Leung, Carl; Roy, Debdulal; Hoogenboom, Bart W

    2014-08-27

    Based on soft-touch atomic force microscopy, a method is described to reconstruct the secondary structure of single extended biomolecules, without the need for crystallization. The method is tested by accurately reproducing the dimensions of the B-DNA crystal structure. Importantly, intramolecular variations in groove depth of the DNA double helix are resolved, which would be inaccessible for methods that rely on ensemble-averaging.

  1. A permutation based simulated annealing algorithm to predict pseudoknotted RNA secondary structures.

    PubMed

    Tsang, Herbert H; Wiese, Kay C

    2015-01-01

    Pseudoknots are RNA tertiary structures which perform essential biological functions. This paper discusses SARNA-Predict-pk, a RNA pseudoknotted secondary structure prediction algorithm based on Simulated Annealing (SA). The research presented here extends previous work of SARNA-Predict and further examines the effect of the new algorithm to include prediction of RNA secondary structure with pseudoknots. An evaluation of the performance of SARNA-Predict-pk in terms of prediction accuracy is made via comparison with several state-of-the-art prediction algorithms using 20 individual known structures from seven RNA classes. We measured the sensitivity and specificity of nine prediction algorithms. Three of these are dynamic programming algorithms: Pseudoknot (pknotsRE), NUPACK, and pknotsRG-mfe. One is using the statistical clustering approach: Sfold and the other five are heuristic algorithms: SARNA-Predict-pk, ILM, STAR, IPknot and HotKnots algorithms. The results presented in this paper demonstrate that SARNA-Predict-pk can out-perform other state-of-the-art algorithms in terms of prediction accuracy. This supports the use of the proposed method on pseudoknotted RNA secondary structure prediction of other known structures. PMID:26558299

  2. Protein secondary-structure description with a coarse-grained model.

    PubMed

    Kneller, Gerald R; Hinsen, Konrad

    2015-07-01

    A coarse-grained geometrical model for protein secondary-structure description and analysis is presented which uses only the positions of the C(α) atoms. A space curve connecting these positions by piecewise polynomial interpolation is constructed and the folding of the protein backbone is described by a succession of screw motions linking the Frenet frames at consecutive C(α) positions. Using the ASTRAL subset of the SCOPe database of protein structures, thresholds are derived for the screw parameters of secondary-structure elements and demonstrate that the latter can be reliably assigned on the basis of a C(α) model. For this purpose, a comparative study with the widely used DSSP (Define Secondary Structure of Proteins) algorithm was performed and it was shown that the parameter distribution corresponding to the ensemble of all pure C(α) structures in the RCSB Protein Data Bank matches that of the ASTRAL database. It is expected that this approach will be useful in the development of structure-refinement techniques for low-resolution data. PMID:26143913

  3. RNApdbee—a webserver to derive secondary structures from pdb files of knotted and unknotted RNAs

    PubMed Central

    Antczak, Maciej; Zok, Tomasz; Popenda, Mariusz; Lukasiak, Piotr; Adamiak, Ryszard W.; Blazewicz, Jacek; Szachniuk, Marta

    2014-01-01

    In RNA structural biology and bioinformatics an access to correct RNA secondary structure and its proper representation is of crucial importance. This is true especially in the field of secondary and 3D RNA structure prediction. Here, we introduce RNApdbee—a new tool that allows to extract RNA secondary structure from the pdb file, and presents it in both textual and graphical form. RNApdbee supports processing of knotted and unknotted structures of large RNAs, also within protein complexes. The method works not only for first but also for high order pseudoknots, and gives an information about canonical and non-canonical base pairs. A combination of these features is unique among existing applications for RNA structure analysis. Additionally, a function of converting between the text notations, i.e. BPSEQ, CT and extended dot-bracket, is provided. In order to facilitate a more comprehensive study, the webserver integrates the functionality of RNAView, MC-Annotate and 3DNA/DSSR, being the most common tools used for automated identification and classification of RNA base pairs. RNApdbee is implemented as a publicly available webserver with an intuitive interface and can be freely accessed at http://rnapdbee.cs.put.poznan.pl/. PMID:24771339

  4. An Adaptive Defect Weighted Sampling Algorithm to Design Pseudoknotted RNA Secondary Structures

    PubMed Central

    Zandi, Kasra; Butler, Gregory; Kharma, Nawwaf

    2016-01-01

    Computational design of RNA sequences that fold into targeted secondary structures has many applications in biomedicine, nanotechnology and synthetic biology. An RNA molecule is made of different types of secondary structure elements and an important RNA element named pseudoknot plays a key role in stabilizing the functional form of the molecule. However, due to the computational complexities associated with characterizing pseudoknotted RNA structures, most of the existing RNA sequence designer algorithms generally ignore this important structural element and therefore limit their applications. In this paper we present a new algorithm to design RNA sequences for pseudoknotted secondary structures. We use NUPACK as the folding algorithm to compute the equilibrium characteristics of the pseudoknotted RNAs, and describe a new adaptive defect weighted sampling algorithm named Enzymer to design low ensemble defect RNA sequences for targeted secondary structures including pseudoknots. We used a biological data set of 201 pseudoknotted structures from the Pseudobase library to benchmark the performance of our algorithm. We compared the quality characteristics of the RNA sequences we designed by Enzymer with the results obtained from the state of the art MODENA and antaRNA. Our results show our method succeeds more frequently than MODENA and antaRNA do, and generates sequences that have lower ensemble defect, lower probability defect and higher thermostability. Finally by using Enzymer and by constraining the design to a naturally occurring and highly conserved Hammerhead motif, we designed 8 sequences for a pseudoknotted cis-acting Hammerhead ribozyme. Enzymer is available for download at https://bitbucket.org/casraz/enzymer. PMID:27499762

  5. An Adaptive Defect Weighted Sampling Algorithm to Design Pseudoknotted RNA Secondary Structures.

    PubMed

    Zandi, Kasra; Butler, Gregory; Kharma, Nawwaf

    2016-01-01

    Computational design of RNA sequences that fold into targeted secondary structures has many applications in biomedicine, nanotechnology and synthetic biology. An RNA molecule is made of different types of secondary structure elements and an important RNA element named pseudoknot plays a key role in stabilizing the functional form of the molecule. However, due to the computational complexities associated with characterizing pseudoknotted RNA structures, most of the existing RNA sequence designer algorithms generally ignore this important structural element and therefore limit their applications. In this paper we present a new algorithm to design RNA sequences for pseudoknotted secondary structures. We use NUPACK as the folding algorithm to compute the equilibrium characteristics of the pseudoknotted RNAs, and describe a new adaptive defect weighted sampling algorithm named Enzymer to design low ensemble defect RNA sequences for targeted secondary structures including pseudoknots. We used a biological data set of 201 pseudoknotted structures from the Pseudobase library to benchmark the performance of our algorithm. We compared the quality characteristics of the RNA sequences we designed by Enzymer with the results obtained from the state of the art MODENA and antaRNA. Our results show our method succeeds more frequently than MODENA and antaRNA do, and generates sequences that have lower ensemble defect, lower probability defect and higher thermostability. Finally by using Enzymer and by constraining the design to a naturally occurring and highly conserved Hammerhead motif, we designed 8 sequences for a pseudoknotted cis-acting Hammerhead ribozyme. Enzymer is available for download at https://bitbucket.org/casraz/enzymer. PMID:27499762

  6. An Adaptive Defect Weighted Sampling Algorithm to Design Pseudoknotted RNA Secondary Structures.

    PubMed

    Zandi, Kasra; Butler, Gregory; Kharma, Nawwaf

    2016-01-01

    Computational design of RNA sequences that fold into targeted secondary structures has many applications in biomedicine, nanotechnology and synthetic biology. An RNA molecule is made of different types of secondary structure elements and an important RNA element named pseudoknot plays a key role in stabilizing the functional form of the molecule. However, due to the computational complexities associated with characterizing pseudoknotted RNA structures, most of the existing RNA sequence designer algorithms generally ignore this important structural element and therefore limit their applications. In this paper we present a new algorithm to design RNA sequences for pseudoknotted secondary structures. We use NUPACK as the folding algorithm to compute the equilibrium characteristics of the pseudoknotted RNAs, and describe a new adaptive defect weighted sampling algorithm named Enzymer to design low ensemble defect RNA sequences for targeted secondary structures including pseudoknots. We used a biological data set of 201 pseudoknotted structures from the Pseudobase library to benchmark the performance of our algorithm. We compared the quality characteristics of the RNA sequences we designed by Enzymer with the results obtained from the state of the art MODENA and antaRNA. Our results show our method succeeds more frequently than MODENA and antaRNA do, and generates sequences that have lower ensemble defect, lower probability defect and higher thermostability. Finally by using Enzymer and by constraining the design to a naturally occurring and highly conserved Hammerhead motif, we designed 8 sequences for a pseudoknotted cis-acting Hammerhead ribozyme. Enzymer is available for download at https://bitbucket.org/casraz/enzymer.

  7. mRNA sequence of three respiratory syncytial virus genes encoding two nonstructural proteins and a 22K structural protein.

    PubMed Central

    Elango, N; Satake, M; Venkatesan, S

    1985-01-01

    An mRNA sequence of two human respiratory syncytial viral nonstructural protein genes and of a gene for a 22,000-molecular-weight (22K) protein was obtained by cDNA cloning and DNA sequencing. Sequences corresponding to the 5' ends of the respective transcripts were deduced directly by primer extension and dideoxy nucleotide sequencing of the mRNAs. The availability of a bicistronic clone (pRSC6) confirmed the gene order for this portion of the genome. Contrary to other unsegmented negative-stranded RNA viruses, a 19-nucleotide intercistronic sequence was present between the NS1 and NS2 genes. The translation of cloned viral sequences in the bicistronic and monocistronic clones (pRSNS1 and pRSNS2) revealed two moderately hydrophobic proteins of 15,568 and 14,703 daltons. Their similarity in molecular size explained our earlier inability to resolve these proteins. A DNA sequence of an additional recombinant plasmid (pRSA2) revealed a long open reading frame encoding a 22,156-dalton protein containing 194 amino acids. It was relatively basic and moderately hydrophobic. A protein of this size was readily translated in vitro from a viral mRNA hybrid selected by this plasmid and corresponded to an unglycosylated 22K protein seen in purified extracellular virus but not associated with detergent- and salt-resistant cores. A second open reading frame of 90 amino acids partially overlapping with the C terminus of the 22K protein was also present within this sequence. This was reminiscent of the viral matrix protein gene which was previously shown by us to contain two overlapping reading frames. The finding of three additional viral transcripts encoding at least three identifiable proteins in human respiratory syncytial virus was a novel departure from the usual genetic organization of paramyxoviruses. The 5' ends of all three transcripts had a 5'NGGGCAAAU sequence that is common to all viral transcripts analyzed so far. Although there was no obvious homology immediately

  8. Male secondary sexual structures and the systematics of the Thereus oppia species group (Lepidoptera, Lycaenidae, Eumaeini).

    PubMed

    Robbins, Robert K; Heredia, María Dolores; Busby, Robert C

    2015-01-01

    The Thereus oppia species group includes species with and without a scent pad, which is a histologically and morphologically characterized male secondary sexual structure on the dorsal surface of the forewing. To assess the hypothesis that these structures are lost evolutionarily, but not regained (Dollo's Law), the taxonomy of this species group is revised. Thereus lomalarga sp. n., and Thereus brocki sp. n., are described. Diagnostic traits, especially male secondary structures, within the Thereus oppia species group are illustrated. Distributional and biological information is summarized for each species. Three species have been reared, and the caterpillars eat Loranthaceae. An inferred phylogeny is consistent with the hypothesis that scent pads in the Thereus oppia species group have been lost evolutionarily twice (in allopatry), and not re-gained. PMID:26448715

  9. Intron positions in actin genes seem unrelated to the secondary structure of the protein.

    PubMed Central

    Weber, K; Kabsch, W

    1994-01-01

    A catalogue of intron positions along the coding sequence was assembled from the large number of actin genes known for different eukaryotes. 36 positions in the amino acid sequence were compared with the known three-dimensional structure of actin. At least 20 but not more than 23 intron positions are at the start or end of a secondary structural element (beta-strand, alpha-helix or 3/10 helix) while eight positions interrupt such an element. Statistical analysis shows that due to the large number of end positions the boundaries of secondary structural elements are not correlated with the intron positions. In addition, the observed intron pattern seems compatible with the null hypothesis, i.e. intron positions are randomly distributed along the actin sequence. Images PMID:8137812

  10. Male secondary sexual structures and the systematics of the Thereus oppia species group (Lepidoptera, Lycaenidae, Eumaeini)

    PubMed Central

    Robbins, Robert K.; Heredia, María Dolores; Busby, Robert C.

    2015-01-01

    Abstract The Thereus oppia species group includes species with and without a scent pad, which is a histologically and morphologically characterized male secondary sexual structure on the dorsal surface of the forewing. To assess the hypothesis that these structures are lost evolutionarily, but not regained (Dollo’s Law), the taxonomy of this species group is revised. Thereus lomalarga sp. n., and Thereus brocki sp. n., are described. Diagnostic traits, especially male secondary structures, within the Thereus oppia species group are illustrated. Distributional and biological information is summarized for each species. Three species have been reared, and the caterpillars eat Loranthaceae. An inferred phylogeny is consistent with the hypothesis that scent pads in the Thereus oppia species group have been lost evolutionarily twice (in allopatry), and not re-gained. PMID:26448715

  11. Secondary structure of the HIV-2 leader RNA comprising the tRNA-primer binding site.

    PubMed Central

    Berkhout, B; Schoneveld, I

    1993-01-01

    The initiation of reverse transcription of a retroviral RNA genome occurs by a tRNA primer bound near the 5' end of the genomic RNA at a position called the primer-binding site (PBS). To understand the molecular basis for this RNA-RNA interaction, the secondary structure of the leader RNA of the human immunodeficiency virus type 2 (HIV-2) RNA was analyzed. In vitro synthesized HIV-2 RNA was probed with various structure-specific enzymes and chemicals. A computer program was then used to predict the secondary structure consistent with these data. In addition, the nucleotide sequences of different HIV-2 isolates were used to screen for the occurrence of covariation among putative base pairs. The primary sequences have diverged rapidly in some HIV-2 isolates, however, some strikingly conserved secondary structure elements were identified. Most nucleotides in the leader region are involved in base pairing. An exception is the PBS sequence, of which 15 out of 18 nucleotides are exposed in an internal loop. These findings suggest that the overall structure of the HIV-2 genome has evolved to facilitate an optimal interaction with its tRNA primer. Images PMID:8464701

  12. Influence of MLS laser radiation on erythrocyte membrane fluidity and secondary structure of human serum albumin.

    PubMed

    Pasternak, Kamila; Nowacka, Olga; Wróbel, Dominika; Pieszyński, Ireneusz; Bryszewska, Maria; Kujawa, Jolanta

    2014-03-01

    The biostimulating activity of low level laser radiation of various wavelengths and energy doses is widely documented in the literature, but the mechanisms of the intracellular reactions involved are not precisely known. The aim of this paper is to evaluate the influence of low level laser radiation from an multiwave locked system (MLS) of two wavelengths (wavelength = 808 nm in continuous emission and 905 nm in pulsed emission) on the human erythrocyte membrane and on the secondary structure of human serum albumin (HSA). Human erythrocytes membranes and HSA were irradiated with laser light of low intensity with surface energy density ranging from 0.46 to 4.9 J cm(-2) and surface energy power density 195 mW cm(-2) (1,000 Hz) and 230 mW cm(-2) (2,000 Hz). Structural and functional changes in the erythrocyte membrane were characterized by its fluidity, while changes in the protein were monitored by its secondary structure. Dose-dependent changes in erythrocyte membrane fluidity were induced by near-infrared laser radiation. Slight changes in the secondary structure of HSA were also noted. MLS laser radiation influences the structure and function of the human erythrocyte membrane resulting in a change in fluidity.

  13. Conserved RNA secondary structures and long-range interactions in hepatitis C viruses.

    PubMed

    Fricke, Markus; Dünnes, Nadia; Zayas, Margarita; Bartenschlager, Ralf; Niepmann, Michael; Marz, Manja

    2015-07-01

    Hepatitis C virus (HCV) is a hepatotropic virus with a plus-strand RNA genome of ∼9.600 nt. Due to error-prone replication by its RNA-dependent RNA polymerase (RdRp) residing in nonstructural protein 5B (NS5B), HCV isolates are grouped into seven genotypes with several subtypes. By using whole-genome sequences of 106 HCV isolates and secondary structure alignments of the plus-strand genome and its minus-strand replication intermediate, we established refined secondary structures of the 5' untranslated region (UTR), the cis-acting replication element (CRE) in NS5B, and the 3' UTR. We propose an alternative structure in the 5' UTR, conserved secondary structures of 5B stem-loop (SL)1 and 5BSL2, and four possible structures of the X-tail at the very 3' end of the HCV genome. We predict several previously unknown long-range interactions, most importantly a possible circularization interaction between distinct elements in the 5' and 3' UTR, reminiscent of the cyclization elements of the related flaviviruses. Based on analogy to these viruses, we propose that the 5'-3' UTR base-pairing in the HCV genome might play an important role in viral RNA replication. These results may have important implications for our understanding of the nature of the cis-acting RNA elements in the HCV genome and their possible role in regulating the mutually exclusive processes of viral RNA translation and replication.

  14. A Dynamic Programming Algorithm for Finding the Optimal Placement of a Secondary Structure Topology in Cryo-EM Data.

    PubMed

    Biswas, Abhishek; Ranjan, Desh; Zubair, Mohammad; He, Jing

    2015-09-01

    The determination of secondary structure topology is a critical step in deriving the atomic structures from the protein density maps obtained from electron cryomicroscopy technique. This step often relies on matching the secondary structure traces detected from the protein density map to the secondary structure sequence segments predicted from the amino acid sequence. Due to inaccuracies in both sources of information, a pool of possible secondary structure positions needs to be sampled. One way to approach the problem is to first derive a small number of possible topologies using existing matching algorithms, and then find the optimal placement for each possible topology. We present a dynamic programming method of Θ(Nq(2)h) to find the optimal placement for a secondary structure topology. We show that our algorithm requires significantly less computational time than the brute force method that is in the order of Θ(q(N) h).

  15. "Well-determined" regions in RNA secondary structure prediction: analysis of small subunit ribosomal RNA.

    PubMed Central

    Zuker, M; Jacobson, A B

    1995-01-01

    Recent structural analyses of genomic RNAs from RNA coliphages suggest that both well-determined base paired helices and well-determined structural domains that are identified by "energy dot plot" analysis using the RNA folding package mfold, are likely to be predicted correctly. To test these observations with another group of large RNAs, we have analyzed 15 ribosomal RNAs. Published secondary structure models that were derived by comparative sequence analysis were used to evaluate the predicted structures. Both the optimal predicted fold and the predicted "energy dot plot" of each sequence were examined. Each prediction was obtained from a single computer run on an entire ribosomal RNA sequence. All predicted base pairs in optimal foldings were examined for agreement with proven base pairs in the comparative models. Our analyses show that the overall correspondence between the predicted and comparative models varied for different RNAs and ranges from a low of 27% to high of 70%, with a mean value of 49%. The correspondence improves to a mean value of 81% when the analysis is limited to well-determined helices. In addition to well-determined helices, large well-determined structural domains can be observed in "energy dot plots" of some 16S ribosomal RNAs. The predicted domains correspond closely with structural domains that are found by the comparative method in the same RNAs. Our analyses also show that measuring the agreement between predicted and comparative secondary structure models underestimates the reliability of structural prediction by mfold. PMID:7544463

  16. Self-Efficacy, School Resources, Job Stressors and Burnout among Spanish Primary and Secondary School Teachers: A Structural Equation Approach

    ERIC Educational Resources Information Center

    Betoret, Fernando Domenech

    2009-01-01

    This study examines the relationship between school resources, teacher self-efficacy, potential multi-level stressors and teacher burnout using structural equation modelling. The causal structure for primary and secondary school teachers was also examined. The sample was composed of 724 primary and secondary Spanish school teachers. The changes…

  17. Minimum-free-energy distribution of RNA secondary structures: Entropic and thermodynamic properties of rare events

    NASA Astrophysics Data System (ADS)

    Wolfsheimer, S.; Hartmann, A. K.

    2010-08-01

    We study the distribution of the minimum free energy (MFE) for the Turner model of pseudoknot free RNA secondary structures over ensembles of random RNA sequences. In particular, we are interested in those rare and intermediate events of unexpected low MFEs. Generalized ensemble Markov-chain Monte Carlo methods allow us to explore the rare-event tail of the MFE distribution down to probabilities such as 10-70 and to study the relationship between the sequence entropy and structural properties for sequence ensembles with fixed MFEs. Entropic and structural properties of those ensembles are compared with natural RNA of the same reduced MFE ( z score).

  18. Determination of Protein Secondary Structure from Infrared Spectra Using Partial Least-Squares Regression.

    PubMed

    Wilcox, Kieaibi E; Blanch, Ewan W; Doig, Andrew J

    2016-07-12

    Infrared (IR) spectra contain substantial information about protein structure. This has previously most often been exploited by using known band assignments. Here, we convert spectral intensities in bins within Amide I and II regions to vectors and apply machine learning methods to determine protein secondary structure. Partial least squares was performed on spectra of 90 proteins in H2O. After preprocessing and removal of outliers, 84 proteins were used for this work. Standard normal variate and second-derivative preprocessing methods on the combined Amide I and II data generally gave the best performance, with root-mean-square values for prediction of ∼12% for α-helix, ∼7% for β-sheet, 7% for antiparallel β-sheet, and ∼8% for other conformations. Analysis of Fourier transform infrared (FTIR) spectra of 16 proteins in D2O showed that secondary structure determination was slightly poorer than in H2O. Interval partial least squares was used to identify the critical regions within spectra for secondary structure prediction and showed that the sides of bands were most valuable, rather than their peak maxima. In conclusion, we have shown that multivariate analysis of protein FTIR spectra can give α-helix, β-sheet, other, and antiparallel β-sheet contents with good accuracy, comparable to that of circular dichroism, which is widely used for this purpose. PMID:27322779

  19. Visualizing the formation of an RNA folding intermediate through a fast highly modular secondary structure switch

    PubMed Central

    Xue, Yi; Gracia, Brant; Herschlag, Daniel; Russell, Rick; Al-Hashimi, Hashim M.

    2016-01-01

    Intermediates play important roles in RNA folding but can be difficult to characterize when short-lived or not significantly populated. By combining 15N relaxation dispersion NMR with chemical probing, we visualized a fast (kex=k1+k−1≈423 s−1) secondary structural switch directed towards a low-populated (∼3%) partially folded intermediate in tertiary folding of the P5abc subdomain of the ‘Tetrahymena' group I intron ribozyme. The secondary structure switch changes the base-pairing register across the P5c hairpin, creating a native-like structure, and occurs at rates of more than two orders of magnitude faster than tertiary folding. The switch occurs robustly in the absence of tertiary interactions, Mg2+ or even when the hairpin is excised from the three-way junction. Fast, highly modular secondary structural switches may be quite common during RNA tertiary folding where they may help smoothen the folding landscape by allowing folding to proceed efficiently via additional pathways. PMID:27292179

  20. Small-angle X-ray scattering: a bridge between RNA secondary structures and three-dimensional topological structures

    SciTech Connect

    Fang, Xianyang; Stagno, Jason R.; Bhandari, Yuba R.; Zuo, Xiaobing; Wang, Yun-Xing

    2015-02-01

    Whereas the structures of small to medium-sized well folded RNA molecules often can be determined by either X-ray crystallography or NMR spectroscopy, obtaining structural information for large RNAs using experimental, computational, or combined approaches remains a major interest and challenge. RNA is very sensitive to small-angle X-ray scattering (SAXS) due to high electron density along phosphate-sugar backbones, whose scattering contribution dominates SAXS intensity. For this reason, SAXS is particularly useful in obtaining global RNA structural information that outlines backbone topologies and, therefore, molecular envelopes. Such information is extremely valuable in bridging the gap between the secondary structures and three-dimensional topological structures of RNAmolecules, particularly those that have proven difficult to study using other structuredetermination methods. Here we review published results of RNA topological structures derived from SAXS data or in combination with other experimental data, as well as details on RNA sample preparation for SAXS experiments.

  1. Crystal Structure of A. aeolicus Argonaute, a Site-Specific DNA-Guided Endoribonuclease, Provides Insights into RISC-Mediated mRNA Cleavage

    PubMed Central

    Yuan, Yu-Ren; Pei, Yi; Ma, Jin-Biao; Kuryavyi, Vitaly; Zhadina, Maria; Meister, Gunter; Chen, Hong-Ying; Dauter, Zbigniew; Tuschl, Thomas; Patel, Dinshaw J.

    2015-01-01

    Summary Argonaute (Ago) proteins constitute a key component of the RNA-induced silencing complex (RISC). We report the crystal structure of Aquifex aeolicus Ago (Aa-Ago) together with binding and cleavage studies, which establish this eubacterial Ago as a bona fide guide DNA strand-mediated site-specific RNA endonuclease. We have generated a stereochemically robust model of the complex, where the guide DNA-mRNA duplex is positioned within a basic channel spanning the bilobal interface, such that the 5′ phosphate of the guide strand can be anchored in a basic pocket, and the mRNA can be positioned for site-specific cleavage by RNase H-type divalent cation-coordinated catalytic Asp residues of the PIWI domain. Domain swap experiments involving chimeras of human Ago (hAgo1) and cleavage-competent hAgo2 reinforce the role of the PIWI domain in “slicer” activity. We propose a four-step Ago-mediated catalytic cleavage cycle model, which provides distinct perspectives into the mechanism of guide strand-mediated mRNA cleavage within the RISC. PMID:16061186

  2. Human retina-specific amine oxidase: genomic structure of the gene (AOC2), alternatively spliced variant, and mRNA expression in retina.

    PubMed

    Imamura, Y; Noda, S; Mashima, Y; Kudoh, J; Oguchi, Y; Shimizu, N

    1998-07-15

    Previously, we reported the isolation of cDNA for human retina-specific amine oxidase (RAO) and the expression of RAO exclusively in retina. Bacterial artificial chromosome clones containing the human RAO gene (AOC2) were mapped to human chromosome 17q21 (Imamura et al., 1997, Genomics 40: 277-283). Here, we report the complete genomic structure of the RAO gene, including 5' flanking sequence, and mRNA expression in retina. The human RAO gene spans 6 kb and is composed of four exons corresponding to the amino acid sequence 1-530, 530-598, 598-641, and 642-729 separated by three introns of 3000, 310, and 351 bp. Screening of a human retina cDNA library revealed the existence of an alternatively spliced cDNA variant with an additional 81 bp at the end of exon 2. The sizes of exons and the locations of exon/intron boundaries in the human RAO gene showed remarkable similarity to those of the human kidney diamine oxidase gene (AOC1). In situ hybridization revealed that mRNA coding for RAO is expressed preferentially in the ganglion cell layer of the mouse retina. We designed four sets of PCR primers to amplify four exons, which will be valuable for analyzing mutations in patients with ocular diseases affecting the retinal ganglion cell layer.

  3. Crystal structure of A. aeolicus argonaute, a site-specific DNA-guided endoribonuclease, provides insights into RISC-mediated mRNA cleavage

    SciTech Connect

    Yuan,Y.; Pei, Y.; Ma, J.; Kuryavyi, V.; Zhadina, M.; Meister, G.; Chen, H.; Dauter, Z.; Tuschi, T.; Patel, D.

    2005-01-01

    Argonaute (Ago) proteins constitute a key component of the RNA-induced silencing complex (RISC). We report the crystal structure of Aquifex aeolicus Ago (Aa-Ago) together with binding and cleavage studies, which establish this eubacterial Ago as a bona fide guide DNA strand-mediated site-specific RNA endonuclease. We have generated a stereochemically robust model of the complex, where the guide DNA-mRNA duplex is positioned within a basic channel spanning the bilobal interface, such that the 5' phosphate of the guide strand can be anchored in a basic pocket, and the mRNA can be positioned for site-specific cleavage by RNase H-type divalent cation-coordinated catalytic Asp residues of the PIWI domain. Domain swap experiments involving chimeras of human Ago (hAgo1) and cleavage-competent hAgo2 reinforce the role of the PIWI domain in 'slicer' activity. We propose a four-step Ago-mediated catalytic cleavage cycle model, which provides distinct perspectives into the mechanism of guide strand-mediated mRNA cleavage within the RISC.

  4. Structural and functional analysis of the interaction between the nucleoporin Nup98 and the mRNA export factor Rae1

    SciTech Connect

    Ren, Yi; Seo, Hyuk-Soo; Blobel, Günter; Hoelz, André

    2010-07-23

    The export of mRNAs is a multistep process, involving the packaging of mRNAs into messenger ribonucleoprotein particles (mRNPs), their transport through nuclear pore complexes, and mRNP remodeling events prior to translation. Ribonucleic acid export 1 (Rae1) and Nup98 are evolutionarily conserved mRNA export factors that are targeted by the vesicular stomatitis virus matrix protein to inhibit host cell nuclear export. Here, we present the crystal structure of human Rae1 in complex with the Gle2-binding sequence (GLEBS) of Nup98 at 1.65 {angstrom} resolution. Rae1 forms a seven-bladed {beta}-propeller with several extensive surface loops. The Nup98 GLEBS motif forms an {approx} 50-{angstrom}-long hairpin that binds with its C-terminal arm to an essentially invariant hydrophobic surface that extends over the entire top face of the Rae1 {beta}-propeller. The C-terminal arm of the GLEBS hairpin is necessary and sufficient for Rae1 binding, and we identify a tandem glutamate element in this arm as critical for complex formation. The Rae1 {center_dot} Nup98{sup GLEBS} surface features an additional conserved patch with a positive electrostatic potential, and we demonstrate that the complex possesses single-stranded RNA-binding capability. Together, these data suggest that the Rae1 {center_dot} Nup98 complex directly binds to the mRNP at several stages of the mRNA export pathway.

  5. Structural and Functional Analysis of the Interaction Between the Nucleoporin Nup98 and the mRNA Export Facto Rae1

    SciTech Connect

    Y Ren; H Seo; G Blobel; A Hoelz

    2011-12-31

    The export of mRNAs is a multistep process, involving the packaging of mRNAs into messenger ribonucleoprotein particles (mRNPs), their transport through nuclear pore complexes, and mRNP remodeling events prior to translation. Ribonucleic acid export 1 (Rae1) and Nup98 are evolutionarily conserved mRNA export factors that are targeted by the vesicular stomatitis virus matrix protein to inhibit host cell nuclear export. Here, we present the crystal structure of human Rae1 in complex with the Gle2-binding sequence (GLEBS) of Nup98 at 1.65 {angstrom} resolution. Rae1 forms a seven-bladed {beta}-propeller with several extensive surface loops. The Nup98 GLEBS motif forms an {approx}50-{angstrom}-long hairpin that binds with its C-terminal arm to an essentially invariant hydrophobic surface that extends over the entire top face of the Rae1 {beta}-propeller. The C-terminal arm of the GLEBS hairpin is necessary and sufficient for Rae1 binding, and we identify a tandem glutamate element in this arm as critical for complex formation. The Rae1 {center_dot} Nup98{sup GLEBS} surface features an additional conserved patch with a positive electrostatic potential, and we demonstrate that the complex possesses single-stranded RNA-binding capability. Together, these data suggest that the Rae1 {center_dot} Nup98 complex directly binds to the mRNP at several stages of the mRNA export pathway.

