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Sample records for 23s ribosomal rna

  1. Helix 69 of E. coli 23S ribosomal RNA as a peptide nucleic acid target.

    PubMed

    Kulik, Marta; Markowska-Zagrajek, Agnieszka; Wojciechowska, Monika; Grzela, Renata; Wituła, Tomasz; Trylska, Joanna

    2017-04-07

    A fragment of 23S ribosomal RNA (nucleotides 1906-1924 in E. coli), termed Helix 69, forms a hairpin that is essential for ribosome function. Helix 69 forms a conformationally flexible inter-subunit connection with helix 44 of 16S ribosomal RNA, and the nucleotide A1913 of Helix 69 influences decoding accuracy. Nucleotides U1911 and U1917 are post-transcriptionally modified with pseudouridines () and U1915 with 3-methyl-. We investigated Helix 69 as a target for a complementary synthetic oligonucleotide - peptide nucleic acid (PNA). We determined thermodynamic properties of Helix 69 and its complexes with PNA. We also verified the performance of PNA targeted at Helix 69 in inhibiting translation in cell-free extracts and growth of E. coli cells. First, we examined the interactions of a PNA oligomer complementary to the G1907-A1919 fragment of Helix 69 with the sequences corresponding to human and bacterial species (with or without pseudouridine modifications). PNA invades the Helix 69 hairpin creating stable complexes and PNA binding to the pseudouridylated bacterial sequence is stronger than to Helix 69 without any modifications. Second, we confirmed the binding of PNA to 23S rRNA and 70S ribosomes. Third, we verified the efficiency of translation inhibition of these PNA oligomers in the cell-free translation/transcription E. coli system, which turned out to be in a similar range as tetracycline. Next, we confirmed that PNA conjugated to the (KFF)3K transporter peptide inhibited E. coli growth in micromolar concentrations. Overall, targeting Helix 69 with PNA or other sequence-specific oligomers could be a promising way to inhibit bacterial translation.

  2. Ribosomal protein L3 bound to 23S precursor rRNA stimulates its maturation by Mini-III ribonuclease.

    PubMed

    Redko, Yulia; Condon, Ciarán

    2009-03-01

    Ribosomal RNAs (rRNAs) are processed from larger primary transcripts in every living system known. The maturation of 23S rRNA in Bacillus subtilis is catalysed by Mini-III, a member of the RNase III family of enzymes that lacks the characteristic double-stranded RNA binding domain of its relatives. We have previously shown that Mini-III processing of 23S precursor rRNA in assembled 50S ribosomal subunits is much more efficient than a substrate with no ribosomal proteins bound, suggesting that one or more large subunit proteins act as a cofactor for Mini-III cleavage. Here we show that this cofactor is ribosomal protein L3. Stimulation of the Mini-III cleavage reaction is through L3 binding to its normal site at the 3' end of 23S rRNA. We present indirect evidence that suggests that L3 acts at the level of substrate, rather than enzyme conformation. We also discuss the potential implication of using ribosomal protein cofactors in rRNA processing for ribosome quality control.

  3. Coregulation of processing and translation: mature 5' termini of Escherichia coli 23S ribosomal RNA form in polysomes.

    PubMed Central

    Srivastava, A K; Schlessinger, D

    1988-01-01

    In Escherichia coli, the final maturation of rRNA occurs in precursor particles, and recent experiments have suggested that ongoing protein synthesis may somehow be required for maturation to occur. The protein synthesis requirement for the formation of the 5' terminus of 23S rRNA has been clarified in vitro by varying the substrate of the reaction. In cell extracts, pre-23S rRNA in free ribosomes was not matured, but that in polysomes was efficiently processed. The reaction occurred in polysomes without the need for an energy source or other additives required for protein synthesis. Furthermore, when polysomes were dissociated into ribosomal subunits, they were no longer substrates for maturation; but the ribosomes became substrates again when they once more were incubated in the conditions for protein synthesis. All of these results are consistent with the notion that protein synthesis serves to form a polysomal complex that is the true substrate for maturation. Ribosomes in polysomes, possibly in the form of 70S initiation complexes, may more easily adopt a conformation that facilitates maturation cleavage. As a result, the rates of ribosome formation and protein synthesis could be coregulated. Images PMID:3050989

  4. The role of 23S ribosomal RNA residue A2451 in peptide bond synthesis revealed by atomic mutagenesis.

    PubMed

    Lang, Kathrin; Erlacher, Matthias; Wilson, Daniel N; Micura, Ronald; Polacek, Norbert

    2008-05-01

    Peptide bond formation is a fundamental reaction in biology, catalyzed by the ribosomal peptidyl-transferase ribozyme. Although all active-site 23S ribosomal RNA nucleotides are universally conserved, atomic mutagenesis suggests that these nucleobases do not carry functional groups directly involved in peptide bond formation. Instead, a single ribose 2'-hydroxyl group at A2451 was identified to be of pivotal importance. Here, we altered the chemical characteristics by replacing its 2'-hydroxyl with selected functional groups and demonstrate that hydrogen donor capability is essential for transpeptidation. We propose that the A2451-2'-hydroxyl directly hydrogen bonds to the P-site tRNA-A76 ribose. This promotes an effective A76 ribose C2'-endo conformation to support amide synthesis via a proton shuttle mechanism. Simultaneously, the direct interaction of A2451 with A76 renders the intramolecular transesterification of the peptide from the 3'- to 2'-oxygen unfeasible, thus promoting effective peptide bond synthesis.

  5. Macrolides and lincomycin susceptibility of Mycoplasma hyorhinis and variable mutation of domain II and V in 23S ribosomal RNA.

    PubMed

    Kobayashi, Hideki; Nakajima, Hiromi; Shimizu, Yuka; Eguchi, Masashi; Hata, Eiji; Yamamoto, Koshi

    2005-08-01

    A total of 151 strains of Mycoplasma hyorhinis isolated from porcine lung lesions (weaned pigs, n=71, and finishers, n=80) were investigated for their in vitro susceptibility to 10 antimicrobial agents. Thirty-one strains (28 from weaned pigs and 3 from finishers) showed resistance to 16-membered macrolide antibiotics and lincomycin. The prevalence of the 16-membered macrolide-resistant M. hyorhinis strain in weaned pigs from Japanese herds has approximately quadrupled in the past 10 years. Several of the 31 strains were examined for mutations in the 23S ribosomal RNA (rRNA). All field strains tested showed a transition of A to G at position 2059 of 23S rRNA-rendered Escherichia coli. On the other hand, individual tylosin- and lincomycin-resistant mutants of M. hyorhinis were selected in vitro from the susceptible type strain BTS7 by 3 to 9 serial passages in subinhibitory concentrations of each antibiotic. The 23S rRNA sequences of both tylosin and lincomycin-resistant mutants were compared with that of the radical BTS7 strain. The BTS7 mutant strain selected by tylosin showed the same transition as the field-isolated strains of A2059G. However, the transition selected in lincomycin showed mutations in domains II and V of 23S rRNA, G2597U, C2611U in domain V, and the addition of an adenine at the pentameric adenine loop in domain II. The strain selected by lincomycin showed an additional point mutation of A2062G selected by tylosin.

  6. Decreased Susceptibility to Macrolide-Lincosamide in Mycoplasma synoviae Is Associated with Mutations in 23S Ribosomal RNA.