  6. Structural and functional analysis of the interaction between the nucleoporin Nup98 and the mRNA export factor Rae1

    PubMed Central

    Ren, Yi; Seo, Hyuk-Soo; Blobel, Günter; Hoelz, André

    2010-01-01

    The export of mRNAs is a multistep process, involving the packaging of mRNAs into messenger ribonucleoprotein particles (mRNPs), their transport through nuclear pore complexes, and mRNP remodeling events prior to translation. Ribonucleic acid export 1 (Rae1) and Nup98 are evolutionarily conserved mRNA export factors that are targeted by the vesicular stomatitis virus matrix protein to inhibit host cell nuclear export. Here, we present the crystal structure of human Rae1 in complex with the Gle2-binding sequence (GLEBS) of Nup98 at 1.65 Å resolution. Rae1 forms a seven-bladed β-propeller with several extensive surface loops. The Nup98 GLEBS motif forms an ≈50-Å-long hairpin that binds with its C-terminal arm to an essentially invariant hydrophobic surface that extends over the entire top face of the Rae1 β-propeller. The C-terminal arm of the GLEBS hairpin is necessary and sufficient for Rae1 binding, and we identify a tandem glutamate element in this arm as critical for complex formation. The Rae1•Nup98GLEBS surface features an additional conserved patch with a positive electrostatic potential, and we demonstrate that the complex possesses single-stranded RNA-binding capability. Together, these data suggest that the Rae1•Nup98 complex directly binds to the mRNP at several stages of the mRNA export pathway. PMID:20498086

  7. Determination of the Secondary Structure of the king Cobra Neurotoxin CM-11.

    PubMed

    Pang, Yu-Xi; Liu, Wei-Dong; Liu, Ai-Zhuo; Pei, Feng-Kui

    1997-01-01

    The king cobra neurotoxin CM-11 is a small protein with 72 amino acid residues. After its complete assignments of (1)H-NMR resonance's were obtained using various 2D-NMR technologies, including DQF-COSY, clean-TOCSY and NOESY, the secondary structure was analysed by studying the various NOEs extracted from the NOESY spectra and the distribution of chemical shifts. The secondary structure was finally determined by MCD as follows: a triple-strand antiparallel beta sheet with I20-W26, R37-A43 and V53-S59 as its beta strands, a short alpha helix formed by W30-G35 and four turns formed by P7-K1O, C14-G17, K50-V53 and D61-N64.

  8. Analysis of the secondary structure of a protein's N-terminal

    NASA Astrophysics Data System (ADS)

    Floare, C. G.; Bogdan, M.; Horovitz, O.; Mocanu, A.; Tomoaia-Cotisel, M.

    2009-08-01

    The major protein component from aleurone cells of barley (Hordeum vulgare L.), PACB, is related to 7S globulins present in other cereals and to the vicilin-type 7S globulins of legumes and cotton seed. It contains 4 subunits of about 20, 25, 40 and 50 kDa molecular weights. The N-terminal sequence of 16 amino acids (over 260 atoms) in the protein was previously determined, and our aim is the prediction of its secondary structure. The empirical Chou-Fasman method was applied in an improved version as well as the empirical DSC method (discrimination of protein secondary structure class) with quite similar results. A molecular dynamics simulation was also performed, using the FF99SB forcefield within AMBER version 9.0. Solvation effects were incorporated using the Born model. The results are compared and a 3D model is proposed.

  9. Sequence and secondary structure of the mitochondrial 16S ribosomal RNA gene of Ixodes scapularis.

    PubMed

    Krakowetz, Chantel N; Chilton, Neil B

    2015-02-01

    The complete DNA sequences and secondary structure of the mitochondrial (mt) 16S ribosomal (r) RNA gene were determined for six Ixodes scapularis adults. There were 44 variable nucleotide positions in the 1252 bp sequence alignment. Most (95%) nucleotide alterations did not affect the integrity of the secondary structure of the gene because they either occurred at unpaired positions or represented compensatory changes that maintained the base pairing in helices. A large proportion (75%) of the intraspecific variation in DNA sequence occurred within Domains I, II and VI of the 16S gene. Therefore, several regions within this gene may be highly informative for studies of the population genetics and phylogeography of I. scapularis, a major vector of pathogens of humans and domestic animals in North America.

  10. Extremely Slow Dynamics of an Abiotic Helical Assembly: Unusual Relevance to the Secondary Structure of Proteins.

    PubMed

    Avinash, M B; Govindaraju, T

    2013-02-21

    Serendipitously, we found that isoleucine methylester functionalized perylenediimide 1 undergoes an extremely slow supramolecular helical assembly over a day's time. Surprisingly, heating led to irreversible chiral denaturation. However, reversible helical assembly could be achieved only in the presence of nondenatured aggregates of 1, which act as seeds. The intriguing functional relevance deduced from 1 was employed to draw parallels with the secondary structure of proteins, envisaging its plausible implications.

  11. A generalized threading model using integer programming that allows for secondary structure element deletion.

    PubMed

    Ellrott, Kyle; Guo, Jun-tao; Olman, Victor; Xu, Ying

    2006-01-01

    Integer programming is a combinatorial optimization method that has been successfully applied to the protein threading problem. We seek to expand the model optimized by this technique to allow for a more accurate description of protein threading. We have developed and implemented an expanded model of integer programming that has the capability to model secondary structure element deletion, which was not possible in previous version of integer programming based optimization. PMID:17503397

  12. TMPyP4, a Stabilizer of Nucleic Acid Secondary Structure, Is a Novel Acetylcholinesterase Inhibitor

    PubMed Central

    Fujiwara, Nana; Mazzola, Michael; Cai, Elizabeth; Wang, Meng; Cave, John W.

    2015-01-01

    The porphyrin compound, TMPyP4 (5,10,15,20-Tetrakis-(N-methyl-4-pyridyl)porphine), is widely used as a photosensitizer and a modulator of nucleic acid secondary structure stability. Our group recently showed in cultured cells and forebrain slice cultures that this compound can also down regulate expression of Tyrosine hydroxylase (Th), which encodes the rate-limiting enzyme in catecholamine biosynthesis, by stabilizing DNA secondary structures in the Th proximal promoter. The current study sought to establish whether treatment with TMPyP4 could modify mouse Th expression levels in vivo. Intraperitoneal administration of low TMPyP4 doses (10mg/kg), similar to those used for photosensitization, did not significantly reduce Th transcript levels in several catecholaminergic regions. Administration of a high dose (40 mg/kg), similar to those used for tumor xenograph reduction, unexpectedly induced flaccid paralysis in an age and sex-dependent manner. In vitro analyses revealed that TMPyP4, but not putative metabolites, inhibited Acetylcholinesterase activity and pre-treatment of TMPyP4 with Hemeoxygenase-2 (HO-2) rescued Acetylcholinesterase function. Age-dependent differences in HO-2 expression levels may account for some of the variable in vivo effects of high TMPyP4 doses. Together, these studies indicate that only low doses of TMPyP4, such as those typically used for photosensitization, are well tolerated in vivo. Thus, despite its widespread use in vitro, TMPyP4 is not ideal for modifying neuronal gene expression in vivo by manipulating nucleic acid secondary structure stability, which highlights the need to identify more clinically suitable compounds that can modulate nucleic acid secondary structure and gene expression. PMID:26402367

  13. TMPyP4, a Stabilizer of Nucleic Acid Secondary Structure, Is a Novel Acetylcholinesterase Inhibitor.

    PubMed

    Fujiwara, Nana; Mazzola, Michael; Cai, Elizabeth; Wang, Meng; Cave, John W

    2015-01-01

    The porphyrin compound, TMPyP4 (5,10,15,20-Tetrakis-(N-methyl-4-pyridyl)porphine), is widely used as a photosensitizer and a modulator of nucleic acid secondary structure stability. Our group recently showed in cultured cells and forebrain slice cultures that this compound can also down regulate expression of Tyrosine hydroxylase (Th), which encodes the rate-limiting enzyme in catecholamine biosynthesis, by stabilizing DNA secondary structures in the Th proximal promoter. The current study sought to establish whether treatment with TMPyP4 could modify mouse Th expression levels in vivo. Intraperitoneal administration of low TMPyP4 doses (10mg/kg), similar to those used for photosensitization, did not significantly reduce Th transcript levels in several catecholaminergic regions. Administration of a high dose (40 mg/kg), similar to those used for tumor xenograph reduction, unexpectedly induced flaccid paralysis in an age and sex-dependent manner. In vitro analyses revealed that TMPyP4, but not putative metabolites, inhibited Acetylcholinesterase activity and pre-treatment of TMPyP4 with Hemeoxygenase-2 (HO-2) rescued Acetylcholinesterase function. Age-dependent differences in HO-2 expression levels may account for some of the variable in vivo effects of high TMPyP4 doses. Together, these studies indicate that only low doses of TMPyP4, such as those typically used for photosensitization, are well tolerated in vivo. Thus, despite its widespread use in vitro, TMPyP4 is not ideal for modifying neuronal gene expression in vivo by manipulating nucleic acid secondary structure stability, which highlights the need to identify more clinically suitable compounds that can modulate nucleic acid secondary structure and gene expression. PMID:26402367

  14. Secondary relaxation dynamics in rigid glass-forming molecular liquids with related structures

    NASA Astrophysics Data System (ADS)

    Li, Xiangqian; Wang, Meng; Liu, Riping; Ngai, Kia L.; Tian, Yongjun; Wang, Li-Min; Capaccioli, Simone

    2015-09-01

    The dielectric relaxation in three glass-forming molecular liquids, 1-methylindole (1MID), 5H-5-Methyl-6,7-dihydrocyclopentapyrazine (MDCP), and Quinaldine (QN) is studied focusing on the secondary relaxation and its relation to the structural α-relaxation. All three glass-formers are rigid and more or less planar molecules with related chemical structures but have dipoles of different strengths at different locations. A strong and fast secondary relaxation is detected in the dielectric spectra of 1MID, while no resolved β-relaxation is observed in MDCP and QN. If the observed secondary relaxation in 1MID is identified with the Johari-Goldstein (JG) β-relaxation, then apparently the relation between the α- and β-relaxation frequencies of 1MID is not in accord with the Coupling Model (CM). The possibility of the violation of the prediction in 1MID as due to either the formation of hydrogen-bond induced clusters or the involvement of intramolecular degree of freedom is ruled out. The violation is explained by the secondary relaxation originating from the in-plane rotation of the dipole located on the plane of the rigid molecule, contributing to dielectric loss at higher frequencies and more intense than the JG β-relaxation generated by the out-of-plane rotation. MDCP has smaller dipole moment located in the plane of the molecule; however, presence of the change of curvature of dielectric loss, ɛ″(f), at some frequency on the high-frequency flank of the α-relaxation reveals the JG β-relaxation in MDCP and which is in accord with the CM prediction. QN has as large an in-plane dipole moment as 1MID, and the absence of the resolved secondary relaxation is explained by the smaller coupling parameter than the latter in the framework of the CM.

  15. Secondary structure encodes a cooperative tertiary folding funnel in the Azoarcus ribozyme

    PubMed Central

    Mustoe, Anthony M.; Al-Hashimi, Hashim M.; Brooks, Charles L.

    2016-01-01

    A requirement for specific RNA folding is that the free-energy landscape discriminate against non-native folds. While tertiary interactions are critical for stabilizing the native fold, they are relatively non-specific, suggesting additional mechanisms contribute to tertiary folding specificity. In this study, we use coarse-grained molecular dynamics simulations to explore how secondary structure shapes the tertiary free-energy landscape of the Azoarcus ribozyme. We show that steric and connectivity constraints posed by secondary structure strongly limit the accessible conformational space of the ribozyme, and that these so-called topological constraints in turn pose strong free-energy penalties on forming different tertiary contacts. Notably, native A-minor and base-triple interactions form with low conformational free energy, while non-native tetraloop/tetraloop–receptor interactions are penalized by high conformational free energies. Topological constraints also give rise to strong cooperativity between distal tertiary interactions, quantitatively matching prior experimental measurements. The specificity of the folding landscape is further enhanced as tertiary contacts place additional constraints on the conformational space, progressively funneling the molecule to the native state. These results indicate that secondary structure assists the ribozyme in navigating the otherwise rugged tertiary folding landscape, and further emphasize topological constraints as a key force in RNA folding. PMID:26481360

  16. The constant region affects antigen binding of antibodies to DNA by altering secondary structure.

    PubMed

    Xia, Yumin; Janda, Alena; Eryilmaz, Ertan; Casadevall, Arturo; Putterman, Chaim

    2013-11-01

    We previously demonstrated an important role of the constant region in the pathogenicity of anti-DNA antibodies. To determine the mechanisms by which the constant region affects autoantibody binding, a panel of isotype-switch variants (IgG1, IgG2a, IgG2b) was generated from the murine PL9-11 IgG3 autoantibody. The affinity of the PL9-11 antibody panel for histone was measured by surface plasmon resonance (SPR). Tryptophan fluorescence was used to determine wavelength shifts of the antibody panel upon binding to DNA and histone. Finally, circular dichroism spectroscopy was used to measure changes in secondary structure. SPR analysis revealed significant differences in histone binding affinity between members of the PL9-11 panel. The wavelength shifts of tryptophan fluorescence emission were found to be dependent on the antibody isotype, while circular dichroism analysis determined that changes in antibody secondary structure content differed between isotypes upon antigen binding. Thus, the antigen binding affinity is dependent on the particular constant region expressed. Moreover, the effects of antibody binding to antigen were also constant region dependent. Alteration of secondary structures influenced by constant regions may explain differences in fine specificity of anti-DNA antibodies between antibodies with similar variable regions, as well as cross-reactivity of anti-DNA antibodies with non-DNA antigens.

  17. CPU-GPU hybrid accelerating the Zuker algorithm for RNA secondary structure prediction applications

    PubMed Central

    2012-01-01

    Background Prediction of ribonucleic acid (RNA) secondary structure remains one of the most important research areas in bioinformatics. The Zuker algorithm is one of the most popular methods of free energy minimization for RNA secondary structure prediction. Thus far, few studies have been reported on the acceleration of the Zuker algorithm on general-purpose processors or on extra accelerators such as Field Programmable Gate-Array (FPGA) and Graphics Processing Units (GPU). To the best of our knowledge, no implementation combines both CPU and extra accelerators, such as GPUs, to accelerate the Zuker algorithm applications. Results In this paper, a CPU-GPU hybrid computing system that accelerates Zuker algorithm applications for RNA secondary structure prediction is proposed. The computing tasks are allocated between CPU and GPU for parallel cooperate execution. Performance differences between the CPU and the GPU in the task-allocation scheme are considered to obtain workload balance. To improve the hybrid system performance, the Zuker algorithm is optimally implemented with special methods for CPU and GPU architecture. Conclusions Speedup of 15.93× over optimized multi-core SIMD CPU implementation and performance advantage of 16% over optimized GPU implementation are shown in the experimental results. More than 14% of the sequences are executed on CPU in the hybrid system. The system combining CPU and GPU to accelerate the Zuker algorithm is proven to be promising and can be applied to other bioinformatics applications. PMID:22369626

  18. Mechanical properties of amyloid-like fibrils defined by secondary structures.

    PubMed

    Bortolini, C; Jones, N C; Hoffmann, S V; Wang, C; Besenbacher, F; Dong, M

    2015-05-01

    Amyloid and amyloid-like fibrils represent a generic class of highly ordered nanostructures that are implicated in some of the most fatal neurodegenerative diseases. On the other hand, amyloids, by possessing outstanding mechanical robustness, have also been successfully employed as functional biomaterials. For these reasons, physical and chemical factors driving fibril self-assembly and morphology are extensively studied - among these parameters, the secondary structures and the pH have been revealed to be crucial, since a variation in pH changes the fibril morphology and net chirality during protein aggregation. It is important to quantify the mechanical properties of these fibrils in order to help the design of effective strategies for treating diseases related to the presence of amyloid fibrils. In this work, we show that by changing pH the mechanical properties of amyloid-like fibrils vary as well. In particular, we reveal that these mechanical properties are strongly related to the content of secondary structures. We analysed and estimated the Young's modulus (E) by comparing the persistence length (Lp) - measured from the observation of TEM images by using statistical mechanics arguments - with the mechanical information provided by peak force quantitative nanomechanical property mapping (PF-QNM). The secondary structure content and the chirality are investigated by means of synchrotron radiation circular dichroism (SR-CD). Results arising from this study could be fruitfully used as a protocol to investigate other medical or engineering relevant peptide fibrils. PMID:25839069

  19. Compensatory evolution of a precursor messenger RNA secondary structure in the Drosophila melanogaster Adh gene

    PubMed Central

    Chen, Ying; Stephan, Wolfgang

    2003-01-01

    Evidence for the evolutionary maintenance of a hairpin structure possibly involved in intron processing had been found in intron 1 of the alcohol dehydrogenase gene (Adh) in diverse Drosophila species. In this study, the putative hairpin structure was evaluated systematically in Drosophila melanogaster by elimination of either side of the stem using site-directed mutagenesis. The effects of these mutations and the compensatory double mutant on intron splicing efficiency and ADH protein production were assayed in Drosophila melanogaster Schneider L2 cells and germ-line transformed adult flies. Mutations that disrupt the putative hairpin structure right upstream of the intron branch point were found to cause a significant reduction in both splicing efficiency and ADH protein production. In contrast, the compensatory double mutant that restores the putative hairpin structure was indistinguishable from the WT in both splicing efficiency and ADH level. It was also observed by mutational analysis that a more stable secondary structure (with a longer stem) in this intron decreases both splicing efficiency and ADH protein production. Implications for RNA secondary structure and intron evolution are discussed. PMID:12972637

  20. Secondary structure of protamine in sperm nuclei: an infrared spectroscopy study

    PubMed Central

    2011-01-01

    Background Protamines are small basic proteins that condense the DNA in mature spermatozoa. Typical protamines are of simple composition and very arginine-rich, usually in the range of 60-80%. Arginine residues are distributed in a number of stretches separated by neutral amino acids. We have used Fourier transform infrared spectroscopy (FTIR) to gain access for the first time to the secondary structure of protamines in sperm nuclei. This technique is particularly well suited to the study of DNA-bound protamine in whole nuclei since it is not affected by turbidity. Results We show that DNA -bound salmon (salmine) and squid protamines contain α-helix, β-turns and a proportion of other structures not stabilized by intramolecular hydrogen bonding. No β-sheet was observed. In salmine, the α-helix amounted to ~20%, while in squid protamine it reached ~40%. In contrast, the structure not stabilized by intermolecular hydrogen bonding was more abundant in salmine (~40%) than in squid protamine (~20%). Both protamines contained ~40% β-turns. The different helical potential of salmine and squid protamine was confirmed by structure predictions and CD in the presence of trifluoroethanol. Conclusion DNA-bound protamine in sperm nuclei contains large amounts of defined secondary structure stabilized by intramolecular hydrogen bonding. Both salmine and squid protamine contain similar amounts of β-turns, but differ in the proportions of α-helix and non-hydrogen bonded conformations. In spite of the large differences in the proportions of secondary structure motifs between salmon and squid protamines, they appear to be equally efficient in promoting tight hexagonal packing of the DNA molecules in sperm nuclei. PMID:21435240

  1. Sheath structure in plasma with two species of positive ions and secondary electrons

    NASA Astrophysics Data System (ADS)

    Xiao-Yun, Zhao; Nong, Xiang; Jing, Ou; De-Hui, Li; Bin-Bin, Lin

    2016-02-01

    The properties of a collisionless plasma sheath are investigated by using a fluid model in which two species of positive ions and secondary electrons are taken into account. It is shown that the positive ion speeds at the sheath edge increase with secondary electron emission (SEE) coefficient, and the sheath structure is affected by the interplay between the two species of positive ions and secondary electrons. The critical SEE coefficients and the sheath widths depend strongly on the positive ion charge number, mass and concentration in the cases with and without SEE. In addition, ion kinetic energy flux to the wall and the impact of positive ion species on secondary electron density at the sheath edge are also discussed. Project supported by the National Natural Science Foundation of China (Grant Nos. 11475220 and 11405208), the Program of Fusion Reactor Physics and Digital Tokamak with the CAS “One-Three-Five” Strategic Planning, the National ITER Program of China (Grant No. 2015GB101003), and the Higher Education Natural Science Research Project of Anhui Province, China (Grant No. 2015KJ009).

  2. Using probe secondary structure information to enhance Affymetrix GeneChip background estimates

    PubMed Central

    Gharaibeh, Raad Z.; Fodor, Anthony A.; Gibas, Cynthia J.

    2007-01-01

    High-density short oligonucleotide microarrays are a primary research tool for assessing global gene expression. Background noise on microarrays comprises a significant portion of the measured raw data. A number of statistical techniques have been developed to correct for this background noise. Here, we demonstrate that probe minimum folding energy and structure can be used to enhance a previously existing model for background noise correction. We estimate that probe secondary structure accounts for up to 3% of all variation on Affymetrix microarrays. PMID:17387043

  3. Lysophosphatidic acid can support the formation of membranous structures and an increase in MBP mRNA levels in differentiating oligodendrocytes

    PubMed Central

    Nogaroli, Luciana; Yuelling, Larra M.; Dennis, Jameel; Gorse, Karen; Payne, Shawn G.; Fuss, Babette

    2009-01-01

    During development, differentiating oligodendrocytes progress in distinct maturation steps from premyelinating to myelinating cells. Such maturing oligodendrocytes express both receptors mediating signaling via extracellular lysophosphatidic acid (LPA) and the major enzyme generating extracellular LPA, namely phosphodiesterase-Iα/autotaxin (PD-Iα/ATX). However, the biological role of extracellular LPA during the maturation of differentiating oligodendrocytes is currently unclear. Here, we demonstrate that application of exogenous LPA induced an increase in the area occupied by the oligodendrocytes’ process network, but only when PD-Iα/ATX expression was down-regulated. This increase in network area was caused primarily by the formation of membranous structures. In addition, LPA increased the number of cells positive for myelin basic protein (MBP). This effect was associated by an increase in the mRNA levels coding for MBP but not myelin oligodendrocyte glycoprotein (MOG). Taken together, these data suggest that LPA may play a crucial role in regulating the later stages of oligodendrocyte maturation. PMID:18594965

  4. VMD-SS: A graphical user interface plug-in to calculate the protein secondary structure in VMD program

    PubMed Central

    Yahyavi, Masoumeh; Falsafi-Zadeh, Sajad; Karimi, Zahra; Kalatarian, Giti; Galehdari, Hamid

    2014-01-01

    The investigation on the types of secondary structure (SS) of a protein is important. The evolution of secondary structures during molecular dynamics simulations is a useful parameter to analyze protein structures. Therefore, it is of interest to describe VMD-SS (a software program) for the identification of secondary structure elements and its trajectories during simulation for known structures available at the Protein Data Bank (PDB). The program helps to calculate (1) percentage SS, (2) SS occurrence in each residue, (3) percentage SS during simulation, and (4) percentage residues in all SS types during simulation. The VMD-SS plug-in was designed using TCL script and stride to calculate secondary structure features. Availability The database is available for free at http://science.scu.ac.ir/HomePage.aspx?TabID=13755 PMID:25258493

  5. Molecular characterization and in situ mRNA localization of the neural recognition molecule J1-160/180: a modular structure similar to tenascin

    PubMed Central

    1993-01-01

    The oligodendrocyte-derived extracellular matrix glycoprotein J1- 160/180 is a recognition molecule expressed exclusively in the central nervous system. J1-160/180 has been shown to be adhesive for astrocytes and repellent towards neurons and growth cones. We report here the complete nucleotide sequence of J1-160/180 in the rat. The predicted amino acid sequence showed a structural architecture very similar to tenascin: a cysteine-rich amino terminal region is followed by 4.5 epidermal growth factor-like repeats, 9 fibronectin type III homologous repeats and a domain homologous to fibrinogen. Sequence comparison analysis revealed highest homology of rat J1-160/180 to mouse tenascin and chicken restrictin with a similarity of 66% and 85%, respectively. The J1-160/180-coding mRNA is derived from a single copy gene. Using the polymerase chain reaction we could show that two J1-160/180 isoforms are generated by alternative splicing of the sixth fibronectin type III homologous repeat. Localization of J1-160/180 mRNA by in situ hybridization in the cerebellum, hippocampus and olfactory bulb confirmed the expression of J1-160/180 by oligodendrocytes with a peak of transcription at 7-14 d after birth, indicating a functional role during myelination. In addition, J1-160/180-specific RNA was found in a small subset of neurons in all three structures of the CNS analyzed. These neurons continue to express J1-160/180 in the adult. PMID:7679676

  6. RNA folding with soft constraints: reconciliation of probing data and thermodynamic secondary structure prediction

    PubMed Central

    Washietl, Stefan; Hofacker, Ivo L.; Stadler, Peter F.; Kellis, Manolis

    2012-01-01

    Thermodynamic folding algorithms and structure probing experiments are commonly used to determine the secondary structure of RNAs. Here we propose a formal framework to reconcile information from both prediction algorithms and probing experiments. The thermodynamic energy parameters are adjusted using ‘pseudo-energies’ to minimize the discrepancy between prediction and experiment. Our framework differs from related approaches that used pseudo-energies in several key aspects. (i) The energy model is only changed when necessary and no adjustments are made if prediction and experiment are consistent. (ii) Pseudo-energies remain biophysically interpretable and hold positional information where experiment and model disagree. (iii) The whole thermodynamic ensemble of structures is considered thus allowing to reconstruct mixtures of suboptimal structures from seemingly contradicting data. (iv) The noise of the energy model and the experimental data is explicitly modeled leading to an intuitive weighting factor through which the problem can be seen as folding with ‘soft’ constraints of different strength. We present an efficient algorithm to iteratively calculate pseudo-energies within this framework and demonstrate how this approach can be used in combination with SHAPE chemical probing data to improve secondary structure prediction. We further demonstrate that the pseudo-energies correlate with biophysical effects that are known to affect RNA folding such as chemical nucleotide modifications and protein binding. PMID:22287623

  7. A novel secondary structure based on fused five-membered rings motif

    PubMed Central

    Dhar, Jesmita; Kishore, Raghuvansh; Chakrabarti, Pinak

    2016-01-01

    An analysis of protein structures indicates the existence of a novel, fused five-membered rings motif, comprising of two residues (i and i + 1), stabilized by interresidue Ni+1–H∙∙∙Ni and intraresidue Ni+1–H∙∙∙O=Ci+1 hydrogen bonds. Fused-rings geometry is the common thread running through many commonly occurring motifs, such as β-turn, β-bulge, Asx-turn, Ser/Thr-turn, Schellman motif, and points to its structural robustness. A location close to the beginning of a β-strand is rather common for the motif. Devoid of side chain, Gly seems to be a key player in this motif, occurring at i, for which the backbone torsion angles cluster at ~(−90°, −10°) and (70°, 20°). The fused-rings structures, distant from each other in sequence, can hydrogen bond with each other, and the two segments aligned to each other in a parallel fashion, give rise to a novel secondary structure, topi, which is quite common in proteins, distinct from two major secondary structures, α-helix and β-sheet. Majority of the peptide segments making topi are identified as aggregation-prone and the residues tend to be conserved among homologous proteins. PMID:27511362

  8. Prediction of Spontaneous Protein Deamidation from Sequence-Derived Secondary Structure and Intrinsic Disorder

    PubMed Central

    Lorenzo, J. Ramiro; Alonso, Leonardo G.; Sánchez, Ignacio E.

    2015-01-01

    Asparagine residues in proteins undergo spontaneous deamidation, a post-translational modification that may act as a molecular clock for the regulation of protein function and turnover. Asparagine deamidation is modulated by protein local sequence, secondary structure and hydrogen bonding. We present NGOME, an algorithm able to predict non-enzymatic deamidation of internal asparagine residues in proteins in the absence of structural data, using sequence-based predictions of secondary structure and intrinsic disorder. Compared to previous algorithms, NGOME does not require three-dimensional structures yet yields better predictions than available sequence-only methods. Four case studies of specific proteins show how NGOME may help the user identify deamidation-prone asparagine residues, often related to protein gain of function, protein degradation or protein misfolding in pathological processes. A fifth case study applies NGOME at a proteomic scale and unveils a correlation between asparagine deamidation and protein degradation in yeast. NGOME is freely available as a webserver at the National EMBnet node Argentina, URL: http://www.embnet.qb.fcen.uba.ar/ in the subpage “Protein and nucleic acid structure and sequence analysis”. PMID:26674530

  9. Prediction of Spontaneous Protein Deamidation from Sequence-Derived Secondary Structure and Intrinsic Disorder.

    PubMed

    Lorenzo, J Ramiro; Alonso, Leonardo G; Sánchez, Ignacio E

    2015-01-01

    Asparagine residues in proteins undergo spontaneous deamidation, a post-translational modification that may act as a molecular clock for the regulation of protein function and turnover. Asparagine deamidation is modulated by protein local sequence, secondary structure and hydrogen bonding. We present NGOME, an algorithm able to predict non-enzymatic deamidation of internal asparagine residues in proteins in the absence of structural data, using sequence-based predictions of secondary structure and intrinsic disorder. Compared to previous algorithms, NGOME does not require three-dimensional structures yet yields better predictions than available sequence-only methods. Four case studies of specific proteins show how NGOME may help the user identify deamidation-prone asparagine residues, often related to protein gain of function, protein degradation or protein misfolding in pathological processes. A fifth case study applies NGOME at a proteomic scale and unveils a correlation between asparagine deamidation and protein degradation in yeast. NGOME is freely available as a webserver at the National EMBnet node Argentina, URL: http://www.embnet.qb.fcen.uba.ar/ in the subpage "Protein and nucleic acid structure and sequence analysis".

  10. Mitochondrial RNase P RNAs in ascomycete fungi: lineage-specific variations in RNA secondary structure.

    PubMed

    Seif, Elias R; Forget, Lise; Martin, Nancy C; Lang, B Franz

    2003-09-01

    The RNA subunit of mitochondrial RNase P (mtP-RNA) is encoded by a mitochondrial gene (rnpB) in several ascomycete fungi and in the protists Reclinomonas americana and Nephroselmis olivacea. By searching for universally conserved structural elements, we have identified previously unknown rnpB genes in the mitochondrial DNAs (mtDNAs) of two fission yeasts, Schizosaccharomyces pombe and Schizosaccharomyces octosporus; in the budding yeast Pichia canadensis; and in the archiascomycete Taphrina deformans. The expression of mtP-RNAs of the predicted size was experimentally confirmed in the two fission yeasts, and their precise 5' and 3' ends were determined by sequencing of cDNAs generated from circularized mtP-RNAs. Comparative RNA secondary structure modeling shows that in contrast to mtP-RNAs of the two protists R. americana and N. olivacea, those of ascomycete fungi all have highly reduced secondary structures. In certain budding yeasts, such as Saccharomycopsis fibuligera, we find only the two most conserved pairings, P1 and P4. A P18 pairing is conserved in Saccharomyces cerevisiae and its close relatives, whereas nearly half of the minimum bacterial consensus structure is retained in the RNAs of fission yeasts, Aspergillus nidulans and Taphrina deformans. The evolutionary implications of the reduction of mtP-RNA structures in ascomycetes will be discussed.

  11. Structural basis for the regulatory function of a complex zinc-binding domain in a replicative arterivirus helicase resembling a nonsense-mediated mRNA decay helicase

    PubMed Central

    Deng, Zengqin; Lehmann, Kathleen C.; Li, Xiaorong; Feng, Chong; Wang, Guoqiang; Zhang, Qi; Qi, Xiaoxuan; Yu, Lin; Zhang, Xingliang; Feng, Wenhai; Wu, Wei; Gong, Peng; Tao, Ye; Posthuma, Clara C.; Snijder, Eric J.; Gorbalenya, Alexander E.; Chen, Zhongzhou

    2014-01-01

    All positive-stranded RNA viruses with genomes >∼7 kb encode helicases, which generally are poorly characterized. The core of the nidovirus superfamily 1 helicase (HEL1) is associated with a unique N-terminal zinc-binding domain (ZBD) that was previously implicated in helicase regulation, genome replication and subgenomic mRNA synthesis. The high-resolution structure of the arterivirus helicase (nsp10), alone and in complex with a polynucleotide substrate, now provides first insights into the structural basis for nidovirus helicase function. A previously uncharacterized domain 1B connects HEL1 domains 1A and 2A to a long linker of ZBD, which further consists of a novel RING-like module and treble-clef zinc finger, together coordinating three Zn atoms. On substrate binding, major conformational changes were evident outside the HEL1 domains, notably in domain 1B. Structural characterization, mutagenesis and biochemistry revealed that helicase activity depends on the extensive relay of interactions between the ZBD and HEL1 domains. The arterivirus helicase structurally resembles the cellular Upf1 helicase, suggesting that nidoviruses may also use their helicases for post-transcriptional quality control of their large RNA genomes. PMID:24369429

  12. Solution structure of a purine rich hexaloop hairpin belonging to PGY/MDR1 mRNA and targeted by antisense oligonucleotides

    PubMed Central

    Joli, Flore; Bouchemal, Nadia; Laigle, Alain; Hartmann, Brigitte; Hantz, Edith

    2006-01-01

    A preferential target of antisense oligonucleotides directed against human PGY/MDR1 mRNA is a hairpin containing a stem with a G•U wobble pair, capped by the purine-rich 5′r(GGGAUG)3′ hexaloop. This hairpin is studied by multidimensional NMR and restrained molecular dynamics, with special emphasis on the conformation of south sugars and non-standard phosphate linkages evidenced in both the stem and the loop. The hairpin is found to be highly structured. The G•U wobble pair, a strong counterion binding site, displays structural particularities that are characteristic of this type of mismatch. The upper part of the stem undergoes distortions that optimize its interactions with the beginning of the loop. The loop adopts a new fold in which the single-stranded GGGA purine tract is structured in A-like conformation stacked in continuity of the stem and displays an extensive hydrogen bonding surface for recognition. The remarkable hairpin stability results from classical inter- and intra-strand interactions reinforced by numerous hydrogen bonds involving unusual backbone conformations and ribose 2′-hydroxyl groups. Overall, this work emphasizes numerous features that account for the well-ordered structure of the whole hairpin and highlights the loop properties that facilitate interaction with antisense oligonucleotides. PMID:17041234

  13. RNA secondary structure prediction by centroids in a Boltzmann weighted ensemble.

    PubMed

    Ding, Ye; Chan, Chi Yu; Lawrence, Charles E

    2005-08-01

    Prediction of RNA secondary structure by free energy minimization has been the standard for over two decades. Here we describe a novel method that forsakes this paradigm for predictions based on Boltzmann-weighted structure ensemble. We introduce the notion of a centroid structure as a representative for a set of structures and describe a procedure for its identification. In comparison with the minimum free energy (MFE) structure using diverse types of structural RNAs, the centroid of the ensemble makes 30.0% fewer prediction errors as measured by the positive predictive value (PPV) with marginally improved sensitivity. The Boltzmann ensemble can be separated into a small number (3.2 on average) of clusters. Among the centroids of these clusters, the "best cluster centroid" as determined by comparison to the known structure simultaneously improves PPV by 46.5% and sensitivity by 21.7%. For 58% of the studied sequences for which the MFE structure is outside the cluster containing the best centroid, the improvements by the best centroid are 62.5% for PPV and 31.4% for sensitivity. These results suggest that the energy well containing the MFE structure under the current incomplete energy model is often different from the one for the unavailable complete model that presumably contains the unique native structure. Centroids are available on the Sfold server at http://sfold.wadsworth.org.

  14. RNA Secondary Structure Modulates FMRP's Bi-Functional Role in the MicroRNA Pathway.

    PubMed

    Kenny, Phillip; Ceman, Stephanie

    2016-01-01

    MicroRNAs act by post-transcriptionally regulating the gene expression of 30%-60% of mammalian genomes. MicroRNAs are key regulators in all cellular processes, though the mechanism by which the cell activates or represses microRNA-mediated translational regulation is poorly understood. In this review, we discuss the RNA binding protein Fragile X Mental Retardation Protein (FMRP) and its role in microRNA-mediated translational regulation. Historically, FMRP is known to function as a translational suppressor. However, emerging data suggests that FMRP has both an agonistic and antagonistic role in regulating microRNA-mediated translational suppression. This bi-functional role is dependent on FMRP's interaction with the RNA helicase Moloney leukemia virus 10 (MOV10), which modifies the structural landscape of bound mRNA, therefore facilitating or inhibiting its association with the RNA-Induced Silencing Complex. PMID:27338369

  15. Perturbation-induced secondary flow structures due to fractured stents in arterial curvatures

    NASA Astrophysics Data System (ADS)

    Bulusu, Kartik V.; Popma, Christopher; Penna, Leanne; Plesniak, Michael W.

    2012-11-01

    An in vitro experimental investigation of secondary flow structures was performed downstream of a model stent that embodied a ``Type-IV'' stent fracture, i.e. complete transverse fracture of elements and element displacement (of 3 diameters). One part of the fractured stent was located in the curved region of a test section comprised of a 180-degree bent tube, and the velocity field measured with PIV. Secondary flow morphologies downstream of the stent were identified with a continuous wavelet transform (CWT) algorithm (PIVlet 1.2) using a 2D Ricker wavelet. A comparison of wavelet transformed vorticity fields of fractured and unfractured model stents is presented under physiological inflow conditions. During systolic deceleration, a breakdown in symmetry of vortical structures occurred with the unfractured stent, but not with the fractured model stent. Potential mechanisms to explain the differences in secondary flow morphologies include redirection of vorticity from the meridional plane of the bend to the normal plane and diffusion of vorticity. Supported by the National Science Foundation, Grant No. CBET-0828903 and GW Center for Biomimetics and Bioinspired Engineering (COBRE).

  16. Crystal Structure of the N-Terminal RNA Recognition Motif of mRNA Decay Regulator AUF1

    PubMed Central

    Choi, Young Jun

    2016-01-01

    AU-rich element binding/degradation factor 1 (AUF1) plays a role in destabilizing mRNAs by forming complexes with AU-rich elements (ARE) in the 3′-untranslated regions. Multiple AUF1-ARE complexes regulate the translation of encoded products related to the cell cycle, apoptosis, and inflammation. AUF1 contains two tandem RNA recognition motifs (RRM) and a Gln- (Q-) rich domain in their C-terminal region. To observe how the two RRMs are involved in recognizing ARE, we obtained the AUF1-p37 protein covering the two RRMs. However, only N-terminal RRM (RRM1) was crystallized and its structure was determined at 1.7 Å resolution. It appears that the RRM1 and RRM2 separated before crystallization. To demonstrate which factors affect the separate RRM1-2, we performed limited proteolysis using trypsin. The results indicated that the intact proteins were cleaved by unknown proteases that were associated with them prior to crystallization. In comparison with each of the monomers, the conformations of the β2-β3 loops were highly variable. Furthermore, a comparison with the RRM1-2 structures of HuR and hnRNP A1 revealed that a dimer of RRM1 could be one of the possible conformations of RRM1-2. Our data may provide a guidance for further structural investigations of AUF1 tandem RRM repeat and its mode of ARE binding. PMID:27437398

  17. Crystal Structure of the N-Terminal RNA Recognition Motif of mRNA Decay Regulator AUF1.

    PubMed

    Choi, Young Jun; Yoon, Je-Hyun; Chang, Jeong Ho

    2016-01-01

    AU-rich element binding/degradation factor 1 (AUF1) plays a role in destabilizing mRNAs by forming complexes with AU-rich elements (ARE) in the 3'-untranslated regions. Multiple AUF1-ARE complexes regulate the translation of encoded products related to the cell cycle, apoptosis, and inflammation. AUF1 contains two tandem RNA recognition motifs (RRM) and a Gln- (Q-) rich domain in their C-terminal region. To observe how the two RRMs are involved in recognizing ARE, we obtained the AUF1-p37 protein covering the two RRMs. However, only N-terminal RRM (RRM1) was crystallized and its structure was determined at 1.7 Å resolution. It appears that the RRM1 and RRM2 separated before crystallization. To demonstrate which factors affect the separate RRM1-2, we performed limited proteolysis using trypsin. The results indicated that the intact proteins were cleaved by unknown proteases that were associated with them prior to crystallization. In comparison with each of the monomers, the conformations of the β2-β3 loops were highly variable. Furthermore, a comparison with the RRM1-2 structures of HuR and hnRNP A1 revealed that a dimer of RRM1 could be one of the possible conformations of RRM1-2. Our data may provide a guidance for further structural investigations of AUF1 tandem RRM repeat and its mode of ARE binding. PMID:27437398

  18. TT2NE: a novel algorithm to predict RNA secondary structures with pseudoknots

    PubMed Central

    Bon, Michaël; Orland, Henri

    2011-01-01

    We present TT2NE, a new algorithm to predict RNA secondary structures with pseudoknots. The method is based on a classification of RNA structures according to their topological genus. TT2NE is guaranteed to find the minimum free energy structure regardless of pseudoknot topology. This unique proficiency is obtained at the expense of the maximum length of sequences that can be treated, but comparison with state-of-the-art algorithms shows that TT2NE significantly improves the quality of predictions. Analysis of TT2NE's incorrect predictions sheds light on the need to study how sterical constraints limit the range of pseudoknotted structures that can be formed from a given sequence. An implementation of TT2NE on a public server can be found at http://ipht.cea.fr/rna/tt2ne.php. PMID:21593129

  19. The effect of structural disorder on the secondary electron emission of graphite

    NASA Astrophysics Data System (ADS)

    Gonzalez, L. A.; Larciprete, R.; Cimino, R.

    2016-09-01

    The dependance of the secondary electron yield (SEY) on the degree of crystallinity of graphite has been investigated during the amorphization of a highly oriented pyrolytic graphite (HOPG) samples by means of Ar+ bombardment. Photoemission and Raman spectroscopies were used to follow the structural damage while the SEY curves were measured from very low energies up to 1000 eV. We found that the increase of lattice defects lowers the contribution of the π electrons in the valence band and loss spectra and smears out the intense modulations in the low energy secondary electron yield (LE-SEY) curve. Raman spectroscopy results showed that ion induced lattice amorphization is confined in a near-surface layer. The evolution of SEY curves was observed with the progressive Ar+ dosage after crystal damage as due to the modification of the electronic transport properties within the damaged near surface layer.

  20. Structural effects of liana presence in secondary tropical dry forests using ground LiDAR

    NASA Astrophysics Data System (ADS)

    Sánchez-Azofeifa, A.; Portillo-Quintero, C.; Durán, S. M.

    2015-10-01

    Lianas, woody vines, are a key component of tropical forest because they may reduce carbon storage potential. Lianas are increasing in density and biomass in tropical forests, but it is unknown what the potential consequences of these increases are for forest dynamics. Lianas may proliferate in disturbed areas, such as regenerating forests, but little is known about the role of lianas in secondary succession. In this study, we evaluated the potential of the ground LiDAR to detect differences in the vertical structure of stands of different ages with and without lianas in tropical dry forests. Specifically, we used a terrestrial laser scanner called VEGNET to assess whether liana presence influences the vertical signature of stands of different ages, and whether successional trajectories as detected by the VEGNET could be altered by liana presence. We deployed the VEGNET ground LiDAR system in 15 secondary forests of different ages early (21 years old since land abandonment), intermediate (32-35 years old) and late stages (> 80 years old) with and without lianas. We compared laser-derived vegetation components such as Plant Area Index (PAI), plant area volume density (PAVD), and the radius of gyration (RG) across forest stands between liana and no-liana treatments. In general forest stands without lianas show a clearer distinction of vertical strata and the vertical height of accumulated PAVD. A significant increase of PAI was found from intermediate to late stages in stands without lianas, but in stands where lianas were present there was not a significant trend. This suggests that lianas may be influencing successional trajectories in secondary forests, and these effects can be captured by terrestrial laser scanners such as the VEGNET. This research contributes to estimate the potential effects of lianas in secondary dry forests and highlight the role of ground LiDAR to monitor structural changes in tropical forests due to liana presence.

  1. In situ protein secondary structure determination in ice: Raman spectroscopy-based process analytical tool for frozen storage of biopharmaceuticals.

    PubMed

    Roessl, Ulrich; Leitgeb, Stefan; Pieters, Sigrid; De Beer, Thomas; Nidetzky, Bernd

    2014-08-01

    A Raman spectroscopy-based method for in situ monitoring of secondary structural composition of proteins during frozen and thawed storage was developed. A set of reference proteins with different α-helix and β-sheet compositions was used for calibration and validation in a chemometric approach. Reference secondary structures were quantified with circular dichroism spectroscopy in the liquid state. Partial least squares regression models were established that enable estimation of secondary structure content from Raman spectra. Quantitative secondary structure determination in ice was accomplished for the first time and correlation with existing (qualitative) protein structural data from the frozen state was achieved. The method can be used in the presence of common stabilizing agents and is applicable in an industrial freezer setup. Raman spectroscopy represents a powerful, noninvasive, and flexibly applicable tool for protein stability monitoring during frozen storage.

  2. Incorporating chemical modification constraints into a dynamic programming algorithm for prediction of RNA secondary structure

    PubMed Central

    Mathews, David H.; Disney, Matthew D.; Childs, Jessica L.; Schroeder, Susan J.; Zuker, Michael; Turner, Douglas H.

    2004-01-01

    A dynamic programming algorithm for prediction of RNA secondary structure has been revised to accommodate folding constraints determined by chemical modification and to include free energy increments for coaxial stacking of helices when they are either adjacent or separated by a single mismatch. Furthermore, free energy parameters are revised to account for recent experimental results for terminal mismatches and hairpin, bulge, internal, and multibranch loops. To demonstrate the applicability of this method, in vivo modification was performed on 5S rRNA in both Escherichia coli and Candida albicans with 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-p-toluene sulfonate, dimethyl sulfate, and kethoxal. The percentage of known base pairs in the predicted structure increased from 26.3% to 86.8% for the E. coli sequence by using modification constraints. For C. albicans, the accuracy remained 87.5% both with and without modification data. On average, for these sequences and a set of 14 sequences with known secondary structure and chemical modification data taken from the literature, accuracy improves from 67% to 76%. This enhancement primarily reflects improvement for three sequences that are predicted with <40% accuracy on the basis of energetics alone. For these sequences, inclusion of chemical modification constraints improves the average accuracy from 28% to 78%. For the 11 sequences with <6% pseudoknotted base pairs, structures predicted with constraints from chemical modification contain on average 84% of known canonical base pairs. PMID:15123812

  3. Importance of the RNA secondary structure for the relative accumulation of clustered viral microRNAs

    PubMed Central

    Contrant, Maud; Fender, Aurélie; Chane-Woon-Ming, Béatrice; Randrianjafy, Ramy; Vivet-Boudou, Valérie; Richer, Delphine; Pfeffer, Sébastien

    2014-01-01

    Micro (mi)RNAs are small non-coding RNAs with key regulatory functions. Recent advances in the field allowed researchers to identify their targets. However, much less is known regarding the regulation of miRNAs themselves. The accumulation of these tiny regulators can be modulated at various levels during their biogenesis from the transcription of the primary transcript (pri-miRNA) to the stability of the mature miRNA. Here, we studied the importance of the pri-miRNA secondary structure for the regulation of mature miRNA accumulation. To this end, we used the Kaposi's sarcoma herpesvirus, which encodes a cluster of 12 pre-miRNAs. Using small RNA profiling and quantitative northern blot analysis, we measured the absolute amount of each mature miRNAs in different cellular context. We found that the difference in expression between the least and most expressed viral miRNAs could be as high as 60-fold. Using high-throughput selective 2′-hydroxyl acylation analyzed by primer extension, we then determined the secondary structure of the long primary transcript. We found that highly expressed miRNAs derived from optimally structured regions within the pri-miRNA. Finally, we confirmed the importance of the local structure by swapping stem-loops or by targeted mutagenesis of selected miRNAs, which resulted in a perturbed accumulation of the mature miRNA. PMID:24831544

  4. Sequence-specific polypeptoids: A diverse family of heteropolymers with stable secondary structure

    PubMed Central

    Kirshenbaum, Kent; Barron, Annelise E.; Goldsmith, Richard A.; Armand, Philippe; Bradley, Erin K.; Truong, Kiet T. V.; Dill, Ken A.; Cohen, Fred E.; Zuckermann, Ronald N.

    1998-01-01

    We have synthesized and characterized a family of structured oligo-N-substituted-glycines (peptoids) up to 36 residues in length by using an efficient solid-phase protocol to incorporate chemically diverse side chains in a sequence-specific fashion. We investigated polypeptoids containing side chains with a chiral center adjacent to the main chain nitrogen. Some of these sequences have stable secondary structure, despite the achirality of the polymer backbone and its lack of hydrogen bond donors. In both aqueous and organic solvents, peptoid oligomers as short as five residues give rise to CD spectra that strongly resemble those of peptide α-helices. Differential scanning calorimetry and CD measurements show that polypeptoid secondary structure is highly stable and that unfolding is reversible and cooperative. Thermodynamic parameters obtained for unfolding are similar to those obtained for the α-helix to coil transitions of peptides. This class of biomimetic polymers may enable the design of self-assembling macromolecules with novel structures and functions. PMID:9539732

  5. The RNAmute web server for the mutational analysis of RNA secondary structures.

    PubMed

    Churkin, Alexander; Gabdank, Idan; Barash, Danny

    2011-07-01

    RNA mutational analysis at the secondary-structure level can be useful to a wide-range of biological applications. It can be used to predict an optimal site for performing a nucleotide mutation at the single molecular level, as well as to analyze basic phenomena at the systems level. For the former, as more sequence modification experiments are performed that include site-directed mutagenesis to find and explore functional motifs in RNAs, a pre-processing step that helps guide in planning the experiment becomes vital. For the latter, mutations are generally accepted as a central mechanism by which evolution occurs, and mutational analysis relating to structure should gain a better understanding of system functionality and evolution. In the past several years, the program RNAmute that is structure based and relies on RNA secondary-structure prediction has been developed for assisting in RNA mutational analysis. It has been extended from single-point mutations to treat multiple-point mutations efficiently by initially calculating all suboptimal solutions, after which only the mutations that stabilize the suboptimal solutions and destabilize the optimal one are considered as candidates for being deleterious. The RNAmute web server for mutational analysis is available at http://www.cs.bgu.ac.il/~xrnamute/XRNAmute.

  6. Effective stiffness and formation of secondary structures in a protein-like model

    NASA Astrophysics Data System (ADS)

    Škrbić, Tatjana; Hoang, Trinh X.; Giacometti, Achille

    2016-08-01

    We use Wang-Landau and replica exchange techniques to study the effect of an increasing stiffness on the formation of secondary structures in protein-like systems. Two possible models are considered. In both models, a polymer chain is formed by tethered beads where non-consecutive backbone beads attract each other via a square-well potential representing the tendency of the chain to fold. In addition, smaller hard spheres are attached to each non-terminal backbone bead along the direction normal to the chain to mimic the steric hindrance of side chains in real proteins. The two models, however, differ in the way bending rigidity is enforced. In the first model, partial overlap between consecutive beads is allowed. This reduces the possible bending angle between consecutive bonds thus producing an effective entropic stiffness that competes with a short-range attraction, and leads to the formation of secondary structures characteristic of proteins. We discuss the low-temperature phase diagram as a function of increasing interpenetration and find a transition from a planar, beta-like structure, to helical shape. In the second model, an energetic stiffness is explicitly introduced by imposing an infinitely large energy penalty for bending above a critical angle between consecutive bonds, and no penalty below it. The low-temperature phase of this model does not show any sign of protein-like secondary structures. At intermediate temperatures, however, where the chain is still in the coil conformation but stiffness is significant, we find the two models to predict a quite similar dependence of the persistence length as a function of the stiffness. This behaviour is rationalized in terms of a simple geometrical mapping between the two models. Finally, we discuss the effect of shrinking side chains to zero and find the above mapping to still hold true.

  7. Effective stiffness and formation of secondary structures in a protein-like model.

    PubMed

    Škrbić, Tatjana; Hoang, Trinh X; Giacometti, Achille

    2016-08-28

    We use Wang-Landau and replica exchange techniques to study the effect of an increasing stiffness on the formation of secondary structures in protein-like systems. Two possible models are considered. In both models, a polymer chain is formed by tethered beads where non-consecutive backbone beads attract each other via a square-well potential representing the tendency of the chain to fold. In addition, smaller hard spheres are attached to each non-terminal backbone bead along the direction normal to the chain to mimic the steric hindrance of side chains in real proteins. The two models, however, differ in the way bending rigidity is enforced. In the first model, partial overlap between consecutive beads is allowed. This reduces the possible bending angle between consecutive bonds thus producing an effective entropic stiffness that competes with a short-range attraction, and leads to the formation of secondary structures characteristic of proteins. We discuss the low-temperature phase diagram as a function of increasing interpenetration and find a transition from a planar, beta-like structure, to helical shape. In the second model, an energetic stiffness is explicitly introduced by imposing an infinitely large energy penalty for bending above a critical angle between consecutive bonds, and no penalty below it. The low-temperature phase of this model does not show any sign of protein-like secondary structures. At intermediate temperatures, however, where the chain is still in the coil conformation but stiffness is significant, we find the two models to predict a quite similar dependence of the persistence length as a function of the stiffness. This behaviour is rationalized in terms of a simple geometrical mapping between the two models. Finally, we discuss the effect of shrinking side chains to zero and find the above mapping to still hold true. PMID:27586943

  8. The Interplay between Adolescent Needs and Secondary School Structures: Fostering Developmentally Responsive Middle and High School Environments across the Transition

    ERIC Educational Resources Information Center

    Ellerbrock, Cheryl R.; Kiefer, Sarah M.

    2013-01-01

    Understanding the developmental responsiveness of secondary school environments may be an important factor in supporting students as they make the transition from one school to the next. Students' needs may or may not be met depending on the nature of the fit between their basic and developmental needs and secondary school structures at the…

  9. Prediction of RNA secondary structures: from theory to models and real molecules

    NASA Astrophysics Data System (ADS)

    Schuster, Peter

    2006-05-01

    RNA secondary structures are derived from RNA sequences, which are strings built form the natural four letter nucleotide alphabet, {AUGC}. These coarse-grained structures, in turn, are tantamount to constrained strings over a three letter alphabet. Hence, the secondary structures are discrete objects and the number of sequences always exceeds the number of structures. The sequences built from two letter alphabets form perfect structures when the nucleotides can form a base pair, as is the case with {GC} or {AU}, but the relation between the sequences and structures differs strongly from the four letter alphabet. A comprehensive theory of RNA structure is presented, which is based on the concepts of sequence space and shape space, being a space of structures. It sets the stage for modelling processes in ensembles of RNA molecules like evolutionary optimization or kinetic folding as dynamical phenomena guided by mappings between the two spaces. The number of minimum free energy (mfe) structures is always smaller than the number of sequences, even for two letter alphabets. Folding of RNA molecules into mfe energy structures constitutes a non-invertible mapping from sequence space onto shape space. The preimage of a structure in sequence space is defined as its neutral network. Similarly the set of suboptimal structures is the preimage of a sequence in shape space. This set represents the conformation space of a given sequence. The evolutionary optimization of structures in populations is a process taking place in sequence space, whereas kinetic folding occurs in molecular ensembles that optimize free energy in conformation space. Efficient folding algorithms based on dynamic programming are available for the prediction of secondary structures for given sequences. The inverse problem, the computation of sequences for predefined structures, is an important tool for the design of RNA molecules with tailored properties. Simultaneous folding or cofolding of two or more RNA

  10. Multithreaded comparative RNA secondary structure prediction using stochastic context-free grammars

    PubMed Central

    2011-01-01

    Background The prediction of the structure of large RNAs remains a particular challenge in bioinformatics, due to the computational complexity and low levels of accuracy of state-of-the-art algorithms. The pfold model couples a stochastic context-free grammar to phylogenetic analysis for a high accuracy in predictions, but the time complexity of the algorithm and underflow errors have prevented its use for long alignments. Here we present PPfold, a multithreaded version of pfold, which is capable of predicting the structure of large RNA alignments accurately on practical timescales. Results We have distributed both the phylogenetic calculations and the inside-outside algorithm in PPfold, resulting in a significant reduction of runtime on multicore machines. We have addressed the floating-point underflow problems of pfold by implementing an extended-exponent datatype, enabling PPfold to be used for large-scale RNA structure predictions. We have also improved the user interface and portability: alongside standalone executable and Java source code of the program, PPfold is also available as a free plugin to the CLC Workbenches. We have evaluated the accuracy of PPfold using BRaliBase I tests, and demonstrated its practical use by predicting the secondary structure of an alignment of 24 complete HIV-1 genomes in 65 minutes on an 8-core machine and identifying several known structural elements in the prediction. Conclusions PPfold is the first parallelized comparative RNA structure prediction algorithm to date. Based on the pfold model, PPfold is capable of fast, high-quality predictions of large RNA secondary structures, such as the genomes of RNA viruses or long genomic transcripts. The techniques used in the parallelization of this algorithm may be of general applicability to other bioinformatics algorithms. PMID:21501497

  11. Landscape and variation of RNA secondary structure across the human transcriptome.

    PubMed

    Wan, Yue; Qu, Kun; Zhang, Qiangfeng Cliff; Flynn, Ryan A; Manor, Ohad; Ouyang, Zhengqing; Zhang, Jiajing; Spitale, Robert C; Snyder, Michael P; Segal, Eran; Chang, Howard Y

    2014-01-30

    In parallel to the genetic code for protein synthesis, a second layer of information is embedded in all RNA transcripts in the form of RNA structure. RNA structure influences practically every step in the gene expression program. However, the nature of most RNA structures or effects of sequence variation on structure are not known. Here we report the initial landscape and variation of RNA secondary structures (RSSs) in a human family trio (mother, father and their child). This provides a comprehensive RSS map of human coding and non-coding RNAs. We identify unique RSS signatures that demarcate open reading frames and splicing junctions, and define authentic microRNA-binding sites. Comparison of native deproteinized RNA isolated from cells versus refolded purified RNA suggests that the majority of the RSS information is encoded within RNA sequence. Over 1,900 transcribed single nucleotide variants (approximately 15% of all transcribed single nucleotide variants) alter local RNA structure. We discover simple sequence and spacing rules that determine the ability of point mutations to impact RSSs. Selective depletion of 'riboSNitches' versus structurally synonymous variants at precise locations suggests selection for specific RNA shapes at thousands of sites, including 3' untranslated regions, binding sites of microRNAs and RNA-binding proteins genome-wide. These results highlight the potentially broad contribution of RNA structure and its variation to gene regulation.

  12. Mechanical properties of amyloid-like fibrils defined by secondary structures

    NASA Astrophysics Data System (ADS)

    Bortolini, C.; Jones, N. C.; Hoffmann, S. V.; Wang, C.; Besenbacher, F.; Dong, M.

    2015-04-01

    Amyloid and amyloid-like fibrils represent a generic class of highly ordered nanostructures that are implicated in some of the most fatal neurodegenerative diseases. On the other hand, amyloids, by possessing outstanding mechanical robustness, have also been successfully employed as functional biomaterials. For these reasons, physical and chemical factors driving fibril self-assembly and morphology are extensively studied - among these parameters, the secondary structures and the pH have been revealed to be crucial, since a variation in pH changes the fibril morphology and net chirality during protein aggregation. It is important to quantify the mechanical properties of these fibrils in order to help the design of effective strategies for treating diseases related to the presence of amyloid fibrils. In this work, we show that by changing pH the mechanical properties of amyloid-like fibrils vary as well. In particular, we reveal that these mechanical properties are strongly related to the content of secondary structures. We analysed and estimated the Young's modulus (E) by comparing the persistence length (Lp) - measured from the observation of TEM images by using statistical mechanics arguments - with the mechanical information provided by peak force quantitative nanomechanical property mapping (PF-QNM). The secondary structure content and the chirality are investigated by means of synchrotron radiation circular dichroism (SR-CD). Results arising from this study could be fruitfully used as a protocol to investigate other medical or engineering relevant peptide fibrils.Amyloid and amyloid-like fibrils represent a generic class of highly ordered nanostructures that are implicated in some of the most fatal neurodegenerative diseases. On the other hand, amyloids, by possessing outstanding mechanical robustness, have also been successfully employed as functional biomaterials. For these reasons, physical and chemical factors driving fibril self-assembly and morphology

  13. Understanding of Relation Structures of Graphical Models by Lower Secondary Students

    NASA Astrophysics Data System (ADS)

    van Buuren, Onne; Heck, André; Ellermeijer, Ton

    2016-10-01

    A learning path has been developed on system dynamical graphical modelling, integrated into the Dutch lower secondary physics curriculum. As part of the developmental research for this learning path, students' understanding of the relation structures shown in the diagrams of graphical system dynamics based models has been investigated. One of our main findings is that only some students understand these structures correctly. Reality-based interpretation of the diagrams can conceal an incorrect understanding of diagram structures. As a result, students seemingly have no problems interpreting the diagrams until they are asked to construct a graphical model. Misconceptions have been identified that are the consequence of the fact that the equations are not clearly communicated by the diagrams or because the icons used in the diagrams mislead novice modellers. Suggestions are made for improvements.

  14. [The effect of crystallization of calcium carbonate on the secondary structure of pepsin].

    PubMed

    Zhu, Shu-fa; Tang, Jun-ming; Ma, Xiao-ming; Guo, Yu-ming; Zhang, Xiu-ying; Yang, Lin

    2003-06-01

    The effect of crystallization of calcium carbonate on the secondary structure of pepsin was studied by Fourier transform infrared spectroscopy, derivative, deconvolution and curve-fitting techniques in this paper. The result shows that the pure protein is composed of 24.38% alpha-helices, 29.91% beta-sheets, 39.22% beta-turns and 6.49% random structures, and the pepsin in the CaCO3/pepsin solution is composed of 2.09% alpha-helices, 93.304% beta-sheets, 4.60% beta-turns and 0.006% random structures. From these data we can see that the alpha-helices decrease and the beta-turns increase with the formation of the crystal of calcium carbonate. The essence of these changes is discussed in the paper.

  15. Rigidity, Secondary Structure, and the Universality of the Boson Peak in Proteins

    PubMed Central

    Perticaroli, Stefania; Nickels, Jonathan D.; Ehlers, Georg; Sokolov, Alexei P.

    2014-01-01

    Complementary neutron- and light-scattering results on nine proteins and amino acids reveal the role of rigidity and secondary structure in determining the time- and lengthscales of low-frequency collective vibrational dynamics in proteins. These dynamics manifest in a spectral feature, known as the boson peak (BP), which is common to all disordered materials. We demonstrate that BP position scales systematically with structural motifs, reflecting local rigidity: disordered proteins appear softer than α-helical proteins; which are softer than β-sheet proteins. Our analysis also reveals a universal spectral shape of the BP in proteins and amino acid mixtures; superimposable on the shape observed in typical glasses. Uniformity in the underlying physical mechanism, independent of the specific chemical composition, connects the BP vibrations to nanometer-scale heterogeneities, providing an experimental benchmark for coarse-grained simulations, structure/rigidity relationships, and engineering of proteins for novel applications. PMID:24940784

  16. Neural-network design applied to protein-secondary-structure predictions

    SciTech Connect

    Yu, R.C.; Head-Gordon, T.

    1995-04-01

    The success of neural networks is often limited by a sparse database of training examples, deficient neural-network architectures, and nonglobal optimization of the network variables. The convolution of these three problems has curtailed the application of network models to protein-structure predictions, where homology modeling or information theory approaches are considered better controlled alternatives. This paper introduces our broad objective of disentangling the three degrading features of neural networks cited above, beginning with improved designs of network architectures used in the prediction of protein secondary structure. This work demonstrates that network architecture design considerations greatly improve generalization and more efficiently extract complex sequence-structure relationships from the existing database, as compared to arbitrary architectures with the same size input window.

  17. Sequence-specific sup 1 H NMR assignments and secondary structure of eglin c

    SciTech Connect

    Hyberts, S.G.; Wagner, G. )

    1990-02-13

    Sequence-specific nuclear magnetic resonance assignments were obtained for eglin c, a polypeptide inhibitor of the granulocytic proteinases elastase and cathepsin G and some other proteinases. The protein consists of a single polypeptide chain of 70 residues. All proton resonances were assigned except for some labile protons of arginine side chains. The patterns of nuclear Overhauser enhancements and coupling constants and the observation of slow hydrogen exchange were used to characterize the secondary structure of the protein. The results indicate that the solution structure of the free inhibitor is very similar to the crystal structure reported for the same protein in the complex with subtilisin Carlsberg. However, a part of the binding loop seems to have a significantly different conformation in the free protein.

  18. Proton NMR assignment and secondary structural elements of human transforming growth factor. alpha

    SciTech Connect

    Brown, S.C.; Mueller, L.; Jeffs, P.W. )

    1989-01-24

    The {sup 1}H NMR spectrum of human transforming growth factor {alpha} (hTGF-{alpha}) has been completely assigned, and secondary structural elements have been identified as a preliminary step in determining the structure of this protein by distance geometry methods. Many of these structural elements closely correspond to those previously found in a truncated human EGF and murine EGF. These include the presence of an antiparallel {beta}-sheet between residues G19 and C34 with a type I {beta}-turn at V25-D28, a type II {beta}-turn at H35-Y38, and another short {beta}-sheet between residues Y38-V39 and H45-A46.