    PubMed

    Lysnyansky, Inna; Gerchman, Irena; Flaminio, Barbara; Catania, Salvatore

    2015-12-01

    The mechanism responsible for acquired decreased susceptibility to macrolides (14-membered erythromycin [Ery], 16-membered tylosin [Ty] and tilmicosin [Tm]) and to lincosamides (lincomycin [Ln]) was investigated in Mycoplasma synoviae, a pathogen that causes respiratory infections and synovitis in chicken and turkey. Sequence analysis of domains II and V of the two 23S rRNA alleles and ribosomal proteins L4 and L22 was performed on 49 M. synoviae isolates, M. synoviae type strain WVU1853, and reference strain FMT showing minimal inhibitory concentrations (MICs) to Ty (≤ 0.015 to 2 μg/ml), Tm (0.03 to ≥ 8 μg/ml), and Ln (0.125 to 8 μg/ml); MICs to Ery ranged from 32 to ≥ 128 μg/ml. Our results showed that the nucleotide substitution G748A (Escherichia coli numbering) in domain II of one or both 23S rRNA alleles may account for a slight increase in MICs to Ty and Tm (up to 0.5 and 2 μg/ml, respectively). No correlation between the presence of G748A and decreased susceptibility to Ln was found. However, the presence of the point mutations A2058G or A2059G in domain V of one or both alleles of the 23S rRNAs was correlated with a more significant decrease in susceptibility to Ty (1-2 μg/ml), Tm (≥ 8 μg/ml), and Ln (≥ 8 μg/ml). All M. synoviae isolates tested had a G2057A transition in the 23S rRNAs consistent with previously described intrinsic resistance to Ery. Mutations G64E (one isolate) and Q90K/H (two isolates) were identified in the L4 and L22 proteins, respectively, but their impact on decreased susceptibility to macrolides and lincomycin was not clear.

  7. Mini-III, a fourth class of RNase III catalyses maturation of the Bacillus subtilis 23S ribosomal RNA.

    PubMed

    Olmedo, Gabriela; Guzmán, Plinio

    2008-06-01

    Ribonuclease III (RNase III) type of enzymes are double-stranded RNA (dsRNA)-specific endoribonucleases that have important roles in RNA maturation and mRNA decay. They are involved in processing precursors of ribosomal RNA (rRNA) in bacteria as well as precursors of short interfering RNAs (siRNAs) and microRNAs (miRNAs) in eukaryotes. RNase III proteins have been grouped in three major classes according to their domain organization. In this issue of Molecular Microbiology, Redko et al. identified a novel class of bacterial RNase III, named Mini-III, consisting only of the RNase III catalytic domain and functioning in the maturation of the 23S rRNA in Bacillus subtilis. Its absence from proteobacteria reveals that this step is mechanistically different from the corresponding step in Escherichia coli. The fact that Mini-III orthologues are present in unicellular photosynthetic eukaryotes and in plants opens new opportunities for functional studies of this type of RNases.

  8. Importance of tRNA interactions with 23S rRNA for peptide bond formation on the ribosome: studies with substrate analogs.

    PubMed

    Beringer, Malte; Rodnina, Marina V

    2007-07-01

    The major enzymatic activity of the ribosome is the catalysis of peptide bond formation. The active site -- the peptidyl transferase center -- is composed of ribosomal RNA (rRNA), and interactions between rRNA and the reactants, peptidyl-tRNA and aminoacyl-tRNA, are crucial for the reaction to proceed rapidly and efficiently. Here, we describe the influence of rRNA interactions with cytidine residues in A-site substrate analogs (C-puromycin or CC-puromycin), mimicking C74 and C75 of tRNA on the reaction. Base-pairing of C75 with G2553 of 23S rRNA accelerates peptide bond formation, presumably by stabilizing the peptidyl transferase center in its productive conformation. When C74 is also present in the substrate analog, the reaction is slowed down considerably, indicating a slow step in substrate binding to the active site, which limits the reaction rate. The tRNA-rRNA interactions lead to a robust reaction that is insensitive to pH changes or base substitutions in 23S rRNA at the active site of the ribosome.

  9. Identification and characterization of an intervening sequence within the 23S ribosomal RNA genes of Edwardsiella ictaluri

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Comparison of the 23S rRNA gene sequences of Edwardsiella tarda and Edwardsiella ictaluri confirmed a close phylogenetic relationship between these two fish pathogen species and a distant relation with the 'core' members of the Enterobacteriaceae family. Analysis of the rrl gene for 23S rRNA in E. i...

  10. Mini-III, an unusual member of the RNase III family of enzymes, catalyses 23S ribosomal RNA maturation in B. subtilis.