  19. Pitch accent alignment in romance: primary and secondary associations with metrical structure.

    PubMed

    Prieto, Pilar; D'Imperio, Mariapaola; Fivela, Barbara Gili

    2005-01-01

    The article describes the contrastive possibilities of alignment of high accents in three Romance varieties, namely, Central Catalan, Neapolitan Italian, and Pisa Italian. The Romance languages analyzed in this article provide crucial evidence that small differences in alignment in rising accents should be encoded phonologically. To account for such facts within the AM model, the article develops the notion of "phonological anchoring" as an extension of the concept of secondary association originally proposed by Pierrehumbert and Beckman (1988), and later adopted by Grice (1995), Grice, Ladd, and Arvaniti (2000), and others to explain the behavior of edge tones. The Romance data represent evidence that not only peripheral edge tones seek secondary associations. We claim that the phonological representation of pitch accents should include two independent mechanisms to encode alignment properties with metrical structure: (1) encoding of the primary phonological association (or affiliation) between the tone and its tone-bearing unit; and (2), for some specific cases, encoding of the secondary phonological anchoring of tones to prosodic edges (moras, syllables, and prosodic words). The Romance data described in the article provide crucial evidence of mora-edge, syllable-edge, and word-edge H tonal associations.

  20. Interplay between desolvation and secondary structure in mediating cosolvent and temperature induced alpha-synuclein aggregation

    NASA Astrophysics Data System (ADS)

    Anderson, V. L.; Webb, W. W.; Eliezer, D.

    2012-10-01

    Both increased temperature and moderate concentrations of fluorinated alcohols enhance aggregation of the Parkinson's disease-associated protein α-synuclein (αS). Here, we investigate the secondary structural rearrangements induced by heating and trifluoroethanol [TFE]. At low TFE concentrations, CD spectra feature a negative peak characteristic of disordered polypeptides near 200 nm and a slight shoulder around 220 nm suggesting some polyproline-II content. Upon heating, these peaks weaken, while a weak negative signal develops at 222 nm. At high TFE concentrations, the spectra show distinct minima at 208 and 222 nm, indicative of considerable α-helical structure, which diminish upon heating. We observe a crossover between the low-TFE and high-TFE behavior near 15% TFE, where we previously showed that a partially helical intermediate is populated. We postulate that the protein is well solvated by water at low TFE concentrations and by TFE at high TFE concentrations, but may become desolvated at the crossover point. We discuss the potential roles and interplay of desolvation and helical secondary structure in driving αS aggregation.

  1. Protein Secondary Structure Prediction Using Local Adaptive Techniques in Training Neural Networks

    NASA Astrophysics Data System (ADS)

    Aik, Lim Eng; Zainuddin, Zarita; Joseph, Annie

    2008-01-01

    One of the most significant problems in computer molecular biology today is how to predict a protein's three-dimensional structure from its one-dimensional amino acid sequence or generally call the protein folding problem and difficult to determine the corresponding protein functions. Thus, this paper involves protein secondary structure prediction using neural network in order to solve the protein folding problem. The neural network used for protein secondary structure prediction is multilayer perceptron (MLP) of the feed-forward variety. The training set are taken from the protein data bank which are 120 proteins while 60 testing set is the proteins which were chosen randomly from the protein data bank. Multiple sequence alignment (MSA) is used to get the protein similar sequence and Position Specific Scoring matrix (PSSM) is used for network input. The training process of the neural network involves local adaptive techniques. Local adaptive techniques used in this paper comprises Learning rate by sign changes, SuperSAB, Quickprop and RPROP. From the simulation, the performance for learning rate by Rprop and Quickprop are superior to all other algorithms with respect to the convergence time. However, the best result was obtained using Rprop algorithm.

  2. Prediction algorithm for amino acid types with their secondary structure in proteins (PLATON) using chemical shifts.

    PubMed

    Labudde, D; Leitner, D; Krüger, M; Oschkinat, H

    2003-01-01

    The algorithm PLATON is able to assign sets of chemical shifts derived from a single residue to amino acid types with its secondary structure (amino acid species). A subsequent ranking procedure using optionally two different penalty functions yields predictions for possible amino acid species for the given set of chemical shifts. This was demonstrated in the case of the alpha-spectrin SH3 domain and applied to 9 further protein data sets taken from the BioMagRes database. A database consisting of reference chemical shift patterns (reference CSPs) was generated from assigned chemical shifts of proteins with known 3D-structure. This reference CSP database is used in our approach for extracting distributions of amino acid types with their most likely secondary structure elements (namely alpha-helix, beta-sheet, and coil) for single amino acids by comparison with query CSPs. Results obtained for the 10 investigated proteins indicates that the percentage of correct amino acid species in the first three positions in the ranking list, ranges from 71.4% to 93.2% for the more favorable penalty function. Where only the top result of the ranking list for these 10 proteins is considered, 36.5% to 83.1% of the amino acid species are correctly predicted. The main advantage of our approach, over other methods that rely on average chemical shift values is the ability to increase database content by incorporating newly derived CSPs, and therefore to improve PLATON's performance over time.

  3. Pan-eukaryote ITS2 homologies revealed by RNA secondary structure

    PubMed Central

    Coleman, Annette W.

    2007-01-01

    For evolutionary comparisons, phylogenetics and evaluation of potential interbreeding taxa of a species, various loci have served for animals and plants and protistans. One [second internal transcribed spacer (ITS2) of the nuclear ribosomal DNA] is highly suitable for all. Its sequence is species specific. It has already been used extensively and very successfully for plants and some protistans, and a few animals (where historically, the mitochondrial genes have dominated species studies). Despite initial impressions that ITS2 is too variable, it has proven to provide useful biological information at higher taxonomic levels, even across all eukaryotes, thanks to the conserved aspects of its transcript secondary structure. The review of all eukaryote groups reveals that ITS2 is expandable, but always retains in its RNA transcript a common core structure of two helices with hallmark characteristics important for ribosomal RNA processing. This aspect of its RNA transcript secondary structure can rescue difficult alignment problems, making the ITS2 a more powerful tool for phylogenetics. Equally important, the recognition of eukaryote-wide homology regions provides extensive and detailed information to test experimental studies of ribosomal rRNA processing. PMID:17459886

  4. A global sampling approach to designing and reengineering RNA secondary structures.

    PubMed

    Levin, Alex; Lis, Mieszko; Ponty, Yann; O'Donnell, Charles W; Devadas, Srinivas; Berger, Bonnie; Waldispühl, Jérôme

    2012-11-01

    The development of algorithms for designing artificial RNA sequences that fold into specific secondary structures has many potential biomedical and synthetic biology applications. To date, this problem remains computationally difficult, and current strategies to address it resort to heuristics and stochastic search techniques. The most popular methods consist of two steps: First a random seed sequence is generated; next, this seed is progressively modified (i.e. mutated) to adopt the desired folding properties. Although computationally inexpensive, this approach raises several questions such as (i) the influence of the seed; and (ii) the efficiency of single-path directed searches that may be affected by energy barriers in the mutational landscape. In this article, we present RNA-ensign, a novel paradigm for RNA design. Instead of taking a progressive adaptive walk driven by local search criteria, we use an efficient global sampling algorithm to examine large regions of the mutational landscape under structural and thermodynamical constraints until a solution is found. When considering the influence of the seeds and the target secondary structures, our results show that, compared to single-path directed searches, our approach is more robust, succeeds more often and generates more thermodynamically stable sequences. An ensemble approach to RNA design is thus well worth pursuing as a complement to existing approaches. RNA-ensign is available at http://csb.cs.mcgill.ca/RNAensign. PMID:22941632

  5. Effects of high hydrostatic pressure on secondary structure and emulsifying behavior of sweet potato protein

    NASA Astrophysics Data System (ADS)

    Mehmood Khan, Nasir; Mu, Tai-Hua; Sun, Hong-Nan; Zhang, Miao; Chen, Jing-Wang

    2015-04-01

    In this study, secondary structures of sweet potato protein (SPP) after high hydrostatic pressure (HHP) treatment (200-600 MPa) were evaluated and emulsifying properties of emulsions with HHP-treated SPP solutions in different pH values (3, 6, and 9) were investigated. Circular dichroism analysis confirmed the modification of the SPP secondary structure. Surface hydrophobicity increased at pH 3 and decreased at 6 and 9. Emulsifying activity index at pH 6 increased with an increase in pressure, whereas emulsifying stability index increased at pH 6 and 9. Oil droplet sizes decreased, while volume frequency distribution of the smaller droplets increased at pH 3 and 6 with the HHP treatment. Emulsion viscosity increased at pH 6 and 9 and pseudo-plastic flow behaviors were not altered for all emulsions produced with HHP-treated SPP. These results suggested that HHP could modify the SPP structure for better emulsifying properties, which could increase the use of SPP emulsion in the food industry.

  6. Molecular topography and secondary structure comparisons of botulinum neurotoxin types A, B and E.

    PubMed

    Singh, B R; DasGupta, B R

    1989-03-16

    Botulinum neurotoxin (NT) serotypes A, B and E differ in microstructure and biological activities. The three NTs were examined for secondary structure parameters (alpha-helix, beta-sheet, beta-turn and random coil content) on the basis of circular dichroism; degree of exposed Tyr residues (second derivative spectroscopy) and state of the Trp residues (fluorescence and fluorescence quantum yield). The proteins are high in beta-pleated sheet content (41-44%) and low in alpha-helical content (21-28%). About 30-36% of the amino acids are in random coils. The beta-sheet contents in the NTs are similar irrespective of their structural forms (i.e. single or dichain forms) or level of toxicity. About 84%, 58% and 61% of Tyr residues of types A, B, and E NT, respectively, were exposed to the solvent (pH 7.2 phosphate buffer). Although the fluorescence emission maximum of Trp residues of type B NT was most blue shifted (331 nm compared to 334 for types A and E NT, and 346 nm for free tryptophan) the fluorescence quantum yields of types A and B were similar and higher than type E. In general the NTs have similar secondary (low alpha-helix and high beta-sheets) and tertiary (exposed tyrosine residues and tryptophan fluorescence quantum yield) structures. Within this generalized picture there are significant differences which might be related to the differences in their biological activities.

  7. Secondary Structure of Corona Proteins Determines the Cell Surface Receptors Used by Nanoparticles

    PubMed Central

    2015-01-01

    Nanoparticles used for biological and biomedical applications encounter a host of extracellular proteins. These proteins rapidly adsorb onto the nanoparticle surface, creating a protein corona. Poly(ethylene glycol) can reduce, but not eliminate, the nonspecific adsorption of proteins. As a result, the adsorbed proteins, rather than the nanoparticle itself, determine the cellular receptors used for binding, the internalization mechanism, the intracellular transport pathway, and the subsequent immune response. Using fluorescence microscopy and flow cytometry, we first characterize a set of polystyrene nanoparticles in which the same adsorbed protein, bovine serum albumin, leads to binding to two different cell surface receptors: native albumin receptors and scavenger receptors. Using a combination of circular dichroism spectroscopy, isothermal titration calorimetry, and fluorescence spectroscopy, we demonstrate that the secondary structure of the adsorbed bovine serum albumin protein controls the cellular receptors used by the protein–nanoparticle complexes. These results show that protein secondary structure is a key parameter in determining the cell surface receptor used by a protein–nanoparticle complex. We expect this link between protein structure and cellular outcomes will provide a molecular basis for the design of nanoparticles for use in biological and biomedical applications. PMID:24779411

  8. Nonrandom gene organization: structural arrangements of specific pre- mRNA transcription and splicing with SC-35 domains

    PubMed Central

    1995-01-01

    This work demonstrates a highly nonrandom distribution of specific genes relative to nuclear domains enriched in splicing factors and poly(A)+ RNA, and provides evidence for the direct involvement of these in pre-mRNA metabolism. As investigated in hundreds of diploid fibroblasts, human collagen I alpha 1 and beta-actin DNA/RNA showed a very high degree of spatial association with SC-35 domains, whereas three nontranscribed genes, myosin heavy chain, neurotensin, and albumin, showed no such preferential association. Collagen I alpha 1 RNA accumulates within the more central region of the domain, whereas beta-actin RNA localizes at the periphery. A novel approach revealed that collagen RNA tracks are polarized, with the entire gene at one end, on the edge of the domain, and the RNA extending into the domain. Intron 26 is spliced within the RNA track at the domain periphery. Transcriptional inhibition studies show both the structure of the domain and the gene's relationship to it are not dependent upon the continued presence of accumulated collagen RNA, and that domains remaining after inhibition are not just storage sites. Results support a model reconciling light and electron microscopic observations which proposes that transcription of some specific genes occurs at the border of domains, which may also function in the assembly or distribution of RNA metabolic components. In contrast to the apparently random dispersal of total undefined hnRNA synthesis through interdomain space, transcription and splicing for some genes occurs preferentially at specific sites, and a high degree of individual pre-mRNA metabolism is compartmentalized with discrete SC-35 domains. PMID:8557734

  9. Comparative structure and biomechanics of plant primary and secondary cell walls.

    PubMed

    Cosgrove, Daniel J; Jarvis, Michael C

    2012-01-01

    Recent insights into the physical biology of plant cell walls are reviewed, summarizing the essential differences between primary and secondary cell walls and identifying crucial gaps in our knowledge of their structure and biomechanics. Unexpected parallels are identified between the mechanism of expansion of primary cell walls during growth and the mechanisms by which hydrated wood deforms under external tension. There is a particular need to revise current "cartoons" of plant cell walls to be more consistent with data from diverse approaches and to go beyond summarizing limited aspects of cell walls, serving instead as guides for future experiments and for the application of new techniques.

  10. Monte Carlo simulation of secondary electron images for real sample structures in scanning electron microscopy.

    PubMed

    Zhang, P; Wang, H Y; Li, Y G; Mao, S F; Ding, Z J

    2012-01-01

    Monte Carlo simulation methods for the study of electron beam interaction with solids have been mostly concerned with specimens of simple geometry. In this article, we propose a simulation algorithm for treating arbitrary complex structures in a real sample. The method is based on a finite element triangular mesh modeling of sample geometry and a space subdivision for accelerating simulation. Simulation of secondary electron image in scanning electron microscopy has been performed for gold particles on a carbon substrate. Comparison of the simulation result with an experiment image confirms that this method is effective to model complex morphology of a real sample.

  11. Comparative structure and biomechanics of plant primary and secondary cell walls

    PubMed Central

    Cosgrove, Daniel J.; Jarvis, Michael C.

    2012-01-01

    Recent insights into the physical biology of plant cell walls are reviewed, summarizing the essential differences between primary and secondary cell walls and identifying crucial gaps in our knowledge of their structure and biomechanics. Unexpected parallels are identified between the mechanism of expansion of primary cell walls during growth and the mechanisms by which hydrated wood deforms under external tension. There is a particular need to revise current “cartoons” of plant cell walls to be more consistent with data from diverse approaches and to go beyond summarizing limited aspects of cell walls, serving instead as guides for future experiments and for the application of new techniques. PMID:22936943

  12. Identify five kinds of simple super-secondary structures with quadratic discriminant algorithm based on the chemical shifts.

    PubMed

    Kou, Gaoshan; Feng, Yonge

    2015-09-01

    The biological function of protein is largely determined by its spatial structure. The research on the relationship between structure and function is the basis of protein structure prediction. However, the prediction of super secondary structure is an important step in the prediction of protein spatial structure. Many algorithms have been proposed for the prediction of protein super secondary structure. However, the parameters used by these methods were primarily based on amino acid sequences. In this paper, we proposed a novel model for predicting five kinds of protein super secondary structures based on the chemical shifts (CSs). Firstly, we analyzed the statistical distribution of chemical shifts of six nuclei in five kinds of protein super secondary structures by using the analysis of variance (ANOVA). Secondly, we used chemical shifts of six nuclei as features, and combined with quadratic discriminant analysis (QDA) to predict five kinds of protein super secondary structures. Finally, we achieved the averaged sensitivity, specificity and the overall accuracy of 81.8%, 95.19%, 82.91%, respectively in seven-fold cross-validation. Moreover, we have performed the prediction by combining the five different chemical shifts as features, the maximum overall accuracy up to 89.87% by using the C,Cα,Cβ,N,Hα of Hα chemical shifts, which are clearly superior to that of the quadratic discriminant analysis (QDA) algorithm by using 20 amino acid compositions (AAC) as feature in the seven-fold cross-validation. These results demonstrated that chemical shifts (CSs) are indeed an outstanding parameter for the prediction of five kinds of super secondary structures. In addition, we compared the prediction of the quadratic discriminant analysis (QDA) with that of support vector machine (SVM) by using the same six CSs as features. The result suggested that the quadratic discriminant analysis method by using chemical shifts as features is a good predictor for protein super

  13. GraphClust: alignment-free structural clustering of local RNA secondary structures

    PubMed Central

    Rose, Dominic; Backofen, Rolf

    2012-01-01

    Motivation: Clustering according to sequence–structure similarity has now become a generally accepted scheme for ncRNA annotation. Its application to complete genomic sequences as well as whole transcriptomes is therefore desirable but hindered by extremely high computational costs. Results: We present a novel linear-time, alignment-free method for comparing and clustering RNAs according to sequence and structure. The approach scales to datasets of hundreds of thousands of sequences. The quality of the retrieved clusters has been benchmarked against known ncRNA datasets and is comparable to state-of-the-art sequence–structure methods although achieving speedups of several orders of magnitude. A selection of applications aiming at the detection of novel structural ncRNAs are presented. Exemplarily, we predicted local structural elements specific to lincRNAs likely functionally associating involved transcripts to vital processes of the human nervous system. In total, we predicted 349 local structural RNA elements. Availability: The GraphClust pipeline is available on request. Contact: backofen@informatik.uni-freiburg.de Supplementary information: Supplementary data are available at Bioinformatics online. PMID:22689765

  14. Formation of C-terminally truncated version of the Taz1 protein employs cleavage-box structure in mRNA

    SciTech Connect

    Gunisova, Stanislava; Bartosova, Zdenka; Kramara, Juraj; Nosek, Jozef; Tomaska, Lubomir

    2010-02-12

    When expressed in various hosts the taz1{sup +} gene encoding the fission yeast telomere-binding protein produces two forms of polypeptides: full-length (Taz1p) and truncated (Taz1p{Delta}C) version lacking almost entire Myb-domain. Whereas Taz1p binds telomeric DNA in vitro, Taz1p{Delta}C forms long filaments unable of DNA binding. The formation of Taz1p{Delta}C is a result of neither site-specific proteolysis, nor premature termination of transcription. In silico analysis of the taz1{sup +} RNA transcript revealed a stem-loop structure at the site of cleavage (cleavage box; CB). In order to explore whether it possesses inherent destabilizing effects, we cloned CB sequence into the open reading frame (ORF) of glutathione-S-transferase (GST) and observed that when expressed in Escherichia coli the engineered gene produced two forms of the reporter protein. The formation of the truncated version of GST was abolished, when CB was replaced with recoded sequence containing synonymous codons thus indicating that the truncation is based on structural properties of taz1{sup +} mRNA.

  15. Identification of miRNA-Mediated Core Gene Module for Glioma Patient Prediction by Integrating High-Throughput miRNA, mRNA Expression and Pathway Structure

    PubMed Central

    Han, Junwei; Shang, Desi; Zhang, Yunpeng; Zhang, Wei; Yao, Qianlan; Han, Lei; Xu, Yanjun; Yan, Wei; Bao, Zhaoshi; You, Gan; Jiang, Tao; Kang, Chunsheng; Li, Xia

    2014-01-01

    The prognosis of glioma patients is usually poor, especially in patients with glioblastoma (World Health Organization (WHO) grade IV). The regulatory functions of microRNA (miRNA) on genes have important implications in glioma cell survival. However, there are not many studies that have investigated glioma survival by integrating miRNAs and genes while also considering pathway structure. In this study, we performed sample-matched miRNA and mRNA expression profilings to systematically analyze glioma patient survival. During this analytical process, we developed pathway-based random walk to identify a glioma core miRNA-gene module, simultaneously considering pathway structure information and multi-level involvement of miRNAs and genes. The core miRNA-gene module we identified was comprised of four apparent sub-modules; all four sub-modules displayed a significant correlation with patient survival in the testing set (P-values≤0.001). Notably, one sub-module that consisted of 6 miRNAs and 26 genes also correlated with survival time in the high-grade subgroup (WHO grade III and IV), P-value = 0.0062. Furthermore, the 26-gene expression signature from this sub-module had robust predictive power in four independent, publicly available glioma datasets. Our findings suggested that the expression signatures, which were identified by integration of miRNA and gene level, were closely associated with overall survival among the glioma patients with various grades. PMID:24809850

  16. Structures of the PIN domains of SMG6 and SMG5 reveal a nuclease within the mRNA surveillance complex

    PubMed Central

    Glavan, Filip; Behm-Ansmant, Isabelle; Izaurralde, Elisa; Conti, Elena

    2006-01-01

    SMG6 and SMG5 are essential factors in nonsense-mediated mRNA decay, a conserved pathway that degrades mRNAs with premature translation termination codons. Both SMG5 and SMG6 have been predicted to contain a C-terminal PIN (PilT N-terminus) domain, present in proteins with ribonuclease activity. We have determined the structures of human SMG5 and SMG6 PIN domains. Although they share a similar overall fold related to ribonucleases of the RNase H family, they have local differences at the putative active site. SMG6 has the canonical triad of acidic residues that are crucial in RNase H for nuclease activity, while SMG5 lacks key catalytic residues. The structural differences are reflected at the functional level. Only the PIN domain of SMG6 has degradation activity on single-stranded RNA in vitro. This difference in catalytic activity is conserved in Drosophila, where an SMG6 with an inactive PIN domain inhibits NMD in a dominant-negative manner. Our findings suggest that the NMD machinery has intrinsic nuclease activity that is likely to contribute to the rapid decay of mRNAs that terminate translation prematurely. PMID:17053788

  17. RNA-protein interactions and secondary structures of cowpea chlorotic mottle virus for in vitro assembly.

    PubMed

    Verduin, B J; Prescott, B; Thomas, G J

    1984-09-11

    Laser Raman spectroscopy of the cowpea chlorotic mottle virus (CCMV) in native (pH 5.0) and partially swollen (pH 7.5) states reveals the presence of small percentages of protonated adenine (less than 15%) and cytosine (less than 7%) bases in the encapsidated RNA molecule of the native virion. The protonated bases are titrated with pH-induced swelling of the virus. Titration of putative COOH groups of aspartic and glutamic side chains of the virion subunit cannot be detected over the same pH range, which suggests that carboxyl anions (CO-2) and protonated bases are both available at pH 5 to stabilize the ribonucleoprotein particles by electrostatic interactions. The highly (95%) ordered secondary structure of encapsidated RNA may undergo a small additional increase (less than 3%) in ordered structure with release from the virion, suggesting at most a marginal structure-distorting influence from protein contacts in the native particle. The Raman spectra of the virion are also compared by difference spectroscopy with spectra of capsids (empty shells devoid of RNA), subunit dimers, and protein-free RNA. The results indicate that the subunit structure is altered by the release of RNA from the virion, as well as by the swelling of the virion. Amino acid residues and protein secondary structures that are affected in these in vitro assembly and disassembly processes are identified from their characteristic Raman lines. Two classes of cysteinyl SH groups, solvent exposed and solvent protected, are revealed for the capsid and virion subunit.(ABSTRACT TRUNCATED AT 250 WORDS)

  18. Secondary structures of short peptide chains in the gas phase: Double resonance spectroscopy of protected dipeptides

    NASA Astrophysics Data System (ADS)

    Chin, Wutharath; Dognon, Jean-Pierre; Canuel, Clélia; Piuzzi, François; Dimicoli, Iliana; Mons, Michel; Compagnon, Isabelle; von Helden, Gert; Meijer, Gerard

    2005-02-01

    The conformational structure of short peptide chains in the gas phase is studied by laser spectroscopy of a series of protected dipeptides, Ac-Xxx-Phe-NH2, Xxx=Gly, Ala, and Val. The combination of laser desorption with supersonic expansion enables us to vaporize the peptide molecules and cool them internally; IR/UV double resonance spectroscopy in comparison to density functional theory calculations on Ac-Gly-Phe-NH2 permits us to identify and characterize the conformers populated in the supersonic expansion. Two main conformations, corresponding to secondary structures of proteins, are found to compete in the present experiments. One is composed of a doubly γ-fold corresponding to the 27 ribbon structure. Topologically, this motif is very close to a β-strand backbone conformation. The second conformation observed is the β-turn, responsible for the chain reversal in proteins. It is characterized by a relatively weak hydrogen bond linking remote NH and CO groups of the molecule and leading to a ten-membered ring. The present gas phase experiment illustrates the intrinsic folding properties of the peptide chain and the robustness of the β-turn structure, even in the absence of a solvent. The β-turn population is found to vary significantly with the residues within the sequence; the Ac-Val-Phe-NH2 peptide, with its two bulky side chains, exhibits the largest β-turn population. This suggests that the intrinsic stabilities of the 27 ribbon and the β-turn are very similar and that weakly polar interactions occurring between side chains can be a decisive factor capable of controlling the secondary structure.

  19. Secondary Airflow Structure around Clustered Shrubs and Its Significance for Vegetated Dune Evolution

    NASA Astrophysics Data System (ADS)

    Luo, Wanyin; Dong, Zhibao; Qian, Guangqiang; Lu, Junfeng

    2016-04-01

    Shrubs have an important significance in aeolian processes due to their disturbance of the local airflow. In the formation of vegetated dunes, there is an iterative interaction between shrub geometry, the structure of the secondary airflow, and the interaction between neighboring shrubs. Understanding the dynamics of vegetated dunes thus requires an insight into the airflow fields around shrubs. Based on aerodynamic and aeolian sand physics theory, this project measured the complex secondary flow field and aeolian sand deposition pattern around single and cluster shrubs with varied densities (i.e., 0.05, 0.08, 0.15, 0.20) and gap ratios (the ratio of the gap spacing between the shrub models to the center-to-center distance for the shrub models, ranged from 1.1 to 1.8 with side-by-side arrangement and 1.2 to 4.3 with tandem arrangement) using the particle image velocimetry system through wind tunnle simulation. The relationship between the secondary airflow structure and the shrub's porosity and arrangement was analyzed quantitatively. Research results revealed that porosity (density) is the key parameter to affect the flow patterns around single shrub. Compared to solid obstacles, bleed flow through the shrubs has great influence on the secondary airflow patterns around itself. Under cluster modes, the distance between two adjacent shrubs has great influence on flow field structures around them. The flow patterns around two side-by-side arranged shrubs can be classified into three kinds of modes, that is: single-bluff-body, biased flow pattern and parallel vortex streets. The flow patterns around two tandem arranged shrubs can be classified into three regimes, that is: the extended body regime, reattachment regime and co-shedding regime. The "shadow zone" with low velocity in the lee of shrubs is the optimal position for sand deposition, but its form, size and orientation would varied with the shrub porosity and gap ratio between them. With the increase of the gap

  20. Sequence-specific 1H assignment and secondary structure of the bacteriocin AS-48 cyclic peptide.

    PubMed

    Langdon, G M; Bruix, M; Gálvez, A; Valdivia, E; Maqueda, M; Rico, M

    1998-07-01

    The bacteriocin AS-48 is a cationic peptide (7149 Da) having a broad antimicrobial spectrum, encoded by the 68 kb conjugative plasmid pMB2 from Enterococcus faecalis S-48. It is a unique peptide since it has a cyclic structure, which is achieved by the formation of a tail-head peptide bond after ribosomal synthesis (Gálvez et al., 1989; Martínez-Bueno et al., 1994; Samyn et al., 1994). Preliminary CD and calorimetric studies (data not shown) pointed towards a highly helical and very stable three dimensional structure. All the information gathered until now indicates that the target of AS-48 is the cytoplasmic membrane in which it opens channels or pores, leading to dissipation of the proton motive force and cell death, which in some cases is also followed by bacterial lysis (Gálvez et al., 1991). This peptide is a suitable tool for studying protein-membrane interactions, and it also offers promising perspectives for biotechnological applications. Knowledge of the 3D structure of AS-48 is a first step in the conduct of further structure-function studies. Here we report the complete 1H NMR assignment of its proton resonances together with the resulting secondary structure pattern as prerequisites for the determination of a high-resolution 3D solution structure.

  1. Accounting for solvent accessibility and secondary structure in protein phylogenetics is clearly beneficial.

    PubMed

    Le, Si Quang; Gascuel, Olivier

    2010-05-01

    Amino acid substitution models are essential to most methods to infer phylogenies from protein data. These models represent the ways in which proteins evolve and substitutions accumulate along the course of time. It is widely accepted that the substitution processes vary depending on the structural configuration of the protein residues. However, this information is very rarely used in phylogenetic studies, though the 3-dimensional structure of dozens of thousands of proteins has been elucidated. Here, we reinvestigate the question in order to fill this gap. We use an improved estimation methodology and a very large database comprising 1471 nonredundant globular protein alignments with structural annotations to estimate new amino acid substitution models accounting for the secondary structure and solvent accessibility of the residues. These models incorporate a confidence coefficient that is estimated from the data and reflects the reliability and usefulness of structural annotations in the analyzed sequences. Our results with 300 independent test alignments show an impressive likelihood gain compared with standard models such as JTT or WAG. Moreover, the use of these models induces significant topological changes in the inferred trees, which should be of primary interest to phylogeneticists. Our data, models, and software are available for download from http://atgc.lirmm.fr/phyml-structure/.

  2. Ambient modal identification of a primary-secondary structure by Fast Bayesian FFT method

    NASA Astrophysics Data System (ADS)

    Au, Siu-Kui; Zhang, Feng-Liang

    2012-04-01

    The Mong Man Wai Building is a seven-storied reinforced concrete structure situated on the campus of the City University of Hong Kong. On its roof a two-storied steel frame has been recently constructed to host a new wind tunnel laboratory. The roof frame and the main building form a primary-secondary structure. The dynamic characteristics of the resulting system are of interest from a structural dynamics point of view. This paper presents work on modal identification of the structure using ambient vibration measurement. An array of tri-axial acceleration data has been obtained using a number of setups to cover all locations of interest with a limited number of sensors. Modal identification is performed using a recently developed Fast Bayesian FFT method. In addition to the most probable modal properties, their posterior uncertainties can also be assessed using the method. The posterior uncertainty of mode shape is assessed by the expected value of the Modal Assurance Criteria (MAC) of the most probable mode shape with a random mode shape consistent with the posterior distribution. The mode shapes of the overall structural system are obtained by assembling those from individual setups using a recently developed least-square method. The identification results reveal a number of interesting features about the structural system and provide important information defining the baseline modal properties of the building. Practical interpretation of the statistics of modal parameters calculated from frequentist and Bayesian context is also discussed.

  3. Membrane Association and Destabilization by Aggregatibacter actinomycetemcomitans Leukotoxin Requires Changes in Secondary Structures

    PubMed Central

    Walters, Michael J.; Brown, Angela C.; Edrington, Thomas C.; Baranwal, Somesh; Du, Yurong; Lally, Edward T.; Boesze-Battaglia, Kathleen

    2013-01-01

    SUMMARY Aggregatibacter actinomycetemcomitans is a common inhabitant of the upper aerodigestive tract of humans and non-human primates and is associated with disseminated infections, including lung and brain abscesses, pediatric infective endocarditis in children, and localized aggressive periodontitis. A. actinomycetemcomitans secretes a repeats-in-toxin protein, leukotoxin, which exclusively kills lymphocyte function-associated antigen-1-bearing cells. The toxin's pathological mechanism is not fully understood; however, experimental evidence indicates that it involves the association with and subsequent destabilization of the target cell's plasma membrane. We have long hypothesized that leukotoxin secondary structure is strongly correlated with membrane association and/or destabilization. In this study, we tested this hypothesis by analyzing lipid-induced changes in leukotoxin conformation. Upon incubation of leukotoxin with lipids that favor leukotoxin-membrane association, we observed an increase in leukotoxin α-helical content that was not observed with lipids that favor membrane destabilization. The change in leukotoxin conformation after incubation with these lipids suggests that membrane binding and membrane destabilization have distinct secondary structural requirements, suggesting that they are independent events. These studies thus provide insight into the mechanism of cell damage that leads to disease progression by A. actinomycetemcomitans. PMID:23678967

  4. Improving the prediction of secondary structure of 'TIM-barrel' enzymes.

    PubMed

    Niermann, T; Kirschner, K

    1991-02-01

    The information contained in aligned sets of homologous protein sequences should improve the score of secondary structure prediction. Seven different enzymes having the (beta/alpha)8 or TIM-barrel fold were used to optimize the prediction with regard to this class of enzymes. The alpha-helix, beta-strand and loop propensities of the Garnier-Osguthorpe-Robson method were averaged at aligned residue positions, leading to a significant improvement over the average score obtained from single sequences. The increased accuracy correlates with the average sequence variability of the aligned set. Further improvements were obtained by using the following averaged properties as weights for the averaged state propensities: amphipathic moment and alpha-helix; hydropathy and beta-strand; chain flexibility and loop. The clustering of conserved residues at the C-terminal ends of the beta-strands was used as an additional positive weight for beta-strand propensity and increased the prediction of otherwise unpredicted beta-strands decisively. The automatic weighted prediction method identifies greater than 95% of the secondary structure elements of the set of seven TIM-barrel enzymes.

  5. Secondary flow structures under simple harmonic inflow in a bent pipe model for curved arteries

    NASA Astrophysics Data System (ADS)

    Glenn, Autumn; Seagrave, Penelope; Shu, Fangjun; Bulusu, Kartik; Plesniak, Michael W.

    2010-11-01

    Inward centrifuging of fluid in the inviscid core of a 180 degree curved pipe leads to Lyne-type vortices under zero-mean harmonic oscillations, along with the formation of vortices in the Stokes' layer, that rotate in the same directional sense as their steady flow counterpart (Dean vortices). Under physiological conditions, the development of the Lyne-type vortices is believed to be influenced by the systolic pulse, and its associated rapid acceleration and deceleration. Experimental data acquired using Particle Image Velocimetry (PIV) for three harmonic waveforms of different frequencies clarify the conditions under which Lyne vortices form. Multiple vortex pairs were observed for all waveforms and frequencies investigated, including Dean and Lyne-type vortex structures at a Womersley number of 4.22, much lower than previously reported. Hence, frequency alone is not an adequate governing parameter to characterize secondary flow structures in pulsatile flows. A regime map of the secondary flow was sought by using an acceleration-based parameter and the Dean number.

  6. Transcriptome-wide interrogation of RNA secondary structure in living cells with icSHAPE

    PubMed Central

    Flynn, Ryan A; Zhang, Qiangfeng Cliff; Spitale, Robert C; Lee, Byron; Mumbach, Maxwell R; Chang, Howard Y

    2016-01-01

    icSHAPE (in vivo click selective 2-hydroxyl acylation and profiling experiment) captures RNA secondary structure at a transcriptome-wide level by measuring nucleotide flexibility at base resolution. Living cells are treated with the icSHAPE chemical NAI-N3 followed by selective chemical enrichment of NAI-N3–modified RNA, which provides an improved signal-to-noise ratio compared with similar methods leveraging deep sequencing. Purified RNA is then reverse-transcribed to produce cDNA, with SHAPE-modified bases leading to truncated cDNA. After deep sequencing of cDNA, computational analysis yields flexibility scores for every base across the starting RNA population. The entire experimental procedure can be completed in ~5 d, and the sequencing and bioinformatics data analysis take an additional 4–5 d with no extensive computational skills required. Comparing in vivo and in vitro icSHAPE measurements can reveal in vivo RNA-binding protein imprints or facilitate the dissection of RNA post-transcriptional modifications. icSHAPE reactivities can additionally be used to constrain and improve RNA secondary structure prediction models. PMID:26766114

  7. Transcriptome-wide interrogation of RNA secondary structure in living cells with icSHAPE.

    PubMed

    Flynn, Ryan A; Zhang, Qiangfeng Cliff; Spitale, Robert C; Lee, Byron; Mumbach, Maxwell R; Chang, Howard Y

    2016-02-01

    icSHAPE (in vivo click selective 2-hydroxyl acylation and profiling experiment) captures RNA secondary structure at a transcriptome-wide level by measuring nucleotide flexibility at base resolution. Living cells are treated with the icSHAPE chemical NAI-N3 followed by selective chemical enrichment of NAI-N3-modified RNA, which provides an improved signal-to-noise ratio compared with similar methods leveraging deep sequencing. Purified RNA is then reverse-transcribed to produce cDNA, with SHAPE-modified bases leading to truncated cDNA. After deep sequencing of cDNA, computational analysis yields flexibility scores for every base across the starting RNA population. The entire experimental procedure can be completed in ∼5 d, and the sequencing and bioinformatics data analysis take an additional 4-5 d with no extensive computational skills required. Comparing in vivo and in vitro icSHAPE measurements can reveal in vivo RNA-binding protein imprints or facilitate the dissection of RNA post-transcriptional modifications. icSHAPE reactivities can additionally be used to constrain and improve RNA secondary structure prediction models.

  8. 18S rRNA secondary structure and phylogenetic position of Peloridiidae (Insecta, hemiptera).

    PubMed

    Ouvrard, D; Campbell, B C; Bourgoin, T; Chan, K L

    2000-09-01

    A secondary structure model for 18S rRNA of peloridiids, relict insects with a present-day circumantarctic distribution, is constructed using comparative sequence analysis, thermodynamic folding, a consensus method using 18S rRNA models of other taxa, and support of helices based on compensatory substitutions. Results show that probable in vivo configuration of 18S rRNA is not predictable using current free-energy models to fold the entire molecule concurrently. This suggests that refinements in free-energy minimization algorithms are needed. Molecular phylogenetic datasets were created using 18S rRNA nucleotide alignments produced by CLUSTAL and rigorous interpretation of homologous position based on certain secondary substructures. Phylogenetic analysis of a hemipteran data matrix of 18S rDNA sequences placed peloridiids sister to Heteroptera. Resolution of affiliations between the three main euhemipteran lineages was unresolved. The peloridiid 18S RNA model presented here provides the most accurate template to date for aligning homologous nucleotides of hemipteran taxa. Using folded 18S rRNA to infer homology of character as morpho-molecular structures or nucleotides and scoring particular sites or substructures is discussed. PMID:10991793

  9. Evolutionary conservation of sequence and secondary structures inCRISPR repeats

    SciTech Connect

    Kunin, Victor; Sorek, Rotem; Hugenholtz, Philip

    2006-09-01

    Clustered Regularly Interspaced Palindromic Repeats (CRISPRs) are a novel class of direct repeats, separated by unique spacer sequences of similar length, that are present in {approx}40% of bacterial and all archaeal genomes analyzed to date. More than 40 gene families, called CRISPR-associated sequences (CAS), appear in conjunction with these repeats and are thought to be involved in the propagation and functioning of CRISPRs. It has been proposed that the CRISPR/CAS system samples, maintains a record of, and inactivates invasive DNA that the cell has encountered, and therefore constitutes a prokaryotic analog of an immune system. Here we analyze CRISPR repeats identified in 195 microbial genomes and show that they can be organized into multiple clusters based on sequence similarity. All individual repeats in any given cluster were inferred to form characteristic RNA secondary structure, ranging from non-existent to pronounced. Stable secondary structures included G:U base pairs and exhibited multiple compensatory base changes in the stem region, indicating evolutionary conservation and functional importance. We also show that the repeat-based classification corresponds to, and expands upon, a previously reported CAS gene-based classification including specific relationships between CRISPR and CAS subtypes.

  10. Proton NMR assignments and secondary structure of the snake venom protein echistatin

    SciTech Connect

    Yuan Chen; Baum, J. ); Pitzenberger, S.M.; Garsky, V.M.; Lumma, P.K.; Sanyal, G. )

    1991-12-17

    The snake venom protein echistatin is a potent inhibitor of platelet aggregation. The inhibitory properties of echistatin have been attributed to the Arg-Gly-Asp sequence at residues 24-26. In this paper, sequence-specific nuclear magnetic resonance assignments are presented for the proton resonances of echistatin in water. The single-chain protein contains 49 amino acids and 4 cystine bridges. All of the backbone amide, C{sub alpha}H, and side-chain resonances, except for the {eta}-NH of the arginines, have been assigned. The secondary structure of the protein was characterized from the pattern of nuclear Overhauser enhancements, from the identification of slowly exchanging amide protons, from {sup 3}J{sub c{alpha}H-NH} coupling constants, and from circular dichroism studies. The data suggest that the secondary structure consists of a type I {beta}-turn, a short {beta}-hairpin, and a short-, irregular, antiparallel {beta}-sheet and that the Arg-Gly-Asp sequence is in a flexible loop connecting two strands of the distorted antiparallel {beta}-sheet.

  11. Direct RNA motif definition and identification from multiple sequence alignments using secondary structure profiles.

    PubMed

    Gautheret, D; Lambert, A

    2001-11-01

    We present here a new approach to the problem of defining RNA signatures and finding their occurrences in sequence databases. The proposed method is based on "secondary structure profiles". An RNA sequence alignment with secondary structure information is used as an input. Two types of weight matrices/profiles are constructed from this alignment: single strands are represented by a classical lod-scores profile while helical regions are represented by an extended "helical profile" comprising 16 lod-scores per position, one for each of the 16 possible base-pairs. Database searches are then conducted using a simultaneous search for helical profiles and dynamic programming alignment of single strand profiles. The algorithm has been implemented into a new software, ERPIN, that performs both profile construction and database search. Applications are presented for several RNA motifs. The automated use of sequence information in both single-stranded and helical regions yields better sensitivity/specificity ratios than descriptor-based programs. Furthermore, since the translation of alignments into profiles is straightforward with ERPIN, iterative searches can easily be conducted to enrich collections of homologous RNAs.

  12. A Tool Preference Choice Method for RNA Secondary Structure Prediction by SVM with Statistical Tests

    PubMed Central

    Hor, Chiou-Yi; Yang, Chang-Biau; Chang, Chia-Hung; Tseng, Chiou-Ting; Chen, Hung-Hsin

    2013-01-01

    The Prediction of RNA secondary structures has drawn much attention from both biologists and computer scientists. Many useful tools have been developed for this purpose. These tools have their individual strengths and weaknesses. As a result, based on support vector machines (SVM), we propose a tool choice method which integrates three prediction tools: pknotsRG, RNAStructure, and NUPACK. Our method first extracts features from the target RNA sequence, and adopts two information-theoretic feature selection methods for feature ranking. We propose a method to combine feature selection and classifier fusion in an incremental manner. Our test data set contains 720 RNA sequences, where 225 pseudoknotted RNA sequences are obtained from PseudoBase, and 495 nested RNA sequences are obtained from RNA SSTRAND. The method serves as a preprocessing way in analyzing RNA sequences before the RNA secondary structure prediction tools are employed. In addition, the performance of various configurations is subject to statistical tests to examine their significance. The best base-pair accuracy achieved is 75.5%, which is obtained by the proposed incremental method, and is significantly higher than 68.8%, which is associated with the best predictor, pknotsRG. PMID:23641141

  13. Cygnus Pressured Cargo Module: Validation of Mathematical Model and Dynamic Qualification of Secondary Structures

    NASA Astrophysics Data System (ADS)

    Bellini, Marina; Luison, Dario; Tizzani, Luca

    2012-07-01

    Thales Alenia Space Italy is in charge to develop build- up, integrate and verify Cygnus Pressurized Cargo Module (PCM). This cargo is characterized by the large amount of payload, wrapped in foam, transferred in soft stowage bags, connected to the structure of support by belts. The paper summarizes the several tests performed to acquire the dynamic properties of bags. On the basis of the empirical results a reliable linear model was generated and used for a successful campaign of qualification of secondary structure. It is also demonstrated that the empirical-linear model of the soft-stowage bag was also the reason of a significant reduction of loads, which allowed achieving more easily the goal of carried mass, for PCM. The validation of PCM by Modal Survey Test is presented as well, emphasizing that the more realistic modeling of the soft bags has made easier the definition of a very simple test configuration.

  14. In solution cation-induced secondary and tertiary structure alterations of human calprotectin.

    PubMed

    Imani, Mehdi; Bahrami, Yaser; Jaliani, Hossein Zarei; Ardestani, Sussan Kaboudanian

    2014-10-01

    Calprotectin (CP) is widely considered to have diverse roles including growth inhibitory and apoptosis induction in a number of tumor cell lines and antimicrobial activities. As CP has been proposed to bind metal ions with high affinity, we have studied its functional and primarily its structural behavior upon Zn(2+) and Mn(2+) chelation solely and along with Ca(2+). We employed fluorescence spectroscopy and circular dichroism to determine the resulting modifications. Based upon our findings it is clear that treating CP with ions effectively weakened its natural growth inhibitory activity. Moreover, structural analysis of Zn(2+) and Mn(2+)-treated CPs indicated remarkable alterations in the regular secondary structures in favor of irregular structures while Zn(2+) and Mn(2+) treatment of CP after incubation with Ca(2+) displayed no remarkable shifts. Tertiary structure investigation using fluorescence spectroscopy showed that CP undergoes conformational changes upon Zn(2+) and Mn(2+) treatment whereby Trp residues of protein is slightly exposed to the hydrophilic environment, compactness of CP is compromised, whereas in Ca(2+)-treated CP, the tertiary structure integrity is intact upon Zn(2+) and Mn(2+) chelation. Interestingly, CP structural modifications upon Zn(2+) and Mn(2+) treatment was significantly comparable, probably due to similar radii and charges of ions. Taken all together, we have concluded that CP maintains its normal nature in Ca(2+)-loaded state when treated with Zn(2+) and Mn(2+) ions. It can be suggested that Ca(2+) not only stabilize CP structure but also helps CP to keep its structure upon metal ions chelation which is involved in host organism defense system.

  15. Probing secondary structures of peptide chains using gas phase laser spectroscopy

    NASA Astrophysics Data System (ADS)

    Mons, Michel

    2006-03-01

    A bottom-up approach involving conformer-specific IR studies of short peptide sequences enables us to map the intramolecular interactions that shape the peptide backbone, in particular those H-bonds that are responsible for stability and formation of secondary structures in proteins, like turns or helices. The combination of laser-desorption of solid samples coupled to the efficient cooling in a supersonic expansion makes it possible to isolate in the gas phase the lowest conformations of the energy landscape of small flexible biomolecules. The low temperature achieved enables spectroscopists to record UV spectra in which the contribution of each conformer populated can be distinguished and the corresponding conformation identified using IR/UV double resonance spectroscopy. Data collected are directly comparable to the best quantum chemistry calculations on these species and therefore constitute a severe test for the theoretical methods used. It will be shown how investigation of sequences with an increasing number of building blocks permits to deduce the robust structural trends of a peptide backbone: i) local conformational preference of the backbone in one-residue chains, ii) in capped dipeptides, the competition between a succession of local conformational preferences and overall folded structures, in which a different type of H-bonding scheme, involving distant H-bonding sites along the backbone, takes place: in particular beta-turns, the secondary structure responsible for chain reversals, and finally iii) evidence for the spontaneous helical folding (short 3-10 helix) of three-residue chains will be presented, illustrating the relative weakness of the H-bonding in these molecular assemblies.

  16. Nucleotide sequence of a crustacean 18S ribosomal RNA gene and secondary structure of eukaryotic small subunit ribosomal RNAs.

    PubMed

    Nelles, L; Fang, B L; Volckaert, G; Vandenberghe, A; De Wachter, R

    1984-12-11

    The primary structure of the gene for 18 S rRNA of the crustacean Artemia salina was determined. The sequence has been aligned with 13 other small ribosomal subunit RNA sequences of eukaryotic, archaebacterial, eubacterial, chloroplastic and plant mitochondrial origin. Secondary structure models for these RNAs were derived on the basis of previously proposed models and additional comparative evidence found in the alignment. Although there is a general similarity in the secondary structure models for eukaryotes and prokaryotes, the evidence seems to indicate a different topology in a central area of the structures.

  17. Secondary Structure Prediction of Protein Constructs Using Random Incremental Truncation and Vacuum-Ultraviolet CD Spectroscopy.

    PubMed

    Pukáncsik, Mária; Orbán, Ágnes; Nagy, Kinga; Matsuo, Koichi; Gekko, Kunihiko; Maurin, Damien; Hart, Darren; Kézsmárki, István; Vertessy, Beata G

    2016-01-01

    A novel uracil-DNA degrading protein factor (termed UDE) was identified in Drosophila melanogaster with no significant structural and functional homology to other uracil-DNA binding or processing factors. Determination of the 3D structure of UDE is excepted to provide key information on the description of the molecular mechanism of action of UDE catalysis, as well as in general uracil-recognition and nuclease action. Towards this long-term aim, the random library ESPRIT technology was applied to the novel protein UDE to overcome problems in identifying soluble expressing constructs given the absence of precise information on domain content and arrangement. Nine constructs of UDE were chosen to decipher structural and functional relationships. Vacuum ultraviolet circular dichroism (VUVCD) spectroscopy was performed to define the secondary structure content and location within UDE and its truncated variants. The quantitative analysis demonstrated exclusive α-helical content for the full-length protein, which is preserved in the truncated constructs. Arrangement of α-helical bundles within the truncated protein segments suggested new domain boundaries which differ from the conserved motifs determined by sequence-based alignment of UDE homologues. Here we demonstrate that the combination of ESPRIT and VUVCD spectroscopy provides a new structural description of UDE and confirms that the truncated constructs are useful for further detailed functional studies. PMID:27273007

  18. Secondary Structure Prediction of Protein Constructs Using Random Incremental Truncation and Vacuum-Ultraviolet CD Spectroscopy

    PubMed Central

    Pukáncsik, Mária; Orbán, Ágnes; Nagy, Kinga; Matsuo, Koichi; Gekko, Kunihiko; Maurin, Damien; Hart, Darren; Kézsmárki, István; Vertessy, Beata G.

    2016-01-01

    A novel uracil-DNA degrading protein factor (termed UDE) was identified in Drosophila melanogaster with no significant structural and functional homology to other uracil-DNA binding or processing factors. Determination of the 3D structure of UDE is excepted to provide key information on the description of the molecular mechanism of action of UDE catalysis, as well as in general uracil-recognition and nuclease action. Towards this long-term aim, the random library ESPRIT technology was applied to the novel protein UDE to overcome problems in identifying soluble expressing constructs given the absence of precise information on domain content and arrangement. Nine constructs of UDE were chosen to decipher structural and functional relationships. Vacuum ultraviolet circular dichroism (VUVCD) spectroscopy was performed to define the secondary structure content and location within UDE and its truncated variants. The quantitative analysis demonstrated exclusive α-helical content for the full-length protein, which is preserved in the truncated constructs. Arrangement of α-helical bundles within the truncated protein segments suggested new domain boundaries which differ from the conserved motifs determined by sequence-based alignment of UDE homologues. Here we demonstrate that the combination of ESPRIT and VUVCD spectroscopy provides a new structural description of UDE and confirms that the truncated constructs are useful for further detailed functional studies. PMID:27273007

  19. A key role for the mRNA leader structure in translational control of ribosomal protein S1 synthesis in γ-proteobacteria

    PubMed Central

    Tchufistova, Ludmila S.; Komarova, Anastassia V.; Boni, Irina V.

    2003-01-01

    The translation initiation region (TIR) of the Escherichia coli rpsA mRNA coding for ribosomal protein S1 is characterized by a remarkable efficiency in driving protein synthesis despite the absence of the canonical Shine–Dalgarno element, and by a strong and specific autogenous repression in the presence of free S1 in trans. The efficient and autoregulated E.coli rpsA TIR comprises not less than 90 nt upstream of the translation start and can be unambiguously folded into three irregular hairpins (HI, HII and HIII) separated by A/U-rich single-stranded regions (ss1 and ss2). Phylogenetic comparison revealed that this specific fold is highly conserved in the γ-subdivision of proteobacteria (but not in other subdivisions), except for the Pseudomonas group. To test phylogenetic predictions experimentally, we have generated rpsA′–′lacZ translational fusions by inserting the rpsA TIRs from various γ-proteobacteria in-frame with the E.coli chromosomal lacZ gene. Measurements of their translation efficiency and negative regulation by excess protein S1 in trans have shown that only those rpsA TIRs which share the structural features with that of E.coli can govern efficient and regulated translation. We conclude that the E.coli-like mechanism for controlling the efficiency of protein S1 synthesis evolved after divergence of Pseudomona PMID:14627832

  20. SeqFold: genome-scale reconstruction of RNA secondary structure integrating high-throughput sequencing data.

    PubMed

    Ouyang, Zhengqing; Snyder, Michael P; Chang, Howard Y

    2013-02-01

    We present an integrative approach, SeqFold, that combines high-throughput RNA structure profiling data with computational prediction for genome-scale reconstruction of RNA secondary structures. SeqFold transforms experimental RNA structure information into a structure preference profile (SPP) and uses it to select stable RNA structure candidates representing the structure ensemble. Under a high-dimensional classification framework, SeqFold efficiently matches a given SPP to the most likely cluster of structures sampled from the Boltzmann-weighted ensemble. SeqFold is able to incorporate diverse types of RNA structure profiling data, including parallel analysis of RNA structure (PARS), selective 2'-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq), fragmentation sequencing (FragSeq) data generated by deep sequencing, and conventional SHAPE data. Using the known structures of a wide range of mRNAs and noncoding RNAs as benchmarks, we demonstrate that SeqFold outperforms or matches existing approaches in accuracy and is more robust to noise in experimental data. Application of SeqFold to reconstruct the secondary structures of the yeast transcriptome reveals the diverse impact of RNA secondary structure on gene regulation, including translation efficiency, transcription initiation, and protein-RNA interactions. SeqFold can be easily adapted to incorporate any new types of high-throughput RNA structure profiling data and is widely applicable to analyze RNA structures in any transcriptome.

  1. Periodicity in DNA primary structure is defined by secondary structure of the coded protein.

    PubMed Central

    Zhurkin, V B

    1981-01-01

    A 10.5-base periodicity found earlier is inherent in both eu- and prokaryotic coding nucleotide sequences. In the case of noncoding eukaryotic sequences no periodicity is found, so the 10.5-base oscillation seemingly does not correlate with the nucleosomal organization of DNA. It is shown that the DNA fragments, coding the alpha-helical protein segments, manifest the pronounced 10.5-base periodicity, while those regions of DNA which code the beta-structure have a 6-base oscillation. The repeating pattern of nucleotide sequences can be used for comparison of the DNA segments with low degree of homology. PMID:7243595

  2. R2R - software to speed the depiction of aesthetic consensus RNA secondary structures

    PubMed Central

    2011-01-01

    Background With continuing identification of novel structured noncoding RNAs, there is an increasing need to create schematic diagrams showing the consensus features of these molecules. RNA structural diagrams are typically made either with general-purpose drawing programs like Adobe Illustrator, or with automated or interactive programs specific to RNA. Unfortunately, the use of applications like Illustrator is extremely time consuming, while existing RNA-specific programs produce figures that are useful, but usually not of the same aesthetic quality as those produced at great cost in Illustrator. Additionally, most existing RNA-specific applications are designed for drawing single RNA molecules, not consensus diagrams. Results We created R2R, a computer program that facilitates the generation of aesthetic and readable drawings of RNA consensus diagrams in a fraction of the time required with general-purpose drawing programs. Since the inference of a consensus RNA structure typically requires a multiple-sequence alignment, the R2R user annotates the alignment with commands directing the layout and annotation of the RNA. R2R creates SVG or PDF output that can be imported into Adobe Illustrator, Inkscape or CorelDRAW. R2R can be used to create consensus sequence and secondary structure models for novel RNA structures or to revise models when new representatives for known RNA classes become available. Although R2R does not currently have a graphical user interface, it has proven useful in our efforts to create 100 schematic models of distinct noncoding RNA classes. Conclusions R2R makes it possible to obtain high-quality drawings of the consensus sequence and structural models of many diverse RNA structures with a more practical amount of effort. R2R software is available at http://breaker.research.yale.edu/R2R and as an Additional file. PMID:21205310

  3. Impact of Microscale and Pilot-Scale Freeze-Drying on Protein Secondary Structures: Sucrose Formulations of Lysozyme and Catalase.

    PubMed

    Peters, Björn-Hendrik; Leskinen, Jari T T; Molnár, Ferdinand; Ketolainen, Jarkko

    2015-11-01

    Microscale (MS) freeze-drying offers rapid process cycles for early-stage formulation development. The effects of the MS approach on the secondary structures of two model proteins, lysozyme and catalase, were compared with pilot-scale (PS) vial freeze-drying. The secondary structures were assessed by attenuated total reflection Fourier transformed infrared spectroscopy. Formulations were made with increasing sucrose-protein ratios. Freeze-drying protocols involved regular cooling without thermal treatment and annealing with MS and PS equipment, and cooling rate variations with the MS. Principal component analysis of smoothed second-derivative amide I spectra revealed sucrose-protein ratio-dependent shifts toward α-helical structures. Transferability of sucrose-protein formulations from MS to PS vial freeze-drying was evidenced at regular cooling rates. Local differences in protein secondary structures between the bottom and top of sucrose-catalase samples could be detected at the sucrose-catalase ratios of 1 and 2, this being related to the initial filling height and ice crystal morphology. Annealing revealed temperature, protein, formulation, and sample location-dependent effects influencing surface morphology at the top, or causing protein secondary structure perturbation at the bottom. With the MS approach, protein secondary structure differences at different cooling rates could be detected for sucrose-lysozyme samples at the sucrose-lysozyme ratio of 1.

  4. Internal transcribed spacer 1 secondary structure analysis reveals a common core throughout the anaerobic fungi (Neocallimastigomycota).

    PubMed

    Koetschan, Christian; Kittelmann, Sandra; Lu, Jingli; Al-Halbouni, Djamila; Jarvis, Graeme N; Müller, Tobias; Wolf, Matthias; Janssen, Peter H

    2014-01-01

    The internal transcribed spacer (ITS) is a popular barcode marker for fungi and in particular the ITS1 has been widely used for the anaerobic fungi (phylum Neocallimastigomycota). A good number of validated reference sequences of isolates as well as a large number of environmental sequences are available in public databases. Its highly variable nature predisposes the ITS1 for low level phylogenetics; however, it complicates the establishment of reproducible alignments and the reconstruction of stable phylogenetic trees at higher taxonomic levels (genus and above). Here, we overcame these problems by proposing a common core secondary structure of the ITS1 of the anaerobic fungi employing a Hidden Markov Model-based ITS1 sequence annotation and a helix-wise folding approach. We integrated the additional structural information into phylogenetic analyses and present for the first time an automated sequence-structure-based taxonomy of the ITS1 of the anaerobic fungi. The methodology developed is transferable to the ITS1 of other fungal groups, and the robust taxonomy will facilitate and improve high-throughput anaerobic fungal community structure analysis of samples from various environments.

  5. Analysis of FMRP mRNA target datasets reveals highly associated mRNAs mediated by G-quadruplex structures formed via clustered WGGA sequences

    PubMed Central

    Suhl, Joshua A.; Chopra, Pankaj; Anderson, Bart R.; Bassell, Gary J.; Warren, Stephen T.

    2014-01-01

    Fragile X syndrome, a common cause of intellectual disability and a well-known cause of autism spectrum disorder, is the result of loss or dysfunction of fragile X mental retardation protein (FMRP), a highly selective RNA-binding protein and translation regulator. A major research priority has been the identification of the mRNA targets of FMRP, particularly as recent studies suggest an excess of FMRP targets among genes implicated in idiopathic autism and schizophrenia. Several large-scale studies have attempted to identify mRNAs bound by FMRP through several methods, each generating a list of putative target genes, leading to distinct hypotheses by which FMRP recognizes its targets; namely, by RNA structure or sequence. However, no in depth analyses have been performed to identify the level of consensus among the studies. Here, we analyze four large FMRP target datasets to generate high-confidence consensus lists, and examine all datasets for sequence elements within the target RNAs to validate reported FMRP binding motifs (GACR, ACUK and WGGA). We found GACR to be highly enriched in FMRP datasets, while ACUK was not. The WGGA pattern was modestly enriched in several, but not all datasets. The previous association between FMRP and G-quadruplexes prompted the analysis of the distribution of WGGA in the target genes. Consistent with the requirements for G-quadruplex formation, we observed highly clustered WGGA motifs in FMRP targets compared with other genes, implicating both RNA structure and sequence in the recognition motif of FMRP. In addition, we generate a list of the top 40 FMRP targets associated with FXS-related phenotypes. PMID:24876161

  6. Conformational analysis and clustering of short and medium size loops connecting regular secondary structures: a database for modeling and prediction.

    PubMed Central

    Donate, L. E.; Rufino, S. D.; Canard, L. H.; Blundell, T. L.

    1996-01-01

    Loops are regions of nonrepetitive conformation connecting regular secondary structures. We identified 2,024 loops of one to eight residues in length, with acceptable main-chain bond lengths and peptide bond angles, from a database of 223 protein and protein-domain structures. Each loop is characterized by its sequence, main-chain conformation, and relative disposition of its bounding secondary structures as described by the separation between the tips of their axes and the angle between them. Loops, grouped according to their length and type of their bounding secondary structures, were superposed and clustered into 161 conformational classes, corresponding to 63% of all loops. Of these, 109 (51% of the loops) were populated by at least four nonhomologous loops or four loops sharing a low sequence identity. Another 52 classes, including 12% of the loops, were populated by at least three loops of low sequence similarity from three or fewer nonhomologous groups. Loop class suprafamilies resulting from variations in the termini of secondary structures are discussed in this article. Most previously described loop conformations were found among the classes. New classes included a 2:4 type IV hairpin, a helix-capping loop, and a loop that mediates dinucleotide-binding. The relative disposition of bounding secondary structures varies among loop classes, with some classes such as beta-hairpins being very restrictive. For each class, sequence preferences as key residues were identified; those most frequently at these conserved positions than in proteins were Gly, Asp, Pro, Phe, and Cys. Most of these residues are involved in stabilizing loop conformation, often through a positive phi conformation or secondary structure capping. Identification of helix-capping residues and beta-breakers among the highly conserved positions supported our decision to group loops according to their bounding secondary structures. Several of the identified loop classes were associated with

  7. Suppression of secondary electron yield by micro-porous array structure

    NASA Astrophysics Data System (ADS)

    Ye, M.; He, Y. N.; Hu, S. G.; Wang, R.; Hu, T. C.; Yang, J.; Cui, W. Z.

    2013-02-01

    We study secondary electron yield (SEY) suppression for metal materials using a roughened surface with a micro-porous array. First, we perform a Monte Carlo simulation of the electron trajectory in a single cylindrical well using a phenomenological model of secondary electron emission and the SEY suppression efficiency of a micro-porous array. The simulation results show that the SEY of a roughened surface is affected significantly by the aspect ratio of the micro-pores and the surface porosity of the metal plate. Then, to verify the simulation results, we produce a micro-porous array on metal plates using photolithography and measure their SEYs. We show that the micro-porous array structure can efficiently suppress the SEY of metal materials, and the measurements agree quantitatively with the corresponding simulation results. Finally, we derive an analytical formula to evaluate easily the SEY suppression efficiency of the Ag micro-porous array. In total, the micro-porous array proposed in this paper offers an alternative to SEY suppression in related areas such as multipactor effects in satellite payloads or electron cloud effects in accelerators.

  8. Suppression of secondary electron yield by micro-porous array structure

    SciTech Connect

    Ye, M.; He, Y. N.; Hu, S. G.; Wang, R.; Hu, T. C.; Yang, J.; Cui, W. Z.

    2013-02-21

    We study secondary electron yield (SEY) suppression for metal materials using a roughened surface with a micro-porous array. First, we perform a Monte Carlo simulation of the electron trajectory in a single cylindrical well using a phenomenological model of secondary electron emission and the SEY suppression efficiency of a micro-porous array. The simulation results show that the SEY of a roughened surface is affected significantly by the aspect ratio of the micro-pores and the surface porosity of the metal plate. Then, to verify the simulation results, we produce a micro-porous array on metal plates using photolithography and measure their SEYs. We show that the micro-porous array structure can efficiently suppress the SEY of metal materials, and the measurements agree quantitatively with the corresponding simulation results. Finally, we derive an analytical formula to evaluate easily the SEY suppression efficiency of the Ag micro-porous array. In total, the micro-porous array proposed in this paper offers an alternative to SEY suppression in related areas such as multipactor effects in satellite payloads or electron cloud effects in accelerators.

  9. [Isolation and structural elucidation of secondary metabolites from marine Streptomyces sp. SCSIO 1934].