    PubMed

    Redko, Yulia; Bechhofer, David H; Condon, Ciarán

    2008-06-01

    The late steps of both 16S and 5S ribosomal RNA maturation in the Gram-positive bacterium Bacillus subtilis have been shown to be catalysed by ribonucleases that are not present in the Gram-negative paradigm, Escherichia coli. Here we present evidence that final maturation of the 5' and 3' extremities of B. subtilis 23S rRNA is also performed by an enzyme that is absent from the Proteobacteria. Mini-III contains an RNase III-like catalytic domain, but curiously lacks the double-stranded RNA binding domain typical of RNase III itself, Dicer, Drosha and other well-known members of this family of enzymes. Cells lacking Mini-III accumulate precursors and alternatively matured forms of 23S rRNA. We show that Mini-III functions much more efficiently on precursor 50S ribosomal subunits than naked pre-23S rRNA in vitro, suggesting that maturation occurs primarily on assembled subunits in vivo. Lastly, we provide a model for how Mini-III recognizes and cleaves double-stranded RNA, despite lacking three of the four RNA binding motifs of RNase III.

  11. Mutations in 23S rRNA and Ribosomal Protein L4 Account for Resistance in Pneumococcal Strains Selected In Vitro by Macrolide Passage

    PubMed Central

    Tait-Kamradt, A.; Davies, T.; Cronan, M.; Jacobs, M. R.; Appelbaum, P. C.; Sutcliffe, J.

    2000-01-01

    The mechanisms responsible for macrolide resistance in Streptococcus pneumoniae mutants, selected from susceptible strains by serial passage in azithromycin, were investigated. These mutants were resistant to 14- and 15-membered macrolides, but resistance could not be explained by any clinically relevant resistance determinant [mef(A), erm(A), erm(B), erm(C), erm(TR), msr(A), mph(A), mph(B), mph(C), ere(A), ere(B)]. An investigation into the sequences of 23S rRNAs in the mutant and parental strains revealed individual changes of C2611A, C2611G, A2058G, and A2059G (Escherichia coli numbering) in four mutants. Mutations at these residues in domain V of 23S rRNA have been noted to confer erythromycin resistance in other species. Not all four 23S rRNA alleles have to contain the mutation to confer resistance. Some of the mutations also confer coresistance to streptogramin B (C2611A, C2611G, and A2058G), 16-membered macrolides (all changes), and clindamycin (A2058G and A2059G). Interestingly, none of these mutations confer high-level resistance to telithromycin (HMR-3647). Further, two of the mutants which had no changes in their 23S rRNA sequences had changes in a highly conserved stretch of amino acids (63KPWRQKGTGRAR74) in ribosomal protein L4. One mutant contained a single amino acid change (G69C), while the other mutant had a 6-base insert, resulting in two amino acids (S and Q) being inserted between amino acids Q67 and K68. To our knowledge, this is the first description of mutations in 23S rRNA genes or ribosomal proteins in macrolide-resistant S. pneumoniae strains. PMID:10898684

  12. Functional interaction between bases C1049 in domain II and G2751 in domain VI of 23S rRNA in Escherichia coli ribosomes

    PubMed Central

    Miyoshi, Tomohiro; Uchiumi, Toshio

    2008-01-01

    The factor-binding center within the Escherichia coli ribosome is comprised of two discrete domains of 23S rRNA: the GTPase-associated region (GAR) in domain II and the sarcin–ricin loop in domain VI. These two regions appear to collaborate in the factor-dependent events that occur during protein synthesis. Current X-ray crystallography of the ribosome shows an interaction between C1049 in the GAR and G2751 in domain VI. We have confirmed this interaction by site-directed mutagenesis and chemical probing. Disruption of this base pair affected not only the chemical modification of some bases in domains II and VI and in helix H89 of domain V, but also ribosome function dependent on both EF-G and EF-Tu. Mutant ribosomes carrying the C1049 to G substitution, which show enhancement of chemical modification at G2751, were used to probe the interactions between the regions around 1049 and 2751. Binding of EF-G-GDP-fusidic acid, but not EF-G-GMP-PNP, to the ribosome protected G2751 from modification. The G2751 protection was also observed after tRNA binding to the ribosomal P and E sites. The results suggest that the interactions between the bases around 1049 and 2751 alter during different stages of the translation process. PMID:18252772

  13. The NMR Structure of an Internal Loop from 23S Ribosomal RNA Differs from its Structure in Crystals of 50S Ribosomal Subunits

    PubMed Central

    Shankar, Neelaabh; Kennedy, Scott D.; Chen, Gang; Krugh, Thomas R.; Turner, Douglas H.