    PubMed

    Niu, Siwen; Li, Sumei; Tian, Xinpeng; Hu, Tao; Ju, Jianhua; Ynag, Xiaohong; Zhang, Si; Zhang, Changsheng

    2011-07-01

    Marine Actinobacteria are emerging as new resources for bioactive natural products with promise in novel drug discovery. In recent years, the richness and diversity of marine Actinobacteria from the South China Sea and their ability in producing bioactive products have been investigated. The objective of this work is to isolate and identify bioactive secondary metabolites from a marine actinobacterium SCSIO 1934 derived from sediments of South China Sea. The strain was identified as a Streptomyces spieces by analyzing its 16S rDNA sequence. Streptomyces sp. SCSIO 1934 was fermented under optimized conditions and seven bioactive secondary metabolites were isolated and purified by chromatographic methods including colum chromatography over silica gel and Sephadex LH-20. Their structures were elucidated as 17-O-demethylgeldanamycin (1), lebstatin (2), 17-O-demethyllebstatin (3), nigericin (4), nigericin sodium salt (5), abierixin (6), respectively, by detailed NMR spectroscopic data (1H, 13C, COSY, HSQC and HMBC). This work provided a new marine actinobacterium Streptomyces sp. SCSIO 1934, capable of producing diverse bioactive natural products.

  10. mRNA imprinting

    PubMed Central

    2011-01-01

    Following its synthesis in the nucleus, mRNA undergoes various stages that are critical for the proper synthesis, localization and possibly functionality of its encoded protein. Recently, we have shown that two RNA polymerase II (Pol II) subunits, Rpb4p and Rpb7p, associate with the nascent transcript co-transcriptionally. This “mRNA imprinting” lasts throughout the mRNA lifetime and is required for proper regulation of all major stages that the mRNA undergoes. Other possible cases of co-transcriptional imprinting are discussed. Since mRNAs can be transported from the synthesizing cell to other cells, we propose that mRNA imprinting can also affect the phenotype of the recipient cells. This can be viewed as “mRNA-based epigenetics.” PMID:21686103

  11. [Study on the effect of high hydrostatic pressure treatment on the secondary structure of mushroom polyphenoloxidase by SRCD and FTIR].

    PubMed

    Yi, Jian-yong; Dong, Peng; Wang, Yong-tao; Jiang, Bin; Liao, Xiao-jun; Hu, Xiao-song; Zhang, Yan

    2012-02-01

    The secondary structure of the mushroom polyphenoloxidase treated by the high hydrostatic pressure (HHP) was analyzed by the synchrotron radiation circular dichroism (SRCD) and Fourier transform infrared spectroscopy (FTIR). The alpha-helix content of mushroom PPO was decreased after HHP treatment, which indicated that the secondary structure of PPO was changed. There was a discrepancy of the result of the secondary structure content between untreated or HHP-treated mushroom PPO analyzed by SRCD and FTIR spectra, and this discrepancy may be due to the different determination temperature, the concentration of the PPO solution and the spectra analysis method etc. The fluorescence spectra showed that the fluorescence intensity of the mushroom PPO was decreased after HHP treatment, and a red shift was observed after HHP treatment, which indicated that the tertiary structure of the enzyme molecule has been modified.

  12. Poly(L-lysine) and Clay Nanocomposite with Desired Matrix Secondary Structure: Effects of Polypeptide Molecular Weight

    SciTech Connect

    Hule,R.; Pochan, D.

    2007-01-01

    Nanocomposites (NC) were formed using cationic poly(L-lysine) (PLL), a semicrystalline polypeptide, that was reinforced by sodium montmorillonite (MMT) clay via solution intercalation technique. By varying solution conditions such as pH, temperature, and polypeptide concentration in the presence of clay platelets, the secondary structure of PLL was controllably altered into {alpha}-helical, {beta}-sheet, and random coil. The high molecular weight polypeptide shows a strong propensity to fold into the {beta}-sheet structure when cast as films, irrespective of the initial secondary structure in solution. Nanocomposite local morphology confirms intercalated MMT platelets with PLL over a wide range of compositions.

  13. Secondary structure and 3D homology modeling of swine leukocyte antigen class 2 (SLA-2) molecules.

    PubMed

    Gao, Feng-Shan; Xu, Chong-bo; Long, Yi-hou; Xia, Chun

    2009-01-01

    No information to date is available to elucidate the structure of swine leukocyte antigen class I (SLA-I) molecule which is comprised by a heavy chain of SLA-I non-covalently associated with a light chain, beta(2)-microglobulin (beta(2)m) proteins. Presently, one of SLA-I gene SLA-2 and beta(2)m gene were expressed as soluble maltose binding proteins (MBP-proteins) in a pMAL-p2X/Escherichia coli TB1 system and identified by western blotting with anti-MBP polyclonal antibodies. The expressed proteins MBP-SLA-2 and MBP-beta(2)m were purified on amylose affinity columns followed by DEAE-Sepharose. The purified products were cleaved by Factor Xa, respectively, and the interest of proteins SLA-2 and beta(2)m were purified on amylose affinity columns followed by separation from MBP on DEAE-Sepharose. The secondary structures of SLA-2 and beta(2)m were analyzed by circular dichroism (CD) spectrophotometry. The three-dimensional (3D) structure of their peptide-binding domain (PBD) was modeled-based sequence homology. The content of the alpha-helix, beta-sheet, turn, and random coil in the SLA-2 protein were 76, 95, 36, and 67aa, respectively. In the 98aa of beta(2)m, the contents of the alpha-helix, beta-sheet, turn, and random coil were 0, 45, 8, and 45aa, respectively. The SLA-2 protein displayed a typical alpha-helix structure while beta(2)m protein displayed a typical beta-sheet structure. Homology modeling of the SLA-2 and beta(2)m proteins demonstrated similarities with the structure of human and mouse MHC (major histocompatibility complex) class I proteins.

  14. Design and analysis of supporting structure between the primary mirror and the secondary mirror on a space telescope

    NASA Astrophysics Data System (ADS)

    Wang, Chenjie; Chai, Wenyi; Feng, Liangjie; Yang, Wengang; Wang, Wei; Fan, Xuewu

    2015-10-01

    Mechanical stability is a significant segment for an on-axis space telescope to assure its assembly accuracy as well as the image quality in the rigorous space environment, supporting structure between the primary mirror and the secondary mirror as a main structure of the on-axis space telescope must be designed reasonably to meet the mission requirements of the space telescope. Meanwhile, in view of the limitation of the satellite launching cost, it is necessary to reduce the weight and power compensation during the supporting structure design based on the satisfaction of telescope performance. Two types of supporting structure for a space telescope are designed, one is three-tripod structure which has three tripods located on the optical bench to support the secondary mirror assemblies and keep the distance between the primary mirror and the secondary mirror, the other is barrel supporting structure which includes a tube and a secondary mirror support with four spider struts. To compare the mechanical performance and launching cost of the two kinds of supporting structure, both structural and thermal analysis model are established. The analysis results indicates that the three-tripod support is lighter, has better mechanical performance and needs less power compensation than the barrel support.

  15. 1H and 13C NMR characterization and secondary structure of the K2 polysaccharide of Klebsiella pneumoniae strain 52145.

    PubMed

    Corsaro, Maria Michela; De Castro, Cristina; Naldi, Teresa; Parrilli, Michelangelo; Tomás, Juan M; Regué, Miguel

    2005-09-26

    The complete (1)H and (13)C NMR characterization of the tetrasaccharide repeating unit from the K2 polysaccharide of Klebsiella pneumoniae strain 52145 is reported. [chemical structure] In addition a model for its secondary structure was suggested on the basis of dynamic and molecular calculations.

  16. Innovative FT-IR Imaging of Protein Film Secondary Structure Before and After Heat Treatment

    SciTech Connect

    Bonwell, E.; Wetzel, D

    2009-01-01

    Changes in the secondary structure of globular protein occur during thermal processing. An infrared reflecting mirrored optical substrate that is unaffected by heat allows recording infrared spectra of protein films in a reflection absorption mode on the stage of an FT-IR microspectrometer. Hydrated films of myoglobin protein cast from solution on the mirrored substrate are interrogated before and after thermal denaturation to allow a direct comparison. Focal plane array imaging of 280 protein films allowed selection of the same area in the image from which to extract spectra. After treatment, 110 of 140 spectra from multiple films showed a dramatic shift from the {alpha}-helix form (1650 {+-} 5 cm{sup -1}) to aggregated forms on either side of the original band. Seventy maxima were near 1625 cm{sup -1}, and 40 shifted in the direction of 1670 cm{sup -1}. The method developed was applied to films cast from two other commercial animal and plant protein sources.

  17. Peptide secondary structure modulates single-walled carbon nanotube fluorescence as a chaperone sensor for nitroaromatics

    PubMed Central

    Heller, Daniel A.; Pratt, George W.; Zhang, Jingqing; Nair, Nitish; Hansborough, Adam J.; Boghossian, Ardemis A.; Reuel, Nigel F.; Barone, Paul W.; Strano, Michael S.

    2011-01-01

    A class of peptides from the bombolitin family, not previously identified for nitroaromatic recognition, allows near-infrared fluorescent single-walled carbon nanotubes to transduce specific changes in their conformation. In response to the binding of specific nitroaromatic species, such peptide–nanotube complexes form a virtual “chaperone sensor,” which reports modulation of the peptide secondary structure via changes in single-walled carbon nanotubes, near-infrared photoluminescence. A split-channel microscope constructed to image quantized spectral wavelength shifts in real time, in response to nitroaromatic adsorption, results in the first single-nanotube imaging of solvatochromic events. The described indirect detection mechanism, as well as an additional exciton quenching-based optical nitroaromatic detection method, illustrate that functionalization of the carbon nanotube surface can result in completely unique sites for recognition, resolvable at the single-molecule level. PMID:21555544

  18. Prot-2S: a new python web tool for protein secondary structure studies.

    PubMed

    Munteanu, Cristian R; Magalhães, Alexandre L

    2009-01-01

    Prot-2S is a bioinformatics web application devised to analyse the protein chain secondary structures (2S) (http:/ /www.requimte.pt:8080/Prot-2S/). The tool is built on the RCSB Protein Data Bank PDB and DSSP application/files and includes calculation/graphical display of amino acid propensities in 2S motifs based on any user amino acid classification/code (for any particular protein chain list). The interface can calculate the 2S composition, display the 2S subsequences and search for DSSP non-standard residues and for pairs/triplets/quadruplets (amino acid patterns in 2S motifs). This work presents some Prot-2S applications showing its usefulness in protein research and as an e-learning tool as well. PMID:19640828

  19. Class Anxiety in Secondary Education: Exploring Structural Relations with Perceived Control, Engagement, Disaffection, and Performance.

    PubMed

    González, Antonio; Faílde Garrido, José María; Rodríguez Castro, Yolanda; Carrera Rodríguez, María Victoria

    2015-09-14

    The aim of this study was to assess the relationships between class-related anxiety with perceived control, teacher-reported behavioral engagement, behavioral disaffection, and academic performance. Participants were 355 compulsory secondary students (9th and 10th grades; Mean age = 15.2 years; SD = 1.8 years). Structural equation models revealed performance was predicted by perceived control, anxiety, disaffection, and engagement. Perceived control predicted anxiety, disaffection, and engagement. Anxiety predicted disaffection and engagement, and partially mediated the effects from control on disaffection (β = -.277, p < .005; CI = -.378, -.197) and engagement (β = .170, p < .002; CI = .103 .258). The negative association between anxiety and performance was mediated by engagement and disaffection (β = -.295, p < .002; CI = -.439, -.182). Anxiety, engagement, and disaffection mediated the effects of control on performance (β = .352, p < .003; CI = .279, .440). The implications of these results are discussed in the light of current theory and educational interventions.

  20. Secondary-structure characterization by far-UV CD of highly purified uncoupling protein 1 expressed in yeast.

    PubMed Central

    Douette, Pierre; Navet, Rachel; Bouillenne, Fabrice; Brans, Alain; Sluse-Goffart, Claudine; Matagne, André; Sluse, Francis E

    2004-01-01

    The rat UCP1 (uncoupling protein 1) is a mitochondrial inner-membrane carrier involved in energy dissipation and heat production. We expressed UCP1 carrying a His6 epitope at its C-terminus in Saccharomyces cerevisiae mitochondria. The recombinant-tagged UCP1 was purified by immobilized metal-ion affinity chromatography to homogeneity (>95%). This made it suitable for subsequent biophysical characterization. Fluorescence resonance energy transfer experiments showed that n-dodecyl-beta-D-maltoside-solubilized UCP1-His6 retained its PN (purine nucleotide)-binding capacity. The far-UV CD spectrum of the functional protein clearly indicated the predominance of alpha-helices in the UCP1 secondary structure. The UCP1 secondary structure exhibited an alpha-helical degree of approx. 68%, which is at least 25% higher than the previously reported estimations based on computational predictions. Moreover, the helical content remained unchanged in free and PN-loaded UCP1. A homology model of the first repeat of UCP1, built on the basis of X-ray-solved close parent, the ADP/ATP carrier, strengthened the CD experimental results. Our experimental and computational results indicate that (i) alpha-helices are the major component of UCP1 secondary structure; (ii) PN-binding mechanism does not involve significant secondary-structure rearrangement; and (iii) UCP1 shares similar secondary-structure characteristics with the ADP/ATP carrier, at least for the first repeat. PMID:14766012

  1. Effect of Secondary Cooling Conditions on Solidification Structure and Central Macrosegregation in Continuously Cast High-Carbon Rectangular Billet

    NASA Astrophysics Data System (ADS)

    Zeng, Jie; Chen, Weiqing

    2015-10-01

    Solidification structures of high carbon rectangular billet with a size of 180 mm × 240 mm in different secondary cooling conditions were simulated using cellular automaton-finite element (CAFE) coupling model. The adequacy of the model was compared with the simulated and the actual macrostructures of 82B steel. Effects of the secondary cooling water intensity on solidification structures including the equiaxed grain ratio and the equiaxed grain compactness were discussed. It was shown that the equiaxed grain ratio and the equiaxed grain compactness changed in the opposite direction at different secondary cooling water intensities. Increasing the secondary cooling water intensity from 0.9 or 1.1 to 1.3 L/kg could improve the equiaxed grain compactness and decrease the equiaxed grain ratio. Besides, the industrial test was conducted to investigate the effect of different secondary cooling water intensities on the center carbon macrosegregation of 82B steel. The optimum secondary cooling water intensity was 0.9 L/kg, while the center carbon segregation degree was 1.10. The relationship between solidification structure and center carbon segregation was discussed based on the simulation results and the industrial test.

  2. Secondary correction of unsatisfactory blepharoplasty: removing multilaminated septal structures and grafting of preaponeurotic fat.

    PubMed

    Kim, Y W; Park, H J; Kim, S

    2000-11-01

    Oriental blepharoplasty, commonly known as a "double eyelid operation," is the most frequently practiced cosmetic procedure in Orientals, who have probably become more fold conscious because of social westernization and an influx of Caucasians into their society. Anatomically, the upper eyelids of an Oriental are considerably different from those of a white person, and nearly half of Orientals have single eyelids. When performing blepharoplasty, an appropriate design and operative technique must be carefully selected, taking into consideration the anatomical characteristics of Koreans to obtain an aesthetically pleasing result. However, the incidence of complications is high. Patients who are faced with unsatisfactory results are often perplexed by the fact that such a commonly performed procedure could have a very high rate of dissatisfaction and that an improvement is not easy. An unfavorable result need not imply a postoperative complication, but only that the result is not acceptable to the patient, whose goal may not be based on good aesthetic principles. The most common sources of dissatisfaction are postoperative asymmetry and high placement of the lid fold. From 1991 to 1998, secondary blepharoplasty was performed on 72 patients by slitting transversely, removing the multilaminated septal structures exposed to the previous operative scar, spreading the preaponeurotic fat that extruded, and removing the septal structures into a space where the scar was eliminated to prevent secondary adhesion. The average age of the patients was 26.5 years, and the average follow-up period was 2 years. No remarkable complication was encountered after operation with this method, and the desired aesthetic improvements were achieved in the majority of the patients.

  3. Linker histone partial phosphorylation: effects on secondary structure and chromatin condensation

    PubMed Central

    Lopez, Rita; Sarg, Bettina; Lindner, Herbert; Bartolomé, Salvador; Ponte, Inma; Suau, Pedro; Roque, Alicia

    2015-01-01

    Linker histones are involved in chromatin higher-order structure and gene regulation. We have successfully achieved partial phosphorylation of linker histones in chicken erythrocyte soluble chromatin with CDK2, as indicated by HPCE, MALDI-TOF and Tandem MS. We have studied the effects of linker histone partial phosphorylation on secondary structure and chromatin condensation. Infrared spectroscopy analysis showed a gradual increase of β-structure in the phosphorylated samples, concomitant to a decrease in α-helix/turns, with increasing linker histone phosphorylation. This conformational change could act as the first step in the phosphorylation-induced effects on chromatin condensation. A decrease of the sedimentation rate through sucrose gradients of the phosphorylated samples was observed, indicating a global relaxation of the 30-nm fiber following linker histone phosphorylation. Analysis of specific genes, combining nuclease digestion and qPCR, showed that phosphorylated samples were more accessible than unphosphorylated samples, suggesting local chromatin relaxation. Chromatin aggregation was induced by MgCl2 and analyzed by dynamic light scattering (DLS). Phosphorylated chromatin had lower percentages in volume of aggregated molecules and the aggregates had smaller hydrodynamic diameter than unphosphorylated chromatin, indicating that linker histone phosphorylation impaired chromatin aggregation. These findings provide new insights into the effects of linker histone phosphorylation in chromatin condensation. PMID:25870416

  4. Linker histone partial phosphorylation: effects on secondary structure and chromatin condensation.

    PubMed

    Lopez, Rita; Sarg, Bettina; Lindner, Herbert; Bartolomé, Salvador; Ponte, Inma; Suau, Pedro; Roque, Alicia

    2015-05-19

    Linker histones are involved in chromatin higher-order structure and gene regulation. We have successfully achieved partial phosphorylation of linker histones in chicken erythrocyte soluble chromatin with CDK2, as indicated by HPCE, MALDI-TOF and Tandem MS. We have studied the effects of linker histone partial phosphorylation on secondary structure and chromatin condensation. Infrared spectroscopy analysis showed a gradual increase of β-structure in the phosphorylated samples, concomitant to a decrease in α-helix/turns, with increasing linker histone phosphorylation. This conformational change could act as the first step in the phosphorylation-induced effects on chromatin condensation. A decrease of the sedimentation rate through sucrose gradients of the phosphorylated samples was observed, indicating a global relaxation of the 30-nm fiber following linker histone phosphorylation. Analysis of specific genes, combining nuclease digestion and qPCR, showed that phosphorylated samples were more accessible than unphosphorylated samples, suggesting local chromatin relaxation. Chromatin aggregation was induced by MgCl2 and analyzed by dynamic light scattering (DLS). Phosphorylated chromatin had lower percentages in volume of aggregated molecules and the aggregates had smaller hydrodynamic diameter than unphosphorylated chromatin, indicating that linker histone phosphorylation impaired chromatin aggregation. These findings provide new insights into the effects of linker histone phosphorylation in chromatin condensation.

  5. Linker histone partial phosphorylation: effects on secondary structure and chromatin condensation.

    PubMed

    Lopez, Rita; Sarg, Bettina; Lindner, Herbert; Bartolomé, Salvador; Ponte, Inma; Suau, Pedro; Roque, Alicia

    2015-05-19

    Linker histones are involved in chromatin higher-order structure and gene regulation. We have successfully achieved partial phosphorylation of linker histones in chicken erythrocyte soluble chromatin with CDK2, as indicated by HPCE, MALDI-TOF and Tandem MS. We have studied the effects of linker histone partial phosphorylation on secondary structure and chromatin condensation. Infrared spectroscopy analysis showed a gradual increase of β-structure in the phosphorylated samples, concomitant to a decrease in α-helix/turns, with increasing linker histone phosphorylation. This conformational change could act as the first step in the phosphorylation-induced effects on chromatin condensation. A decrease of the sedimentation rate through sucrose gradients of the phosphorylated samples was observed, indicating a global relaxation of the 30-nm fiber following linker histone phosphorylation. Analysis of specific genes, combining nuclease digestion and qPCR, showed that phosphorylated samples were more accessible than unphosphorylated samples, suggesting local chromatin relaxation. Chromatin aggregation was induced by MgCl2 and analyzed by dynamic light scattering (DLS). Phosphorylated chromatin had lower percentages in volume of aggregated molecules and the aggregates had smaller hydrodynamic diameter than unphosphorylated chromatin, indicating that linker histone phosphorylation impaired chromatin aggregation. These findings provide new insights into the effects of linker histone phosphorylation in chromatin condensation. PMID:25870416

  6. Content-Related Knowledge of Biology Teachers from Secondary Schools: Structure and learning opportunities

    NASA Astrophysics Data System (ADS)

    Großschedl, Jörg; Mahler, Daniela; Kleickmann, Thilo; Harms, Ute

    2014-09-01

    Teachers' content-related knowledge is a key factor influencing the learning progress of students. Different models of content-related knowledge have been proposed by educational researchers; most of them take into account three categories: content knowledge, pedagogical content knowledge, and curricular knowledge. As there is no consensus about the empirical separability (i.e. empirical structure) of content-related knowledge yet, a total of 134 biology teachers from secondary schools completed three tests which were to capture each of the three categories of content-related knowledge. The empirical structure of content-related knowledge was analyzed by Rasch analysis, which suggests content-related knowledge to be composed of (1) content knowledge, (2) pedagogical content knowledge, and (3) curricular knowledge. Pedagogical content knowledge and curricular knowledge are highly related (rlatent = .70). The latent correlations between content knowledge and pedagogical content knowledge (rlatent = .48)-and curricular knowledge, respectively (rlatent = .35)-are moderate to low (all ps < .001). Beyond the empirical structure of content-related knowledge, different learning opportunities for teachers were investigated with regard to their relationship to content knowledge, pedagogical content knowledge, and curricular knowledge acquisition. Our results show that an in-depth training in teacher education, professional development, and teacher self-study are positively related to particular categories of content-related knowledge. Furthermore, our results indicate that teaching experience is negatively related to curricular knowledge, compared to no significant relationship with content knowledge and pedagogical content knowledge.

  7. The RNAsnp web server: predicting SNP effects on local RNA secondary structure.

    PubMed

    Sabarinathan, Radhakrishnan; Tafer, Hakim; Seemann, Stefan E; Hofacker, Ivo L; Stadler, Peter F; Gorodkin, Jan

    2013-07-01

    The function of many non-coding RNA genes and cis-regulatory elements of messenger RNA largely depends on the structure, which is in turn determined by their sequence. Single nucleotide polymorphisms (SNPs) and other mutations may disrupt the RNA structure, interfere with the molecular function and hence cause a phenotypic effect. RNAsnp is an efficient method to predict the effect of SNPs on local RNA secondary structure based on the RNA folding algorithms implemented in the Vienna RNA package. The SNP effects are quantified in terms of empirical P-values, which, for computational efficiency, are derived from extensive pre-computed tables of distributions of substitution effects as a function of gene length and GC content. Here, we present a web service that not only provides an interface for RNAsnp but also features a graphical output representation. In addition, the web server is connected to a local mirror of the UCSC genome browser database that enables the users to select the genomic sequences for analysis and visualize the results directly in the UCSC genome browser. The RNAsnp web server is freely available at: http://rth.dk/resources/rnasnp/.

  8. Mod-seq: A High-Throughput Method for Probing RNA Secondary Structure.

    PubMed

    Lin, Yizhu; May, Gemma E; Joel McManus, C

    2015-01-01

    It has become increasingly clear that large RNA molecules, especially long noncoding RNAs, function in almost all gene regulatory processes (Cech & Steitz, 2014). Many large RNAs appear to be structural scaffolds for assembly of important RNA/protein complexes. However, the structures of most large cellular RNA molecules are currently unknown (Hennelly & Sanbonmatsu, 2012). While chemical probing can reveal single-stranded regions of RNA, traditional approaches to identify sites of chemical modification are time consuming. Mod-seq is a high-throughput method used to map chemical modification sites on RNAs of any size, including complex mixtures of RNA. In this protocol, we describe preparation of Mod-seq high-throughput sequencing libraries from chemically modified RNA. We also describe a software package "Mod-seeker," which is a compilation of scripts written in Python, for the analysis of Mod-seq data. Mod-seeker returns statistically significant modification sites, which can then be used to aid in secondary structure prediction. PMID:26068740

  9. Role of loops connecting secondary structure elements in the stabilization of proteins isolated from thermophilic organisms

    PubMed Central

    Balasco, Nicole; Esposito, Luciana; Simone, Alfonso De; Vitagliano, Luigi

    2013-01-01

    It has been recently discovered that the connection of secondary structure elements (ββ-unit, βα- and αβ-units) in proteins follows quite stringent principles regarding the chirality and the orientation of the structural units (Koga et al., Nature 2012;491:222–227). By exploiting these rules, a number of protein scaffolds endowed with a remarkable thermal stability have been designed (Koga et al., Nature 2012;491:222–227). By using structural databases of proteins isolated from either mesophilic or thermophilic organisms, we here investigate the influence of supersecondary associations on the thermal stability of natural proteins. Our results suggest that β-hairpins of proteins from thermophilic organisms are very frequently characterized by shortenings of the loops. Interestingly, this shortening leads to states that display a very strong preference for the most common connectivity of the strands observed in native protein hairpins. The abundance of selective states in these proteins suggests that they may achieve a high stability by adopting a strategy aimed to reduce the possible conformations of the unfolded ensemble. In this scenario, our data indicate that the shortening is effective if it increases the adherence to these rules. We also show that this mechanism may operate in the stabilization of well-known protein folds (thioredoxin and RNase A). These findings suggest that future investigations aimed at defining mechanism of protein stabilization should also consider these effects. PMID:23661276

  10. Rat neuropeptide Y precursor gene expression. mRNA structure, tissue distribution, and regulation by glucocorticoids, cyclic AMP, and phorbol ester.

    PubMed

    Higuchi, H; Yang, H Y; Sabol, S L

    1988-05-01

    Rat brain neuropeptide Y precursor (prepro-NPY) cDNA clones were isolated and sequenced in order to study regulation of the prepro-NPY gene. Rat prepro-NPY (98 amino acid residues) contains a 36-residue NPY sequence, followed by a proteolysis/amidation site Gly-Lys-Arg, followed by a 30-residue COOH-terminal sequence. The strong evolutionary conservation of rat and human sequences of NPY (100%) and COOH-terminal peptide (93%) suggests that both peptides have important biological functions. In the rat central nervous system, prepro-NPY mRNA (800 bases) is most abundant in the striatum and cortex and moderately abundant in the hippocampus, hypothalamus, and spinal cord. The rat adrenal, spleen, heart, and lung have significant levels of prepro-NPY mRNA. Regulation of the prepro-NPY mRNA abundance was studied in several rodent neural cell lines. PC12 rat pheochromocytoma and N18TG-2 mouse neuroblastoma cells possess low basal levels of prepro-NPY mRNA, while NG108-15 hybrid cells possess high levels. Treatment of PC12 cells with a glucocorticoid such as dexamethasone or elevation of cAMP by forskolin increased the prepro-NPY mRNA level 2-3-fold or 3-10-fold, respectively. In N18TG-2 cells dexamethasone and forskolin synergistically increased prepro-NPY mRNA 7-fold. Treatment of PC12 cells with the protein kinase C activator phorbol 12-myristate 13-acetate alone elevated prepro-NPY mRNA marginally, but the phorbol ester plus forskolin elicited 20-70-fold increases, which were further enhanced to over 200-fold by dexamethasone and the calcium ionophore A23187. These results indicate that NPY gene expression can be positively regulated by synergistic actions of glucocorticoids, cAMP elevation, and protein kinase C activation.

  11. Identification of novel DNA repair proteins via primary sequence, secondary structure, and homology

    PubMed Central

    Brown, JB; Akutsu, Tatsuya

    2009-01-01

    Background DNA repair is the general term for the collection of critical mechanisms which repair many forms of DNA damage such as methylation or ionizing radiation. DNA repair has mainly been studied in experimental and clinical situations, and relatively few information-based approaches to new extracting DNA repair knowledge exist. As a first step, automatic detection of DNA repair proteins in genomes via informatics techniques is desirable; however, there are many forms of DNA repair and it is not a straightforward process to identify and classify repair proteins with a single optimal method. We perform a study of the ability of homology and machine learning-based methods to identify and classify DNA repair proteins, as well as scan vertebrate genomes for the presence of novel repair proteins. Combinations of primary sequence polypeptide frequency, secondary structure, and homology information are used as feature information for input to a Support Vector Machine (SVM). Results We identify that SVM techniques are capable of identifying portions of DNA repair protein datasets without admitting false positives; at low levels of false positive tolerance, homology can also identify and classify proteins with good performance. Secondary structure information provides improved performance compared to using primary structure alone. Furthermore, we observe that machine learning methods incorporating homology information perform best when data is filtered by some clustering technique. Analysis by applying these methodologies to the scanning of multiple vertebrate genomes confirms a positive correlation between the size of a genome and the number of DNA repair protein transcripts it is likely to contain, and simultaneously suggests that all organisms have a non-zero minimum number of repair genes. In addition, the scan result clusters several organisms' repair abilities in an evolutionarily consistent fashion. Analysis also identifies several functionally unconfirmed

  12. Improved prediction of RNA secondary structure by integrating the free energy model with restraints derived from experimental probing data

    PubMed Central

    Wu, Yang; Shi, Binbin; Ding, Xinqiang; Liu, Tong; Hu, Xihao; Yip, Kevin Y.; Yang, Zheng Rong; Mathews, David H.; Lu, Zhi John

    2015-01-01

    Recently, several experimental techniques have emerged for probing RNA structures based on high-throughput sequencing. However, most secondary structure prediction tools that incorporate probing data are designed and optimized for particular types of experiments. For example, RNAstructure-Fold is optimized for SHAPE data, while SeqFold is optimized for PARS data. Here, we report a new RNA secondary structure prediction method, restrained MaxExpect (RME), which can incorporate multiple types of experimental probing data and is based on a free energy model and an MEA (maximizing expected accuracy) algorithm. We first demonstrated that RME substantially improved secondary structure prediction with perfect restraints (base pair information of known structures). Next, we collected structure-probing data from diverse experiments (e.g. SHAPE, PARS and DMS-seq) and transformed them into a unified set of pairing probabilities with a posterior probabilistic model. By using the probability scores as restraints in RME, we compared its secondary structure prediction performance with two other well-known tools, RNAstructure-Fold (based on a free energy minimization algorithm) and SeqFold (based on a sampling algorithm). For SHAPE data, RME and RNAstructure-Fold performed better than SeqFold, because they markedly altered the energy model with the experimental restraints. For high-throughput data (e.g. PARS and DMS-seq) with lower probing efficiency, the secondary structure prediction performances of the tested tools were comparable, with performance improvements for only a portion of the tested RNAs. However, when the effects of tertiary structure and protein interactions were removed, RME showed the highest prediction accuracy in the DMS-accessible regions by incorporating in vivo DMS-seq data. PMID:26170232

  13. Improved prediction of RNA secondary structure by integrating the free energy model with restraints derived from experimental probing data.

    PubMed

    Wu, Yang; Shi, Binbin; Ding, Xinqiang; Liu, Tong; Hu, Xihao; Yip, Kevin Y; Yang, Zheng Rong; Mathews, David H; Lu, Zhi John

    2015-09-01

    Recently, several experimental techniques have emerged for probing RNA structures based on high-throughput sequencing. However, most secondary structure prediction tools that incorporate probing data are designed and optimized for particular types of experiments. For example, RNAstructure-Fold is optimized for SHAPE data, while SeqFold is optimized for PARS data. Here, we report a new RNA secondary structure prediction method, restrained MaxExpect (RME), which can incorporate multiple types of experimental probing data and is based on a free energy model and an MEA (maximizing expected accuracy) algorithm. We first demonstrated that RME substantially improved secondary structure prediction with perfect restraints (base pair information of known structures). Next, we collected structure-probing data from diverse experiments (e.g. SHAPE, PARS and DMS-seq) and transformed them into a unified set of pairing probabilities with a posterior probabilistic model. By using the probability scores as restraints in RME, we compared its secondary structure prediction performance with two other well-known tools, RNAstructure-Fold (based on a free energy minimization algorithm) and SeqFold (based on a sampling algorithm). For SHAPE data, RME and RNAstructure-Fold performed better than SeqFold, because they markedly altered the energy model with the experimental restraints. For high-throughput data (e.g. PARS and DMS-seq) with lower probing efficiency, the secondary structure prediction performances of the tested tools were comparable, with performance improvements for only a portion of the tested RNAs. However, when the effects of tertiary structure and protein interactions were removed, RME showed the highest prediction accuracy in the DMS-accessible regions by incorporating in vivo DMS-seq data.