    2014-01-01

    Internal loops play an important role in structure and folding of RNA and in RNA recognition by other molecules such as proteins and ligands. An understanding of internal loops with propensities to form a particular structure will help predict RNA structure, recognition, and function. The structures of internal loops 5'1009CUAAG10133'3'1168GAAGC11645' and 5'998CUAAG10023'3'1157GAAGC11535' from helix 40 of the large subunit rRNA in Deinococcus radiodurans and Escherichia coli, respectively, are phylogenetically conserved, suggesting functional relevance. The energetics and NMR solution structure of the loop were determined in the duplex, 5'1GGCUAAGAC93'3'18CCGAAGCUG105' The internal loop forms a different structure in solution than in the crystal structures of the ribosomal subunits. In particular, the crystal structures have a bulged out adenine at the equivalent of position A15 and a reverse Hoogsteen UA pair (trans Watson-Crick/Hoogsteen UA) at the equivalent of U4 and A14, whereas the solution structure has a single hydrogen bond UA pair (cis Watson-Crick/sugar edge A15U4) between U4 and A15 and a sheared AA pair (trans Hoogsteen/sugar edge A14A5) between A5 and A14. There is cross-strand stacking between A6 and A14 (A6/A14/A15 stacking pattern) in the NMR structure. All three structures have a sheared GA pair (trans Hoogsteen/sugar edge A6G13) at the equivalent of A6 and G13. The internal loop has contacts with ribosomal protein L20 and other parts of the RNA in the crystal structures. These contacts presumably provide the free energy to rearrange the base pairing in the loop. Evidently, molecular recognition of this internal loop involves induced fit binding, which could confer several advantages. The predicted thermodynamic stability of the loop agrees with the experimental value, even though the thermodynamic model assumes a Watson–Crick UA pair. PMID:17002278

  14. Crystallization of the two-domain N-terminal fragment of the archaeal ribosomal protein L10(P0) in complex with a specific fragment of 23S rRNA

    SciTech Connect

    Kravchenko, O. V.; Mitroshin, I. V.; Gabdulkhakov, A. G.; Nikonov, S. V.; Garber, M. B.

    2011-07-15

    Lateral L12-stalk (P1-stalk in Archaea, P1/P2-stalk in eukaryotes) is an obligatory morphological element of large ribosomal subunits in all organisms studied. This stalk is composed of the complex of ribosomal proteins L10(P0) and L12(P1) and interacts with 23S rRNA through the protein L10(P0). L12(P1)-stalk is involved in the formation of GTPase center of the ribosome and plays an important role in the ribosome interaction with translation factors. High mobility of this stalk puts obstacles in determination of its structure within the intact ribosome. Crystals of a two-domain N-terminal fragment of ribosomal protein L10(P0) from the archaeon Methanococcus jannaschii in complex with a specific fragment of rRNA from the same organism have been obtained. The crystals diffract X-rays at 3.2 Angstrom-Sign resolution.

  15. Determination of the nucleotide sequence of the 23S ribosomal RNA and flanking spacers of an Enterococcus faecium strain, reveals insertion-deletion events in the ribosomal spacer 1 of enterococci.

    PubMed

    Naimi, A; Beck, G; Monique, M; Lefèbvre, G; Branlanti, C

    1999-02-01

    The usefulness of 16S-23S (ITS1) and 23S-5S (ITS2) ribosomal spacer nucleotide sequence determination, as a complementary approach to the biochemical tests traditionally used for enterococcal species identification, is shown by its application to the identification of a strain, E27, isolated from a natural bacteria mixture used for cheese production. Using combined approaches we showed, unambiguously, that strain E27 belongs to the Enterococcus faecium species. However, its ITS1 region has an interesting peculiarity. In our previous study of ITS1s from various enterococcal species (NAIMI et al., 1997, Microbiology 143, 823-834), the ITS1s of the two E. faecium strains studied, were found to contain an additional 115-nt long stem-loop structure as compared to the ITS1s of other enterococci, only one out of the 3 ITS1s of E. hirae ATCC 9790, was found to contain a similar 107-nt long stem-loop structure. The ITS1 of strain E27 is 100% identical to that of E. faecium ATCC 19434T, except that the 115-nt additional fragment is absent. This strongly suggests the existence of lateral DNA transfer or DNA recombination events at a hot spot position of the ITS1s from E. faecium and E. hirae. Small and large ITS1 nucleotide sequence determination for strain E27 generalized the notion of two kinds of ITSs in enterococci: one with a tRNA(Ala) gene, one without tRNA gene. To complete strain E27 characterization, its 23S rRNA sequence was established. This is the first complete 23S rRNA nucleotide sequence determined for an enterococcal species.

  16. Specific binding of tRNAMet to 23S rRNA of Escherichia coli.

    PubMed Central

    Dahlberg, J E; Kintner, C; Lund, E

    1978-01-01

    tRNAMetf binds to 23S rRNA of Escherichia coli, forming a complex with a melting temperature of 75 degrees (in 0.6 M NaCl). The regions within the RNAs that bind to each other have been isolated and their nucleotide sequences have been determined. The interacting region in tRNAMetf is 17 nucleotides long, extending from G5 in the acceptor stem to D21 (D = 5.6-dihydrouridine) in the D loop. The sequence in 23S rRNA is complementary to that sequence except for an extra Up in the middle and allowing a Gp.D base pair. We propose that association of these two sequences may play a role in initiation of protein synthesis by tRNAMetf. In addition, part of this sequence in 23S rRNA may also stabilize tRNA binding to the ribosome during elongation of nascent polypeptides. Images PMID:349554

  17. [Ribosomal RNA Evolution

    NASA Technical Reports Server (NTRS)

    1997-01-01

    It is generally believed that an RNA World existed at an early stage in the history of life. During this early period, RNA molecules are seen to be potentially involved in both catalysis and the storage of genetic information. Translation presents several interrelated themes of inquiry for exobiology. First, it is essential, for understanding the very origin of life, how peptides and eventually proteins might have come to be made on the early Earth in a template directed manner. Second, it is necessary to understand how a machinery of similar complexity to that found in the ribosomes of modern organisms came to exist by the time of the last common ancestor (as detected by 16S rRNA sequence studies). Third, the ribosomal RNAs themselves likely had a very early origin and studies of their history may be very informative about the nature of the RNA World. Moreover, studies of these RNAs will contribute to a better understanding of the potential roles of RNA in early evolution.During the past year we have ave conducted a comparative study of four completely sequenced bacterial genoames. We have focused initially on conservation of gene order. The second component of the project continues to build on the model system for studying the validity of variant 5S rRNA sequences in the vicinity of the modern Vibrio proteolyticus 5S rRNA that we established earlier. This system has made it possible to conduct a detailed and extensive analysis of a local portion of the sequence space. These core methods have been used to construct numerous mutants during the last several years. Although it has been a secondary focus, this work has continued over the last year such that we now have in excess of 125 V. proteolyticus derived constructs which have been made and characterized. We have also continued high resolution NMR work on RNA oligomers originally initiated by G. Kenneth Smith who was funded by a NASA Graduate Student Researcher's Fellowship Award until May of 1996. Mr. Smith

  18. Sequestration of Ribosome during Protein Aggregate Formation: Contribution of ribosomal RNA

    PubMed Central

    Pathak, Bani K.; Mondal, Surojit; Banerjee, Senjuti; Ghosh, Amar Nath; Barat, Chandana

    2017-01-01

    An understanding of the mechanisms underlying protein aggregation and cytotoxicity of the protein aggregates is crucial in the prevention of several diseases in humans. Ribosome, the cellular protein synthesis machine is capable of acting as a protein folding modulator. The peptidyltransferase center residing in the domain V of large ribosomal subunit 23S rRNA is the centre for the protein folding ability of the ribosome and is also the cellular target of several antiprion compounds. Our in vitro studies unexpectedly reveal that the partial unfolding or aggregation of lysozyme under reducing conditions in presence of the ribosome can induce aggregation of ribosomal components. Electrostatic interactions complemented by specific rRNA-protein interaction drive the ribosome-protein aggregation process. Under similar conditions the rRNA, especially the large subunit rRNA and in vitro transcribed RNA corresponding to domain V of 23S rRNA (bDV RNA) stimulates lysozyme aggregation leading to RNA-protein aggregate formation. Protein aggregation during the refolding of non-disulfide containing protein BCAII at high concentrations also induces ribosome aggregation. BCAII aggregation was also stimulated in presence of the large subunit rRNA. Our observations imply that the specific sequestration of the translation machine by aggregating proteins might contribute to their cytotoxicity. PMID:28169307