  14. Structural domains within the HIV-1 mRNA and the ribosomal protein S25 influence cap-independent translation initiation.

    PubMed

    Carvajal, Felipe; Vallejos, Maricarmen; Walters, Beth; Contreras, Nataly; Hertz, Marla I; Olivares, Eduardo; Cáceres, Carlos J; Pino, Karla; Letelier, Alejandro; Thompson, Sunnie R; López-Lastra, Marcelo

    2016-07-01

    The 5' leader of the HIV-1 genomic RNA is a multifunctional region that folds into secondary/tertiary structures that regulate multiple processes during viral replication including translation initiation. In this work, we examine the internal ribosome entry site (IRES) located in the 5' leader that drives translation initiation of the viral Gag protein under conditions that hinder cap-dependent translation initiation. We show that activity of the HIV-1 IRES relies on ribosomal protein S25 (eS25). Additionally, a mechanistic and mutational analysis revealed that the HIV-1 IRES is modular in nature and that once the 40S ribosomal subunit is recruited to the IRES, translation initiates without the need of ribosome scanning. These findings elucidate a mechanism of initiation by the HIV-1 IRES whereby a number of highly structured sites present within the HIV-1 5' leader leads to the recruitment of the 40S subunit directly at the site of initiation of protein synthesis. PMID:27191820

  15. Investigations of primary and secondary impact structures on the moon and laboratory experiments to study the ejecta of secondary particles. Ph.D. Thesis - Ruprecht Karl Univ.

    NASA Technical Reports Server (NTRS)

    Koenig, B.

    1977-01-01

    Young lunar impact structures were investigated by using lunar orbiter, Apollo Metric and panorama photographs. Measurements on particularly homogeneous areas low in secondary craters made possible an expansion of primary crater distribution to small diameters. This is now sure for a range between 20m or = D or = 20km and this indicates that the size and velocity distribution of the impacting bodies in the last 3 billion years has been constant. A numerical approximation in the form of a 7th degree polynomial was obtained for the distribution.

  16. Investigating the secondary structures of long oligonucleotides using attenuated-total-reflection surface-enhanced Raman spectroscopy

    NASA Astrophysics Data System (ADS)

    Chiu, K.-C.; Yih, J.-N.; Yu, L.-Y.; Chen, S.-J.

    2006-08-01

    This study utilizes a surface-enhanced Raman spectroscopy (SERS) based on the attenuated-total-reflection method to investigate the secondary structures of long oligonucleotides and their influence on the DNA hybridization. It is found that the ring-breathing modes of adenine, thymine, guanine, and cytosine in Raman fingerprint associated with three 60mer oligonucleotides with prominent secondary structures are lower than those observed for the two oligonucleotides with no obvious secondary structures. It is also determined that increasing the DNA hybridization temperature from 35°C to 45°C reduces secondary structure effects. The kinetics of biomolecular interaction analysis can be performed by using surface plasmons resonance biosensor, but the structural information of the oligonucleotides can not observed directly. The SERS spectrum provides the structural information of the oligonucleotides with the help of a silver colloidal nanoparticle monolayer by control of the size and distribution of the nanoparticles adapted as a Raman active substrate. Also, the detection limit of the DNA Raman signal has been successfully improved to reach sub-micro molarity of DNA concentration.

  17. Improving prediction of secondary structure, local backbone angles, and solvent accessible surface area of proteins by iterative deep learning.

    PubMed

    Heffernan, Rhys; Paliwal, Kuldip; Lyons, James; Dehzangi, Abdollah; Sharma, Alok; Wang, Jihua; Sattar, Abdul; Yang, Yuedong; Zhou, Yaoqi

    2015-01-01

    Direct prediction of protein structure from sequence is a challenging problem. An effective approach is to break it up into independent sub-problems. These sub-problems such as prediction of protein secondary structure can then be solved independently. In a previous study, we found that an iterative use of predicted secondary structure and backbone torsion angles can further improve secondary structure and torsion angle prediction. In this study, we expand the iterative features to include solvent accessible surface area and backbone angles and dihedrals based on Cα atoms. By using a deep learning neural network in three iterations, we achieved 82% accuracy for secondary structure prediction, 0.76 for the correlation coefficient between predicted and actual solvent accessible surface area, 19° and 30° for mean absolute errors of backbone φ and ψ angles, respectively, and 8° and 32° for mean absolute errors of Cα-based θ and τ angles, respectively, for an independent test dataset of 1199 proteins. The accuracy of the method is slightly lower for 72 CASP 11 targets but much higher than those of model structures from current state-of-the-art techniques. This suggests the potentially beneficial use of these predicted properties for model assessment and ranking.

  18. Improving prediction of secondary structure, local backbone angles, and solvent accessible surface area of proteins by iterative deep learning

    PubMed Central

    Heffernan, Rhys; Paliwal, Kuldip; Lyons, James; Dehzangi, Abdollah; Sharma, Alok; Wang, Jihua; Sattar, Abdul; Yang, Yuedong; Zhou, Yaoqi

    2015-01-01

    Direct prediction of protein structure from sequence is a challenging problem. An effective approach is to break it up into independent sub-problems. These sub-problems such as prediction of protein secondary structure can then be solved independently. In a previous study, we found that an iterative use of predicted secondary structure and backbone torsion angles can further improve secondary structure and torsion angle prediction. In this study, we expand the iterative features to include solvent accessible surface area and backbone angles and dihedrals based on Cα atoms. By using a deep learning neural network in three iterations, we achieved 82% accuracy for secondary structure prediction, 0.76 for the correlation coefficient between predicted and actual solvent accessible surface area, 19° and 30° for mean absolute errors of backbone φ and ψ angles, respectively, and 8° and 32° for mean absolute errors of Cα-based θ and τ angles, respectively, for an independent test dataset of 1199 proteins. The accuracy of the method is slightly lower for 72 CASP 11 targets but much higher than those of model structures from current state-of-the-art techniques. This suggests the potentially beneficial use of these predicted properties for model assessment and ranking. PMID:26098304

  19. Improving prediction of secondary structure, local backbone angles, and solvent accessible surface area of proteins by iterative deep learning.

    PubMed

    Heffernan, Rhys; Paliwal, Kuldip; Lyons, James; Dehzangi, Abdollah; Sharma, Alok; Wang, Jihua; Sattar, Abdul; Yang, Yuedong; Zhou, Yaoqi

    2015-01-01

    Direct prediction of protein structure from sequence is a challenging problem. An effective approach is to break it up into independent sub-problems. These sub-problems such as prediction of protein secondary structure can then be solved independently. In a previous study, we found that an iterative use of predicted secondary structure and backbone torsion angles can further improve secondary structure and torsion angle prediction. In this study, we expand the iterative features to include solvent accessible surface area and backbone angles and dihedrals based on Cα atoms. By using a deep learning neural network in three iterations, we achieved 82% accuracy for secondary structure prediction, 0.76 for the correlation coefficient between predicted and actual solvent accessible surface area, 19° and 30° for mean absolute errors of backbone φ and ψ angles, respectively, and 8° and 32° for mean absolute errors of Cα-based θ and τ angles, respectively, for an independent test dataset of 1199 proteins. The accuracy of the method is slightly lower for 72 CASP 11 targets but much higher than those of model structures from current state-of-the-art techniques. This suggests the potentially beneficial use of these predicted properties for model assessment and ranking. PMID:26098304

  20. Protein-associated water and secondary structure effect removal of blood proteins from metallic substrates.

    PubMed

    Anand, Gaurav; Zhang, Fuming; Linhardt, Robert J; Belfort, Georges

    2011-03-01

    Removing adsorbed protein from metals has significant health and industrial consequences. There are numerous protein-adsorption studies using model self-assembled monolayers or polymeric substrates but hardly any high-resolution measurements of adsorption and removal of proteins on industrially relevant transition metals. Surgeons and ship owners desire clean metal surfaces to reduce transmission of disease via surgical instruments and minimize surface fouling (to reduce friction and corrosion), respectively. A major finding of this work is that, besides hydrophobic interaction adhesion energy, water content in an adsorbed protein layer and secondary structure of proteins determined the access and hence ability to remove adsorbed proteins from metal surfaces with a strong alkaline-surfactant solution (NaOH and 5 mg/mL SDS in PBS at pH 11). This is demonstrated with three blood proteins (bovine serum albumin, immunoglobulin, and fibrinogen) and four transition metal substrates and stainless steel (platinum (Pt), gold (Au), tungsten (W), titanium (Ti), and 316 grade stainless steel (SS)). All the metallic substrates were checked for chemical contaminations like carbon and sulfur and were characterized using X-ray photoelectron spectroscopy (XPS). While Pt and Au surfaces were oxide-free (fairly inert elements), W, Ti, and SS substrates were associated with native oxide. Difference measurements between a quartz crystal microbalance with dissipation (QCM-D) and surface plasmon resonance spectroscopy (SPR) provided a measure of the water content in the protein-adsorbed layers. Hydrophobic adhesion forces, obtained with atomic force microscopy, between the proteins and the metals correlated with the amount of the adsorbed protein-water complex. Thus, the amount of protein adsorbed decreased with Pt, Au, W, Ti and SS, in this order. Neither sessile contact angle nor surface roughness of the metal substrates was useful as predictors here. All three globular proteins

  1. Protein-associated water and secondary structure effect removal of blood proteins from metallic substrates.

    PubMed

    Anand, Gaurav; Zhang, Fuming; Linhardt, Robert J; Belfort, Georges

    2011-03-01

    Removing adsorbed protein from metals has significant health and industrial consequences. There are numerous protein-adsorption studies using model self-assembled monolayers or polymeric substrates but hardly any high-resolution measurements of adsorption and removal of proteins on industrially relevant transition metals. Surgeons and ship owners desire clean metal surfaces to reduce transmission of disease via surgical instruments and minimize surface fouling (to reduce friction and corrosion), respectively. A major finding of this work is that, besides hydrophobic interaction adhesion energy, water content in an adsorbed protein layer and secondary structure of proteins determined the access and hence ability to remove adsorbed proteins from metal surfaces with a strong alkaline-surfactant solution (NaOH and 5 mg/mL SDS in PBS at pH 11). This is demonstrated with three blood proteins (bovine serum albumin, immunoglobulin, and fibrinogen) and four transition metal substrates and stainless steel (platinum (Pt), gold (Au), tungsten (W), titanium (Ti), and 316 grade stainless steel (SS)). All the metallic substrates were checked for chemical contaminations like carbon and sulfur and were characterized using X-ray photoelectron spectroscopy (XPS). While Pt and Au surfaces were oxide-free (fairly inert elements), W, Ti, and SS substrates were associated with native oxide. Difference measurements between a quartz crystal microbalance with dissipation (QCM-D) and surface plasmon resonance spectroscopy (SPR) provided a measure of the water content in the protein-adsorbed layers. Hydrophobic adhesion forces, obtained with atomic force microscopy, between the proteins and the metals correlated with the amount of the adsorbed protein-water complex. Thus, the amount of protein adsorbed decreased with Pt, Au, W, Ti and SS, in this order. Neither sessile contact angle nor surface roughness of the metal substrates was useful as predictors here. All three globular proteins

  2. Determination of Endosperm Protein Secondary Structure in Hard Wheat Breeding Lines using Synchrotron Infrared Microspectroscopy

    SciTech Connect

    Wetzel, D.; Bonwell, E; Fritz, T; Fritz, A

    2008-01-01

    One molecular aspect of mature hard wheat protein quality for breadmaking is the relative amount of endosperm protein in the {alpha}-helix form compared with that in other secondary structure forms including {beta}-sheet. Modeling of {alpha}-helix and {beta}-sheet absorption bands that contribute to the amide I band at 1650 cm{sup -1} was applied to more than 1500 spectra in this study. The microscopic view of wheat endosperm is dominated by many large starch granules with protein in between. The spectrum produced from in situ microspectroscopy of this mixture is dominated by carbohydrate bands from the large starch granules that fill up the field. The high spatial resolution achievable with synchrotron infrared microspectroscopy enables revealing good in situ spectra of the protein located interstitially. Synchrotron infrared microspectroscopic mapping of 4 {mu}m thick frozen sections of endosperm in the subaleurone region provides spectra from a large number of pixels. Pixels with protein-dominated spectra are sorted out from among adjacent pixels to minimize the starch absorption and scattering contributions. Subsequent data treatment to extract information from the amide I band requires a high signal to noise ratio. Although spectral interference of the carbohydrate band on the amide band is not a problem, the scattering produced by the large starch granules diminishes the signal to noise ratio throughout the spectrum. High density mapping was done on beamlines U2B and U10B at the National Synchrotron Light Source at Brookhaven National Laboratory, Upton, NY. Mapping with a single masked spot size of 5.5 {mu}m diameter or confocal 5 {mu}mX5{mu}m spot size, respectively, on the two beamlines used produced spectra for new breeding lines under current consideration. Appropriate data treatment allows calculation of a numerical estimate of the {alpha}-helix population relative to other secondary protein structures from the position and shape of the amide I

  3. Determination of Endosperm Protein Secondary Structure in Hard Wheat Breeding Lines using Synchrotron Infrared Microspectroscopy

    SciTech Connect

    Bonwell,E.; Fisher, T.; Fritz, A.; Wetzel, D.

    2008-01-01

    One molecular aspect of mature hard wheat protein quality for breadmaking is the relative amount of endosperm protein in the a-helix form compared with that in other secondary structure forms including {beta}-sheet. Modeling of a-helix and {beta}-sheet absorption bands that contribute to the amide I band at 1650 cm-1 was applied to more than 1500 spectra in this study. The microscopic view of wheat endosperm is dominated by many large starch granules with protein in between. The spectrum produced from in situ microspectroscopy of this mixture is dominated by carbohydrate bands from the large starch granules that fill up the field. The high spatial resolution achievable with synchrotron infrared microspectroscopy enables revealing good in situ spectra of the protein located interstitially. Synchrotron infrared microspectroscopic mapping of 4 {mu}m thick frozen sections of endosperm in the subaleurone region provides spectra from a large number of pixels. Pixels with protein-dominated spectra are sorted out from among adjacent pixels to minimize the starch absorption and scattering contributions. Subsequent data treatment to extract information from the amide I band requires a high signal to noise ratio. Although spectral interference of the carbohydrate band on the amide band is not a problem, the scattering produced by the large starch granules diminishes the signal to noise ratio throughout the spectrum. High density mapping was done on beamlines U2B and U10B at the National Synchrotron Light Source at Brookhaven National Laboratory, Upton, NY. Mapping with a single masked spot size of 5.5 {mu}m diameter or confocal 5 {mu}m x 5 {mu}m spot size, respectively, on the two beamlines used produced spectra for new breeding lines under current consideration. Appropriate data treatment allows calculation of a numerical estimate of the a-helix population relative to other secondary protein structures from the position and shape of the amide I absorption band. Current

  4. Fine Structure in the Secondary Electron Emission Peak for Diamond Crystal with (100) Negative Electron Affinity Surface

    NASA Technical Reports Server (NTRS)

    Asnin, V. M.; Krainsky, I. L.

    1998-01-01

    A fine structure was discovered in the low-energy peak of the secondary electron emission spectra of the diamond surface with negative electron affinity. We studied this structure for the (100) surface of the natural type-IIb diamond crystal. We have found that the low-energy peak consists of a total of four maxima. The relative energy positions of three of them could be related to the electron energy minima near the bottom of the conduction band. The fourth peak, having the lowest energy, was attributed to the breakup of the bulk exciton at the surface during the process of secondary electron emission.

  5. Prediction of protein secondary structure based on residue pair types and conformational states using dynamic programming algorithm.

    PubMed

    Sadeghi, Mehdi; Parto, Sahar; Arab, Shahriar; Ranjbar, Bijan

    2005-06-20

    We have used a statistical approach for protein secondary structure prediction based on information theory and simultaneously taking into consideration pairwise residue types and conformational states. Since the prediction of residue secondary structure by one residue window sliding make ambiguity in state prediction, we used a dynamic programming algorithm to find the path with maximum score. A score system for residue pairs in particular conformations is derived for adjacent neighbors up to ten residue apart in sequence. The three state overall per-residue accuracy, Q3, of this method in a jackknife test with dataset created from PDBSELECT is more than 70%.

  6. Including secondary structure, fossils and molecular dating in the centipede tree of life.

    PubMed

    Murienne, Jerome; Edgecombe, Gregory D; Giribet, Gonzalo

    2010-10-01

    A well-corroborated morphological scheme of interrelationships for centipedes, once broadly accepted, has been in conflict with molecular data with respect to deep branching events. Expanded taxonomic coverage compared to previous analyses adds longer fragments for 28S rRNA and a structural alignment as part of a sample of four genes (two nuclear ribosomal and two mitochondrial) for 111 extant species; these sequence data are combined with morphology under parsimony and maximum likelihood, exploring both traditional multiple sequence alignment and direct optimization approaches. Novel automated procedures to incorporate secondary structure information are also explored. The molecular data in combination yield trees that are highly congruent with morphology as regards the monophyly of all centipede orders as well as the major groups within each of the large orders. Regardless of the optimality criterion or alignment strategy, the Tasmanian/New Zealand Craterostigmomorpha is resolved in a different position by the molecular data than by morphology. Addition of morphology overturns the placement of Craterostigmomorpha in favour of the traditional morphological resolution and eliminates the need to posit major character reversals with respect to developmental mode and maternal care. Calibration of the tree with Palaeozoic and Mesozoic fossils for a relaxed clock analysis corroborates the palaeontological signal that divergences between centipede orders date to the Silurian and earliest Devonian, and familial divergences are likewise almost wholly Palaeozoic.

  7. Determination of the secondary structure of group II bulge loops using the fluorescent probe 2-aminopurine

    PubMed Central

    Dishler, Abigael L.; McMichael, Elizabeth L.; Serra, Martin J.

    2015-01-01

    Eleven RNA hairpins containing 2-aminopurine (2-AP) in either base-paired or single nucleotide bulge loop positions were optically melted in 1 M NaCl; and, the thermodynamic parameters ΔH°, ΔS°, ΔG°37, and TM for each hairpin were determined. Substitution of 2-AP for an A (adenosine) at a bulge position (where either the 2-AP or A is the bulge) in the stem of a hairpin, does not affect the stability of the hairpin. For group II bulge loops such as AA/U, where there is ambiguity as to which of the A residues is paired with the U, hairpins with 2-AP substituted for either the 5′ or 3′ position in the hairpin stem have similar stability. Fluorescent melts were performed to monitor the environment of the 2-AP. When the 2-AP was located distal to the hairpin loop on either the 5′ or 3′ side of the hairpin stem, the change in fluorescent intensity upon heating was indicative of an unpaired nucleotide. A database of phylogenetically determined RNA secondary structures was examined to explore the presence of naturally occurring bulge loops embedded within a hairpin stem. The distribution of bulge loops is discussed and related to the stability of hairpin structures. PMID:25805856

  8. Enzyme stability, thermodynamics and secondary structures of α-amylase as probed by the CD spectroscopy.

    PubMed

    Kikani, B A; Singh, S P

    2015-11-01

    An amylase of a thermophilic bacterium, Bacillus sp. TSSC-3 (GenBank Number, EU710557) isolated from the Tulsi Shyam hot spring reservoir (Gujarat, India) was purified to the homogeneity in a single step on phenyl sepharose 6FF. The molecular weight of the enzyme was 25kD, while the temperature and pH optima for the enzyme catalysis were 80°C and 7, respectively. The purified enzyme was highly thermostable with broad pH stability and displayed remarkable resistance against surfactants, chelators, urea, guanidine HCl and various solvents as well. The stability and changes in the secondary structure of the enzyme under various extreme conditions were determined by the circular dichroism (CD) spectroscopy. The stability trends and the changes in the α-helices and β-sheets were analyzed by Mean Residual Ellipticity (MRE) and K2D3. The CD data confirmed the structural stability of the enzyme under various harsh conditions, yet it indicated reduced α-helix content and increased β-sheets upon denaturation. The thermodynamic parameters; deactivation rate constant, half-life, changes in entropy, enthalpy, activation energy and Gibb's free energy indicated that the enzyme-substrate reactions were highly stable. The overall profile of the enzyme: high thermostability, alkalitolerance, calcium independent nature, dextrose equivalent values and resistance against chemical denaturants, solvents and surfactants suggest its commercial applications.

  9. Secondary structure and conformational change of mushroom polyphenol oxidase during thermosonication treatment by using FTIR spectroscopy.

    PubMed

    Baltacıoğlu, Hande; Bayındırlı, Alev; Severcan, Feride

    2017-01-01

    To understand the conformational changes of mushroom PPO, the secondary structural change of the enzyme during thermosonication treatment at different power (60, 80 and 100%), temperature (20-60°C) and time (0-30min) combinations was investigated by using FTIR spectroscopy and compared with the change in enzyme activity. The enzyme inactivation higher than 99% was obtained at 100% amplitude at 60°C for 10min. FTIR studies showed that marked spectral changes were noted after ultrasound treatment at 20°C. The α-helix and β-sheet contents decreased, while aggregated β-sheet, turns and random coil contents increased as temperature increased up to 60°C during thermosonication treatment for 10min indicating protein denaturation. Aggregated bands located at 1683 and 1616cm(-1) became evident after ultrasound treatment at 40°C. When temperature was lowered back to 25°C, from ultrasound treatment at 60°C, these bands were still observed, indicating the irreversible change in the structure. PMID:27507504

  10. Bimane Fluorescence Scanning Suggests Secondary Structure near the S3-S4 Linker of BK Channels*

    PubMed Central

    Semenova, Nina P.; Abarca-Heidemann, Karin; Loranc, Eva; Rothberg, Brad S.

    2009-01-01

    Gating of large conductance Ca2+-activated K+ channels (BK or maxi-K channels) is controlled by a Ca2+-sensor, formed by the channel cytoplasmic C-terminal domain, and a voltage sensor, formed by its S0-S4 transmembrane helices. Here we analyze structural properties of a portion of the BK channel voltage sensing domain, the S3-S4 linker, using fluorescence lifetime spectroscopy. Single residues in the S3-S4 linker region were substituted with cysteine, and the cysteine-substituted mutants were expressed in CHO cells and covalently labeled with the sulfhydryl-reactive fluorophore monobromo-trimethylammonio-bimane (qBBr). qBBr fluorescence is quenched by tryptophan and, to a lesser extent, tyrosine side chains. We found that qBBr fluorescence in several of the labeled cysteine-substituted channels shows position-specific quenching, as indicated by increase of the brief lifetime component of the qBBr fluorescence decay. Quenching was reduced with the mutation W203F (in the S4 segment), suggesting that Trp-203 acts as a quenching group. Our results suggest a working hypothesis for the secondary structure of the BK channel S3-S4 region, and places residues Leu-204, Gly-205, and Leu-206 within the extracellular end of the S4 helix. PMID:19244238

  11. A Deep Learning Network Approach to ab initio Protein Secondary Structure Prediction

    PubMed Central

    Spencer, Matt; Eickholt, Jesse; Cheng, Jianlin

    2014-01-01

    Ab initio protein secondary structure (SS) predictions are utilized to generate tertiary structure predictions, which are increasingly demanded due to the rapid discovery of proteins. Although recent developments have slightly exceeded previous methods of SS prediction, accuracy has stagnated around 80% and many wonder if prediction cannot be advanced beyond this ceiling. Disciplines that have traditionally employed neural networks are experimenting with novel deep learning techniques in attempts to stimulate progress. Since neural networks have historically played an important role in SS prediction, we wanted to determine whether deep learning could contribute to the advancement of this field as well. We developed an SS predictor that makes use of the position-specific scoring matrix generated by PSI-BLAST and deep learning network architectures, which we call DNSS. Graphical processing units and CUDA software optimize the deep network architecture and efficiently train the deep networks. Optimal parameters for the training process were determined, and a workflow comprising three separately trained deep networks was constructed in order to make refined predictions. This deep learning network approach was used to predict SS for a fully independent test data set of 198 proteins, achieving a Q3 accuracy of 80.7% and a Sov accuracy of 74.2%. PMID:25750595

  12. Assessing secondary structure of a dyed wool fibre by means of FTIR and FTR spectroscopies

    NASA Astrophysics Data System (ADS)

    Pielesz, A.; Freeman, H. S.; Wesełucha-Birczyńska, A.; Wysocki, M.; Włochowicz, A.

    2003-06-01

    The paper describes changes in the structure of a wool fibre dyed with model azo dyes. These were direct dyes, non-genotoxic derivatives of carcinogenic benzidine, synthesized specially for the purpose of the experiment. The non-mutagenic benzidine derivatives were: 2,2'-dimethyl-5,5'-dipropoxybenzidine and 5,5'-dipropoxybenzidine. Using FTIR, changes in secondary structure of fibres were assessed in three measuring ranges: 3600-3000, 1700-1400 and 1000-1300 cm -1. The dyes were found to distinctively affect wave-number shifts of amide A, amide I bands and in the fingerprint area around 1050 cm -1. It seems that these three areas are related to the sites in which dyes bind with wool fibre keratin. In FTR spectra, the focus was on assessing the changes of peptide bond configuration in the area of amide I, disulfide area of cystine and the area of the interaction between dyes and wool fibre keratin, i.e. 1250-1600 cm -1. For analysis, three kinds of materials were selected: (1) raw wool fibres, (2) fibres subjected to deuteration and treated with formic acid, (3) wool fabric. Each of them was dyed with the model azo dyes. The results obtained by both spectroscopies allow for identifying the functional groups responsible for the binding of dyes with keratin fibre.

  13. A Deep Learning Network Approach to ab initio Protein Secondary Structure Prediction.

    PubMed

    Spencer, Matt; Eickholt, Jesse; Jianlin Cheng

    2015-01-01

    Ab initio protein secondary structure (SS) predictions are utilized to generate tertiary structure predictions, which are increasingly demanded due to the rapid discovery of proteins. Although recent developments have slightly exceeded previous methods of SS prediction, accuracy has stagnated around 80 percent and many wonder if prediction cannot be advanced beyond this ceiling. Disciplines that have traditionally employed neural networks are experimenting with novel deep learning techniques in attempts to stimulate progress. Since neural networks have historically played an important role in SS prediction, we wanted to determine whether deep learning could contribute to the advancement of this field as well. We developed an SS predictor that makes use of the position-specific scoring matrix generated by PSI-BLAST and deep learning network architectures, which we call DNSS. Graphical processing units and CUDA software optimize the deep network architecture and efficiently train the deep networks. Optimal parameters for the training process were determined, and a workflow comprising three separately trained deep networks was constructed in order to make refined predictions. This deep learning network approach was used to predict SS for a fully independent test dataset of 198 proteins, achieving a Q3 accuracy of 80.7 percent and a Sov accuracy of 74.2 percent.

  14. Strategies for Selection from Protein Libraries Composed of de Novo Designed Secondary Structure Modules

    NASA Astrophysics Data System (ADS)

    Matsuura, Tomoaki; Plückthun, Andreas

    2004-02-01

    As more and more protein structures are determined, it has become clear that there is only a limited number of protein folds in nature. To explore whether the protein folds found in nature are the only solutions to the protein folding problem, or that a lack of evolutionary pressure causes the paucity of different protein folds found, we set out to construct protein libraries without any restriction on topology. We generated different libraries (all α-helix, all β-strand and α-helix plus β-strand) with an average length of 100 amino acid residues, composed of designed secondary structure modules (α-helix, β-strand and β-turn) in various proportions, based primarily on the patterning of polar and non-polar residues. From the analysis of proteins chosen randomly from the libraries, we found that a substantial portion of pure α-helical proteins show properties similar to native proteins. Using these libraries as a starting point, we aim to establish a selection system which allows us to enrich proteins with favorable folding properties (non-aggregating, compactly folded) from the libraries. We have developed such a method based on ribosome display. This selection is based on two concepts: (1) misfolded proteins are more sensitive to proteolysis, (2) misfolded and/or aggregated proteins are more hydrophobic. We show that by applying each of these selection criteria proteins that are compactly folded and soluble can be enriched over insoluble and random coil proteins.

  15. Bimane fluorescence scanning suggests secondary structure near the S3-S4 linker of BK channels.

    PubMed

    Semenova, Nina P; Abarca-Heidemann, Karin; Loranc, Eva; Rothberg, Brad S

    2009-04-17

    Gating of large conductance Ca(2+)-activated K(+) channels (BK or maxi-K channels) is controlled by a Ca(2+)-sensor, formed by the channel cytoplasmic C-terminal domain, and a voltage sensor, formed by its S0-S4 transmembrane helices. Here we analyze structural properties of a portion of the BK channel voltage sensing domain, the S3-S4 linker, using fluorescence lifetime spectroscopy. Single residues in the S3-S4 linker region were substituted with cysteine, and the cysteine-substituted mutants were expressed in CHO cells and covalently labeled with the sulfhydryl-reactive fluorophore monobromo-trimethylammonio-bimane (qBBr). qBBr fluorescence is quenched by tryptophan and, to a lesser extent, tyrosine side chains. We found that qBBr fluorescence in several of the labeled cysteine-substituted channels shows position-specific quenching, as indicated by increase of the brief lifetime component of the qBBr fluorescence decay. Quenching was reduced with the mutation W203F (in the S4 segment), suggesting that Trp-203 acts as a quenching group. Our results suggest a working hypothesis for the secondary structure of the BK channel S3-S4 region, and places residues Leu-204, Gly-205, and Leu-206 within the extracellular end of the S4 helix.

  16. Functional dissection of nuclear envelope mRNA translocation system: effects of phorbol ester and a monoclonal antibody recognizing cytoskeletal structures.

    PubMed

    Schröder, H C; Diehl-Seifert, B; Rottmann, M; Messer, R; Bryson, B A; Agutter, P S; Müller, W E

    1988-03-01

    Unidirectional transport of poly(A)-containing mRNA [poly(A)+ mRNA] through the nuclear envelope pore complex is thought to be an energy (ATP or GTP)-dependent process which involves a nuclear envelope nucleoside triphosphatase (NTPase). In the intact envelope, this enzyme is regulatable by poly(A) binding and by poly(A)-dependent phosphorylation/dephosphorylation of other components of the mRNA translocation system, which are as yet unidentified. Monoclonal antibodies (mAbs) were elicited against the poly(A) binding nuclear envelope fraction isolated from rat liver. The mAbs were screened for their modulatory effects on mRNA transport in vitro. One stable clone decreased the efflux of rapidly labeled RNA and of one specific mRNA (ovalbumin) from isolated nuclei. It increased the binding of poly(A) to the envelope and increased the maximal catalytic rate of the NTPase, but it did not alter the apparent Km of the enzyme or the extent of its stimulation by poly(A). The nuclear envelope-associated protein kinase that down-regulates the NTPase was inhibited by the antibody, while other protein kinases were not affected. Because both the NTPase and mRNA efflux were inhibited by the tumor promoter, 12-O-tetradecanoylphorbol 13-acetate, the sensitive kinase is probably protein kinase C. Protein kinase C was found to be associated with the isolated nuclear envelope. The antibody reacted with both a Mr 83,000 and a Mr 65,000 nuclear envelope polypeptide from rat liver and other tissues. By immunofluorescence microscopy in CV-1 cells, the antibody localized to the nuclear envelope and, in addition, to cytoplasmic filaments which show some superposition with the microfilament network.

  17. Synthesis and characterization of nitroaromatic peptoids: fine tuning peptoid secondary structure through monomer position and functionality.