  19. Eukaryotic ribosomes that lack a 5.8S RNA

    NASA Technical Reports Server (NTRS)

    Vossbrinck, C. R.; Woese, C. R.

    1986-01-01

    The 5.8S ribosomal RNA is believed to be a universal eukaryotic characteristic. It has no (size) counterpart among the prokaryotes, although its sequence is homologous with the first 150 or so nucleotides of the prokaryotic large subunit (23S) ribosomal RNA. An exception to this rule is reported here. The microsporidian Vairimorpha necatrix is a eukaryote that has no 5.8S rRNA. As in the prokaryotes, it has a single large subunit rRNA, whose 5-prime region corresponds to the 5.8S rRNA.

  20. AMPLIFICATION OF RIBOSOMAL RNA SEQUENCES

    EPA Science Inventory

    This book chapter offers an overview of the use of ribosomal RNA sequences. A history of the technology traces the evolution of techniques to measure bacterial phylogenetic relationships and recent advances in obtaining rRNA sequence information. The manual also describes procedu...

  1. Sequence homologies between eukaryotic 5.8S rRNA and the 5' end of prokaryotic 23S rRNa: evidences for a common evolutionary origin.

    PubMed Central

    Jacq, B

    1981-01-01

    The question of the evolutionary origin of eukaryotic 5.8S rRNA was re-examined after the recent publication of the E. coli 23S rRNA sequence (26,40). A region of the 23S RNA located at its 5' end was found to be approximately 50% homologous to four different eukaryotic 5.8S rRNAs. A computer comparison analysis indicates that no other region of the E. coli ribosomal transcription unit (greater than 5 000 nucleotides in length) shares a comparable homology with 5.8S rRNA. Homology between the 5' end of e. coli 23S and four different eukaryotic 5.8S rRNAs falls within the same range as that between E. coli 5S RNA from the same four eukaryotic species. All these data strongly suggest that the 5' end of prokaryotic 23S rRNA and eukaryotic 5.8S RNA have a common evolutionary origin. Secondary structure models are proposed for the 5' region of E. coli 23S RNA. Images PMID:7024907

  2. Essential role of conserved DUF177A protein in plastid 23S rRNA accumulation and plant embryogenesis

    PubMed Central

    Yang, Jiani; Suzuki, Masaharu; McCarty, Donald R.

    2016-01-01

    DUF177 proteins are nearly universally conserved in bacteria and plants except the Chlorophyceae algae. Thus far, duf177 mutants in bacteria have not established a function. In contrast, duf177a mutants have embryo lethal phenotypes in maize and Arabidopsis. In maize inbred W22, duf177a mutant embryos arrest at an early transition stage, whereas the block is suppressed in the B73 inbred background, conditioning an albino seedling phenotype. Background-dependent embryo lethal phenotypes are characteristic of maize plastid gene expression mutants. Consistent with the plastid gene expression hypothesis, quantitative real-time PCR revealed a significant reduction of 23S rRNA in an Escherichia coli duf177 knockout. Plastid 23S rRNA contents of duf177a mutant tissues were also markedly reduced compared with the wild-type, whereas plastid 16S, 5S, and 4.5S rRNA contents were less affected, indicating that DUF177 is specifically required for accumulation of prokaryote-type 23S rRNA. An AtDUF177A–green fluorescent protein (GFP) transgene controlled by the native AtDUF177A promoter fully complemented the Arabidopsis atduf177a mutant. Transient expression of AtDUF177A–GFP in Nicotiana benthamiana leaves showed that the protein was localized in chloroplasts. The essential role of DUF177A in chloroplast–ribosome formation is reminiscent of IOJAP, another highly conserved ribosome-associated protein, suggesting that key mechanisms controlling ribosome formation in plastids evolved from non-essential pathways for regulation of the prokaryotic ribosome. PMID:27574185