    PubMed

    Fowler, Sarah A; Luechapanichkul, Rinrada; Blackwell, Helen E

    2009-02-20

    N-substituted glycine oligomers, or peptoids, have emerged as an important class of foldamers for the study of biomolecular interactions and for potential use as therapeutic agents. However, the design of peptoids with well-defined conformations a priori remains a formidable challenge. New approaches are required to address this problem, and the systematic study of the role of individual monomer units in the global peptoid folding process represents one strategy. Here, we report our efforts toward this approach through the design, synthesis, and characterization of peptoids containing nitroaromatic monomer units. This work required the synthesis of a new chiral amine building block, (S)-1-(2-nitrophenyl)ethanamine (s2ne), which could be readily installed into peptoids using standard solid-phase peptoid synthesis techniques. We designed a series of peptoid nonamers that allowed us to probe the effects of this relatively electron-deficient and sterically encumbered alpha-chiral side chain on peptoid structure, namely, the peptoid threaded loop and helix. Circular dichroism spectroscopy of the peptoids revealed that the nitroaromatic monomer has a significant effect on peptoid secondary structure. Specifically, the threaded loop structure was disrupted in a nonamer containing alternating N-(S)-1-phenylethylglycine (Nspe) and Ns2ne monomers, and the major conformation was helical instead. Indeed, placement of a single Ns2ne at the N-terminal position of (Nspe)(9) resulted in a destabilized form of the threaded loop structure relative to the homononamer (Nspe)(9). Conversely, we observed that incorporation of N-(S)-1-(4-nitrophenyl)ethylglycine (Nsnp, a p-nitro monomer) at the N-terminal position stabilized the threaded loop structure relative to (Nspe)(9). Additional experiments revealed that nitroaromatic side chains can influence peptoid nonamer folding by modulating the strength of key intramolecular hydrogen bonds in the peptoid threaded loop structure. Steric

  18. Protein kinase A stimulates binding of multiple proteins to a U-rich domain in the 3'-untranslated region of lactate dehydrogenase A mRNA that is required for the regulation of mRNA stability.

    PubMed

    Tian, D; Huang, D; Brown, R C; Jungmann, R A

    1998-10-23

    We have explored the molecular basis of the cAMP-induced stabilization of lactate dehydrogenase A (LDH-A) mRNA and identified four cytoplasmic proteins of 96, 67, 52, and 50 kDa that specifically bind to a 30-nucleotide uridine-rich sequence in the LDH 3'-untranslated region with a predicted stem-loop structure. Mutational analysis revealed that specific protein binding is dependent upon an intact primary nucleotide sequence in the loop as well as integrity of the adjoining double-stranded stem structure, thus indicating a high degree of primary and secondary structure specificity. The critical stem-loop region is located between nucleotides 1473 and 1502 relative to the mRNA cap site and contains a previously identified cAMP-stabilizing region (CSR) required for LDH-A mRNA stability regulation by the protein kinase A pathway. The 3'-untranslated region binding activity of the proteins is up-regulated after protein kinase A activation, whereas protein dephosphorylation is associated with a loss of binding activity. These results imply a cause and effect relationship between LDH-A mRNA stabilization and CSR-phosphoprotein binding activity. We propose that the U-rich CSR is a recognition signal for CSR-binding proteins and for an mRNA processing pathway that specifically stabilizes LDH mRNA in response to activation of the protein kinase A signal transduction pathway.

  19. Residual Structure of Streptococcus mutans Biofilm following Complete Disinfection Favors Secondary Bacterial Adhesion and Biofilm Re-Development

    PubMed Central

    Ohsumi, Tatsuya; Takenaka, Shoji; Wakamatsu, Rika; Sakaue, Yuuki; Narisawa, Naoki; Senpuku, Hidenobu; Ohshima, Hayato; Terao, Yutaka; Okiji, Takashi

    2015-01-01

    Chemical disinfection of oral biofilms often leaves biofilm structures intact. This study aimed to examine whether the residual structure promotes secondary bacterial adhesion. Streptococcus mutans biofilms generated on resin-composite disks in a rotating disc reactor were disinfected completely with 70% isopropyl alcohol, and were again cultured in the same reactor after resupplying with the same bacterial solution. Specimens were subjected to fluorescence confocal laser scanning microscopy, viable cell counts and PCR-Invader assay in order to observe and quantify secondarily adhered cells. Fluorescence microscopic analysis, particularly after longitudinal cryosectioning, demonstrated stratified patterns of viable cells on the disinfected biofilm structure. Viable cell counts of test specimens were significantly higher than those of controls, and increased according to the amount of residual structure and culture period. Linear regression analysis exhibited a high correlation between viable and total cell counts. It was concluded that disinfected biofilm structures favored secondary bacterial adhesion. PMID:25635770

  20. Imaging the 3D structure of secondary osteons in human cortical bone using phase-retrieval tomography

    NASA Astrophysics Data System (ADS)

    Arhatari, B. D.; Cooper, D. M. L.; Thomas, C. D. L.; Clement, J. G.; Peele, A. G.

    2011-08-01

    By applying a phase-retrieval step before carrying out standard filtered back-projection reconstructions in tomographic imaging, we were able to resolve structures with small differences in density within a densely absorbing sample. This phase-retrieval tomography is particularly suited for the three-dimensional segmentation of secondary osteons (roughly cylindrical structures) which are superimposed upon an existing cortical bone structure through the process of turnover known as remodelling. The resulting images make possible the analysis of the secondary osteon structure and the relationship between an osteon and the surrounding tissue. Our observations have revealed many different and complex 3D structures of osteons that could not be studied using previous methods. This work was carried out using a laboratory-based x-ray source, which makes obtaining these sorts of images readily accessible.

  1. Lost in folding space? Comparing four variants of the thermodynamic model for RNA secondary structure prediction

    PubMed Central

    2011-01-01

    Background Many bioinformatics tools for RNA secondary structure analysis are based on a thermodynamic model of RNA folding. They predict a single, "optimal" structure by free energy minimization, they enumerate near-optimal structures, they compute base pair probabilities and dot plots, representative structures of different abstract shapes, or Boltzmann probabilities of structures and shapes. Although all programs refer to the same physical model, they implement it with considerable variation for different tasks, and little is known about the effects of heuristic assumptions and model simplifications used by the programs on the outcome of the analysis. Results We extract four different models of the thermodynamic folding space which underlie the programs RNAFOLD, RNASHAPES, and RNASUBOPT. Their differences lie within the details of the energy model and the granularity of the folding space. We implement probabilistic shape analysis for all models, and introduce the shape probability shift as a robust measure of model similarity. Using four data sets derived from experimentally solved structures, we provide a quantitative evaluation of the model differences. Conclusions We find that search space granularity affects the computed shape probabilities less than the over- or underapproximation of free energy by a simplified energy model. Still, the approximations perform similar enough to implementations of the full model to justify their continued use in settings where computational constraints call for simpler algorithms. On the side, we observe that the rarely used level 2 shapes, which predict the complete arrangement of helices, multiloops, internal loops and bulges, include the "true" shape in a rather small number of predicted high probability shapes. This calls for an investigation of new strategies to extract high probability members from the (very large) level 2 shape space of an RNA sequence. We provide implementations of all four models, written in a

  2. Understanding the effect of secondary structure on molecular interactions of poly-L-lysine with different substrates by SFA.

    PubMed

    Binazadeh, Mojtaba; Faghihnejad, Ali; Unsworth, Larry D; Zeng, Hongbo

    2013-10-14

    Nonspecific adsorption of proteins on biomaterial surfaces challenges the widespread application of engineered materials, and understanding the impact of secondary structure of proteins and peptides on their adsorption process is of both fundamental and practical importance in bioengineering. In this work, poly-L-lysine (PLL)-based α-helices and β-sheets were chosen as a model system to investigate the effect of secondary structure on peptide interactions with substrates of various surface chemistries. Circular dichroism (CD) was used to confirm the presence of both α-helix and β-sheet structured PLL in aqueous solutions and upon adsorption to quartz, where these secondary structures seemed to be preserved. Atomic force microscopy (AFM) imaging showed different surface patterns for adsorbed α-helix and β-sheet PLL. Interactions between PLL of different secondary structures and various substrates (i.e., PLL, Au, mica, and poly(ethylene glycol) (PEG)) were directly measured using a surface forces apparatus (SFA). It was found that β-sheet PLL films showed higher adsorbed layer thicknesses in general. Adhesion energies of β-sheet versus Au and β-sheet versus β-sheet were considerably higher than that of α-helix versus Au and α-helix versus α-helix systems, respectively. Au and β-sheet PLL interactions seemed to be more dependent on the salt concentration than that of α-helix, while the presence of a grafted PEG layer greatly diminished any attraction with either PLL structure. The molecular interaction mechanism of peptide in different secondary structures is discussed in terms of Derjaguin-Landau-Verwey-Overbeek (DLVO) theory, Alexander-de Gennes (AdG) steric model and hydrogen bonding, which provides important insight into the fundamental understanding of the interaction mechanism between proteins and biomaterials. PMID:24032485

  3. Peptoid oligomers with alpha-chiral, aromatic side chains: effects of chain length on secondary structure.

    PubMed

    Wu, C W; Sanborn, T J; Zuckermann, R N; Barron, A E

    2001-04-01

    Oligomeric N-substituted glycines or "peptoids" with alpha-chiral, aromatic side chains can adopt stable helices in organic or aqueous solution, despite their lack of backbone chirality and their inability to form intrachain hydrogen bonds. Helical ordering appears to be stabilized by avoidance of steric clash as well as by electrostatic repulsion between backbone carbonyls and pi clouds of aromatic rings in the side chains. Interestingly, these peptoid helices exhibit intense circular dichroism (CD) spectra that closely resemble those of peptide alpha-helices. Here, we have utilized CD to systematically study the effects of oligomer length, concentration, and temperature on the chiral secondary structure of organosoluble peptoid homooligomers ranging from 3 to 20 (R)-N-(1-phenylethyl)glycine (Nrpe) monomers in length. We find that a striking evolution in CD spectral features occurs for Nrpe oligomers between 4 and 12 residues in length, which we attribute to a chain length-dependent population of alternate structured conformers having cis versus trans amide bonds. No significant changes are observed in CD spectra of oligomers between 13 and 20 monomers in length, suggesting a minimal chain length of about 13 residues for the formation of stable poly(Nrpe) helices. Moreover, no dependence of circular dichroism on concentration is observed for an Nrpe hexamer, providing evidence that these helices remain monomeric in solution. In light of these new data, we discuss chain length-related factors that stabilize organosoluble peptoid helices of this class, which are important for the design of helical, biomimetic peptoids sharing this structural motif.

  4. Inducible expression of Pisum sativum xyloglucan fucosyltransferase in the pea root cap meristem, and effects of antisense mRNA expression on root cap cell wall structural integrity.

    PubMed

    Wen, Fushi; Celoy, Rhodesia M; Nguyen, Trang; Zeng, Weiqing; Keegstra, Kenneth; Immerzeel, Peter; Pauly, Markus; Hawes, Martha C

    2008-07-01

    Mitosis and cell wall synthesis in the legume root cap meristem can be induced and synchronized by the nondestructive removal of border cells from the cap periphery. Newly synthesized cells can be examined microscopically as they differentiate progressively during cap development, and ultimately detach as a new population of border cells. This system was used to demonstrate that Pisum sativum L. fucosyl transferase (PsFut1) mRNA expression is strongly expressed in root meristematic tissues, and is induced >2-fold during a 5-h period when mitosis in the root cap meristem is increased. Expression of PsFut1 antisense mRNA in pea hairy roots under the control of the CaMV35S promoter, which exhibits meristem localized expression in pea root caps, resulted in a 50-60% reduction in meristem localized endogenous PsFut1 mRNA expression measured using whole mount in situ hybridization. Changes in gross levels of cell wall fucosylated xyloglucan were not detected, but altered surface localization patterns were detected using whole mount immunolocalization with CCRC-M1, an antibody that recognizes fucosylated xyloglucan. Emerging hairy roots expressing antisense PsFut1 mRNA appeared normal macroscopically but scanning electron microscopy of tissues with altered CCRC-M1 localization patterns revealed wrinkled, collapsed cell surfaces. As individual border cells separated from the cap periphery, cell death occurred in correlation with extrusion of cellular contents through breaks in the wall.

  5. Orientation Determination of Protein Helical Secondary Structure Using Linear and Nonlinear Vibrational Spectroscopy

    PubMed Central

    Nguyen, Khoi Tan; Le Clair, Stéphanie V.; Ye, Shuji; Chen, Zhan

    2009-01-01

    In this paper, we systematically presented the orientation determination of protein helical secondary structures using vibrational spectroscopic methods, particularly the nonlinear Sum Frequency Generation (SFG) vibrational spectroscopy, along with linear vibrational spectroscopic techniques such as infrared spectroscopy and Raman scattering. SFG amide I signals can be collected using different polarization combinations of the input laser beams and output signal beam to measure the second order nonlinear optical susceptibility components of the helical amide I modes, which are related to their molecular hyperpolarizability elements through the orientation distribution of these helices. The molecular hyperpolarizability elements of amide I modes of a helix can be calculated based on the infrared transition dipole moment and Raman polarizability tensor of the helix; these quantities are determined by using the bond additivity model to sum over the individual infrared dipole transition moments and Raman polarizability tensors, respectively, of the peptide units (or the amino acid residues). The computed overall infrared transition dipole moment and Raman polarizability tensor of a helix can be validated by experimental data using polarized infrared and polarized Raman spectroscopy on samples with well-aligned helical structures. From the deduced SFG hyperpolarizability elements and measured SFG second order nonlinear susceptibility components, orientation information regarding helical structures can be determined. Even though such orientation information can also be measured using polarized infrared or polarized Raman amide I signals, SFG has a much lower detection limit, which can be used to study the orientation of a helix when its surface coverage is much lower than a monolayer. In addition, the combination of different vibrational spectroscopic techniques, e.g., SFG and Attenuated Total Reflectance – Fourier Transform Infrared spectroscopy, provides more

  6. Computational and molecular analysis of conserved influenza A virus RNA secondary structures involved in infectious virion production.

    PubMed

    Kobayashi, Yuki; Dadonaite, Bernadeta; van Doremalen, Neeltje; Suzuki, Yoshiyuki; Barclay, Wendy S; Pybus, Oliver G

    2016-09-01

    As well as encoding viral proteins, genomes of RNA viruses harbor secondary and tertiary RNA structures that have been associated with functions essential for successful replication and propagation. Here, we identified stem-loop structures that are extremely conserved among 1,884 M segment sequences of influenza A virus (IAV) strains from various subtypes and host species using computational and evolutionary methods. These structures were predicted within the 3' and 5' ends of the coding regions of M1 and M2, respectively, where packaging signals have been previously proposed to exist. These signals are thought to be required for the incorporation of a single copy of 8 different negative-strand RNA segments (vRNAs) into an IAV particle. To directly test the functionality of conserved stem-loop structures, we undertook reverse genetic experiments to introduce synonymous mutations designed to disrupt secondary structures predicted at 3 locations and found them to attenuate infectivity of recombinant virus. In one mutant, predicted to disrupt stem loop structure at nucleotide positions 219-240, attenuation was more evident at increased temperature and was accompanied by an increase in the production of defective virus particles. Our results suggest that the conserved secondary structures predicted in the M segment are involved in the production of infectious viral particles during IAV replication.

  7. Observed Lesson Structure during the First Year of Secondary Education: Exploration of Change and Link with Academic Engagement

    ERIC Educational Resources Information Center

    Maulana, Ridwan; Opdenakker, Marie-Christine; Stroet, Kim; Bosker, Roel

    2012-01-01

    This study investigates whether lesson structure (LS) matters and which components are important for academic engagement during the first grade of secondary education. Data from videoed lessons of 10 Dutch and 12 Indonesian teachers analyzed using an observation protocol show that six LS components are found, that between class and over…

  8. DNA secondary structure of the released strand stimulates WRN helicase action on forked duplexes without coordinate action of WRN exonuclease

    SciTech Connect

    Ahn, Byungchan; Bohr, Vilhelm A.

    2011-08-12

    Highlights: {yields} In this study, we investigated the effect of a DNA secondary structure on the two WRN activities. {yields} We found that a DNA secondary structure of the displaced strand during unwinding stimulates WRN helicase without coordinate action of WRN exonuclease. {yields} These results imply that WRN helicase and exonuclease activities can act independently. -- Abstract: Werner syndrome (WS) is an autosomal recessive premature aging disorder characterized by aging-related phenotypes and genomic instability. WS is caused by mutations in a gene encoding a nuclear protein, Werner syndrome protein (WRN), a member of the RecQ helicase family, that interestingly possesses both helicase and exonuclease activities. Previous studies have shown that the two activities act in concert on a single substrate. We investigated the effect of a DNA secondary structure on the two WRN activities and found that a DNA secondary structure of the displaced strand during unwinding stimulates WRN helicase without coordinate action of WRN exonuclease. These results imply that WRN helicase and exonuclease activities can act independently, and we propose that the uncoordinated action may be relevant to the in vivo activity of WRN.

  9. Effect of Programmed Instruction on Students' Attitude towards Structure of the Atom and the Periodic Table among Kenyan Secondary Schools

    ERIC Educational Resources Information Center

    Wangila, M. J.; Martin, W.; Ronald, M.

    2015-01-01

    This study examined the effect of Programmed Instruction on students' attitude towards Structure of the Atom and the Periodic Table (SAPT) among mixed (co-educational) secondary schools of Butere district, Kakamega county, Kenya. The quasi-experimental research design was adopted, using the nonrandomized Solomon four-group as a model. The sample…

  10. Decentralization and Structural Change in Secondary Education in Argentina: The Case of the Province of Buenos Aires

    ERIC Educational Resources Information Center

    Acedo, Clementina; Gorostiaga, Jorge M.; Senen-Gonzalez, Silvia

    2007-01-01

    Over the past decade, Argentina created and implemented a compulsory lower-secondary education level, within an ambitious educational reform programme. This article addresses the reform at the national level, diverse provincial responses, and the particular way that the powerful province of Buenos Aires appropriated the structural change. The…

  11. Inclusion of persistence length-based secondary structure in replica field theoretic models of heteropolymer freezing

    NASA Astrophysics Data System (ADS)

    Weber, Jeffrey K.; Pande, Vijay S.

    2013-09-01

    The protein folding problem has long represented a "holy grail" in statistical physics due to its physical complexity and its relevance to many human diseases. While past theoretical work has yielded apt descriptions of protein folding landscapes, recent large-scale simulations have provided insights into protein folding that were impractical to obtain from early theories. In particular, the role that non-native contacts play in protein folding, and their relation to the existence of misfolded, β-sheet rich trap states on folding landscapes, has emerged as a topic of interest in the field. In this paper, we present a modified model of heteropolymer freezing that includes explicit secondary structural characteristics which allow observations of "intramolecular amyloid" states to be probed from a theoretical perspective. We introduce a variable persistence length-based energy penalty to a model Hamiltonian, and we illustrate how this modification alters the phase transitions present in the theory. We find, in particular, that inclusion of this variable persistence length increases both generic freezing and folding temperatures in the model, allowing both folding and glass transitions to occur in a more highly optimized fashion. We go on to discuss how these changes might relate to protein evolution, misfolding, and the emergence of intramolecular amyloid states.

  12. Line-Based Object Recognition using Hausdorff Distance: From Range Images to Molecular Secondary Structure

    SciTech Connect

    Guerra, C; Pascucci, V

    2004-12-13

    Object recognition algorithms are fundamental tools in automatic matching of geometric shapes within a background scene. Many approaches have been proposed in the past to solve the object recognition problem. Two of the key aspects that distinguish them in terms of their practical usability are: (i) the type of input model description and (ii) the comparison criteria used. In this paper we introduce a novel scheme for 3D object recognition based on line segment representation of the input shapes and comparison using the Hausdor distance. This choice of model representation provides the flexibility to apply the scheme in different application areas. We define several variants of the Hausdor distance to compare the models within the framework of well defined metric spaces. We present a matching algorithm that efficiently finds a pattern in a 3D scene. The algorithm approximates a minimization procedure of the Hausdor distance. The output error due to the approximation is guaranteed to be within a known constant bound. Practical results are presented for two classes of objects: (i) polyhedral shapes extracted from segmented range images and (ii) secondary structures of large molecules. In both cases the use of our approximate algorithm allows to match correctly the pattern in the background while achieving the efficiency necessary for practical use of the scheme. In particular the performance is improved substantially with minor degradation of the quality of the matching.

  13. Fine-grained parallelism accelerating for RNA secondary structure prediction with pseudoknots based on FPGA.

    PubMed

    Xia, Fei; Jin, Guoqing

    2014-06-01

    PKNOTS is a most famous benchmark program and has been widely used to predict RNA secondary structure including pseudoknots. It adopts the standard four-dimensional (4D) dynamic programming (DP) method and is the basis of many variants and improved algorithms. Unfortunately, the O(N(6)) computing requirements and complicated data dependency greatly limits the usefulness of PKNOTS package with the explosion in gene database size. In this paper, we present a fine-grained parallel PKNOTS package and prototype system for accelerating RNA folding application based on FPGA chip. We adopted a series of storage optimization strategies to resolve the "Memory Wall" problem. We aggressively exploit parallel computing strategies to improve computational efficiency. We also propose several methods that collectively reduce the storage requirements for FPGA on-chip memory. To the best of our knowledge, our design is the first FPGA implementation for accelerating 4D DP problem for RNA folding application including pseudoknots. The experimental results show a factor of more than 50x average speedup over the PKNOTS-1.08 software running on a PC platform with Intel Core2 Q9400 Quad CPU for input RNA sequences. However, the power consumption of our FPGA accelerator is only about 50% of the general-purpose micro-processors.

  14. Essential nucleotide sequences and secondary structure elements of the hairpin ribozyme.

    PubMed Central

    Berzal-Herranz, A; Joseph, S; Chowrira, B M; Butcher, S E; Burke, J M

    1993-01-01

    In vitro selection experiments have been used to isolate active variants of the 50 nt hairpin catalytic RNA motif following randomization of individual ribozyme domains and intensive mutagenesis of the ribozyme-substrate complex. Active and inactive variants were characterized by sequencing, analysis of RNA cleavage activity in cis and in trans, and by substrate binding studies. Results precisely define base-pairing requirements for ribozyme helices 3 and 4, and identify eight essential nucleotides (G8, A9, A10, G21, A22, A23, A24 and C25) within the catalytic core of the ribozyme. Activity and substrate binding assays show that point mutations at these eight sites eliminate cleavage activity but do not significantly decrease substrate binding, demonstrating that these bases contribute to catalytic function. The mutation U39C has been isolated from different selection experiments as a second-site suppressor of the down mutants G21U and A43G. Assays of the U39C mutation in the wild-type ribozyme and in a variety of mutant backgrounds show that this variant is a general up mutation. Results from selection experiments involving populations totaling more than 10(10) variants are summarized, and consensus sequences including 16 essential nucleotides and a secondary structure model of four short helices, encompassing 18 bp for the ribozyme-substrate complex are derived. Images PMID:8508779

  15. An essential secondary structure in the 3' untranslated region of the mouse hepatitis virus genome.

    PubMed

    Hsue, B; Masters, P S

    1998-01-01

    The 3' untranslated regions (3' UTRs) of coronaviruses contain the signals necessary for negative strand RNA synthesis and may also harbor elements essential for positive strand replication and subgenomic RNA transcription. The 3' UTRs of mouse hepatitis virus (MHV) and bovine coronavirus (BCV) are more than 30% divergent. In an effort to learn what parts of these regions might be functionally interchangeable, we attempted to replace the 3' UTR of MHV with its BCV counterpart by targeted RNA recombination. Initially, we tried to substitute the 3' 267 nucleotides (nt) of the 301 nt MHV 3' UTR with the corresponding region of the BCV 3' UTR. This exchange did not yield viable recombinant viruses, and the donor DI RNA was shown to be unable to replicate with MHV as a helper virus. Subsequent analysis revealed that the entire BCV 3' UTR could be inserted into the MHV genome in place of the entire MHV 3' UTR. It resulted that the failure of the initial attempted substitution was due to the inadvertent disruption of an essential conserved bulged stem-loop secondary structure in the MHV and BCV 3' URTs immediately downstream of the N gene stop codon.

  16. Comparative Genome Structure, Secondary Metabolite, and Effector Coding Capacity across Cochliobolus Pathogens

    SciTech Connect

    Condon, Bradford J.; Leng, Yueqiang; Wu, Dongliang; Bushley, Kathryn E.; Ohm, Robin A.; Otillar, Robert; Martin, Joel; Schackwitz, Wendy; Grimwood, Jane; MohdZainudin, NurAinlzzati; Xue, Chunsheng; Wang, Rui; Manning, Viola A.; Dhillon, Braham; Tu, Zheng Jin; Steffenson, Brian J.; Salamov, Asaf; Sun, Hui; Lowry, Steve; LaButti, Kurt; Han, James; Copeland, Alex; Lindquist, Erika; Barry, Kerrie; Schmutz, Jeremy; Baker, Scott E.; Ciuffetti, Lynda M.; Grigoriev, Igor V.; Zhong, Shaobin; Turgeon, B. Gillian

    2013-01-24

    The genomes of five Cochliobolus heterostrophus strains, two Cochliobolus sativus strains, three additional Cochliobolus species (Cochliobolus victoriae, Cochliobolus carbonum, Cochliobolus miyabeanus), and closely related Setosphaeria turcica were sequenced at the Joint Genome Institute (JGI). The datasets were used to identify SNPs between strains and species, unique genomic regions, core secondary metabolism genes, and small secreted protein (SSP) candidate effector encoding genes with a view towards pinpointing structural elements and gene content associated with specificity of these closely related fungi to different cereal hosts. Whole-genome alignment shows that three to five of each genome differs between strains of the same species, while a quarter of each genome differs between species. On average, SNP counts among field isolates of the same C. heterostrophus species are more than 25 higher than those between inbred lines and 50 lower than SNPs between Cochliobolus species. The suites of nonribosomal peptide synthetase (NRPS), polyketide synthase (PKS), and SSP encoding genes are astoundingly diverse among species but remarkably conserved among isolates of the same species, whether inbred or field strains, except for defining examples that map to unique genomic regions. Functional analysis of several strain-unique PKSs and NRPSs reveal a strong correlation with a role in virulence.

  17. Comparative Genome Structure, Secondary Metabolite, and Effector Coding Capacity across Cochliobolus Pathogens

    PubMed Central

    Bushley, Kathryn E.; Ohm, Robin A.; Otillar, Robert; Martin, Joel; Schackwitz, Wendy; Grimwood, Jane; MohdZainudin, NurAinIzzati; Xue, Chunsheng; Wang, Rui; Manning, Viola A.; Dhillon, Braham; Tu, Zheng Jin; Steffenson, Brian J.; Salamov, Asaf; Sun, Hui; Lowry, Steve; LaButti, Kurt; Han, James; Copeland, Alex; Lindquist, Erika; Barry, Kerrie; Schmutz, Jeremy; Baker, Scott E.; Ciuffetti, Lynda M.; Grigoriev, Igor V.; Zhong, Shaobin; Turgeon, B. Gillian

    2013-01-01

    The genomes of five Cochliobolus heterostrophus strains, two Cochliobolus sativus strains, three additional Cochliobolus species (Cochliobolus victoriae, Cochliobolus carbonum, Cochliobolus miyabeanus), and closely related Setosphaeria turcica were sequenced at the Joint Genome Institute (JGI). The datasets were used to identify SNPs between strains and species, unique genomic regions, core secondary metabolism genes, and small secreted protein (SSP) candidate effector encoding genes with a view towards pinpointing structural elements and gene content associated with specificity of these closely related fungi to different cereal hosts. Whole-genome alignment shows that three to five percent of each genome differs between strains of the same species, while a quarter of each genome differs between species. On average, SNP counts among field isolates of the same C. heterostrophus species are more than 25× higher than those between inbred lines and 50× lower than SNPs between Cochliobolus species. The suites of nonribosomal peptide synthetase (NRPS), polyketide synthase (PKS), and SSP–encoding genes are astoundingly diverse among species but remarkably conserved among isolates of the same species, whether inbred or field strains, except for defining examples that map to unique genomic regions. Functional analysis of several strain-unique PKSs and NRPSs reveal a strong correlation with a role in virulence. PMID:23357949

  18. Identification of WD40 repeats by secondary structure-aided profile-profile alignment.

    PubMed

    Wang, Chuan; Dong, Xiaobao; Han, Lei; Su, Xiao-Dong; Zhang, Ziding; Li, Jinyan; Song, Jiangning

    2016-06-01

    A WD40 protein typically contains four or more repeats of ~40 residues ended with the Trp-Asp dipeptide, which folds into β-propellers with four β strands in each repeat. They often function as scaffolds for protein-protein interactions and are involved in numerous fundamental biological processes. Despite their important functional role, the "velcro" closure of WD40 propellers and the diversity of WD40 repeats make their identification a difficult task. Here we develop a new WD40 Repeat Recognition method (WDRR), which uses predicted secondary structure information to generate candidate repeat segments, and further employs a profile-profile alignment to identify the correct WD40 repeats from candidate segments. In particular, we design a novel alignment scoring function that combines dot product and BLOSUM62, thereby achieving a great balance of sensitivity and accuracy. Taking advantage of these strategies, WDRR could effectively reduce the false positive rate and accurately identify more remote homologous WD40 repeats with precise repeat boundaries. We further use WDRR to re-annotate the Pfam families in the β-propeller clan (CL0186) and identify a number of WD40 repeat proteins with high confidence across nine model organisms. The WDRR web server and the datasets are available at http://protein.cau.edu.cn/wdrr/.

  19. GADIS: Algorithm for designing sequences to achieve target secondary structure profiles of intrinsically disordered proteins.

    PubMed

    Harmon, Tyler S; Crabtree, Michael D; Shammas, Sarah L; Posey, Ammon E; Clarke, Jane; Pappu, Rohit V

    2016-09-01

    Many intrinsically disordered proteins (IDPs) participate in coupled folding and binding reactions and form alpha helical structures in their bound complexes. Alanine, glycine, or proline scanning mutagenesis approaches are often used to dissect the contributions of intrinsic helicities to coupled folding and binding. These experiments can yield confounding results because the mutagenesis strategy changes the amino acid compositions of IDPs. Therefore, an important next step in mutagenesis-based approaches to mechanistic studies of coupled folding and binding is the design of sequences that satisfy three major constraints. These are (i) achieving a target intrinsic alpha helicity profile; (ii) fixing the positions of residues corresponding to the binding interface; and (iii) maintaining the native amino acid composition. Here, we report the development of a G: enetic A: lgorithm for D: esign of I: ntrinsic secondary S: tructure (GADIS) for designing sequences that satisfy the specified constraints. We describe the algorithm and present results to demonstrate the applicability of GADIS by designing sequence variants of the intrinsically disordered PUMA system that undergoes coupled folding and binding to Mcl-1. Our sequence designs span a range of intrinsic helicity profiles. The predicted variations in sequence-encoded mean helicities are tested against experimental measurements. PMID:27503953

  20. [Study on the effect of electric field on the secondary structure of lipase by circular dichroism].

    PubMed

    Yao, Zhan-quan; Ao, Dun-ge-ri-le; Xu, Qiang; Yang, Ti-qiang

    2006-12-01

    After the lipase was treated with electric field for five minutes, the effect of electric field on the secondary structure of lipase was studied by circular dichroism (CD). The results showed that different electric field strength in the range from 0. 5 to 6. 0 kV x cm(-1) has a different effect on the relative contents of alpha-helix, beta-sheet, beta-turn and random coil of the lipase, and whose changes were non-monotonous with the raising of electric field. In general, the electric treatment tends to transform the alpha-helix and beta-sheet into beta-turn and random coil. The decrease in alpha-helix and beta-sheet ranged respectively from 4. 6% to 48. 0% and from 13. 2% to 35. 1%, and the increase in beta-turn and random coil ranged respectively from 2. 8% to 33. 3% and from 0. 9% to 48. 1%. The result of this study has important meaning to explain the biological effect of electric treatment seeds.

  1. A novel method for protein secondary structure prediction using dual-layer SVM and profiles.

    PubMed

    Guo, Jian; Chen, Hu; Sun, Zhirong; Lin, Yuanlie

    2004-03-01

    A high-performance method was developed for protein secondary structure prediction based on the dual-layer support vector machine (SVM) and position-specific scoring matrices (PSSMs). SVM is a new machine learning technology that has been successfully applied in solving problems in the field of bioinformatics. The SVM's performance is usually better than that of traditional machine learning approaches. The performance was further improved by combining PSSM profiles with the SVM analysis. The PSSMs were generated from PSI-BLAST profiles, which contain important evolution information. The final prediction results were gen