  3. Cleavage of the sarcin–ricin loop of 23S rRNA differentially affects EF-G and EF-Tu binding

    PubMed Central

    García-Ortega, Lucía; Álvarez-García, Elisa; Gavilanes, José G.; Martínez-del-Pozo, Álvaro; Joseph, Simpson

    2010-01-01

    Ribotoxins are potent inhibitors of protein biosynthesis and inactivate ribosomes from a variety of organisms. The ribotoxin α-sarcin cleaves the large 23S ribosomal RNA (rRNA) at the universally conserved sarcin–ricin loop (SRL) leading to complete inactivation of the ribosome and cellular death. The SRL interacts with translation factors that hydrolyze GTP, and it is important for their binding to the ribosome, but its precise role is not yet understood. We studied the effect of α-sarcin on defined steps of translation by the bacterial ribosome. α-Sarcin-treated ribosomes showed no defects in mRNA and tRNA binding, peptide-bond formation and sparsomycin-dependent translocation. Cleavage of SRL slightly affected binding of elongation factor Tu ternary complex (EF-Tu•GTP•tRNA) to the ribosome. In contrast, the activity of elongation factor G (EF-G) was strongly impaired in α-sarcin-treated ribosomes. Importantly, cleavage of SRL inhibited EF-G binding, and consequently GTP hydrolysis and mRNA–tRNA translocation. These results suggest that the SRL is more critical in EF-G than ternary complex binding to the ribosome implicating different requirements in this region of the ribosome during protein elongation. PMID:20215430

  4. Restriction Profiling of 23S Microheterogenic Ribosomal Repeats for Detection and Characterizing of E. coli and Their Clonal, Pathogenic, and Phylogroups

    PubMed Central

    Jayasree Rajagopalan Nair, Parvathi

    2015-01-01

    Correlating ribosomal microheterogenicity with unique restriction profiles can prove to be an efficacious and cost-effective approach compared with sequencing for microbial identification. An attempt to peruse restriction profiling of 23S ribosomal assemblage was ventured; digestion patterns with Bfa I discriminated E. coli from its colony morphovars, while Hae III profiles assisted in establishing distinct clonal groups. Among the gene pool of 399 ribosomal sequences extrapolated from 57 E. coli genomes, varying degree of predominance (I > III > IV > II) of Hae III pattern was observed. This was also corroborated in samples collected from clinical, commensal, and environmental origin. K-12 and its descendants showed type I pattern whereas E. coli-B and its descendants exhibited type IV, both of these patterns being exclusively present in E. coli. A near-possible association between phylogroups and Hae III profiles with presumable correlation between the clonal groups and different pathovars was established. The generic nature, conservation, and barcode gap of 23S rRNA gene make it an ideal choice and substitute to 16S rRNA gene, the most preferred region for molecular diagnostics in bacteria. PMID:26885397

  5. The "DEAD box" protein DbpA interacts specifically with the peptidyltransferase center in 23S rRNA.

    PubMed Central

    Nicol, S M; Fuller-Pace, F V

    1995-01-01

    The Escherichia coli DEAD (Asp-Glu-Ala-Asp) box protein DbpA is a putative RNA helicase and established RNA-dependent ATPase and is the only member of the DEAD box protein family for which a specific RNA substrate, bacterial 23S rRNA, has been identified. We have investigated the nature of this specificity in depth and have localized by deletion mutagenesis and PCR a single region of 93 bases (bases 2496-2588) in 23S rRNA that is both necessary and sufficient for complete activation of ATPase activity of DbpA. This target region forms part of the peptidyltransferase center and includes many bases involved in interaction with the 3' terminal adenosines of both A- and P-site tRNAs. Deletion of stem loops within the 93-base segment abolished ATPase activation. Similarly, point mutations that disrupt base pairing within stem structures ablated stimulation of ATPase activity. These data are consistent with roles for DbpA either in establishing and/or maintaining the correct three-dimensional structure of the peptidyltransferase center in 23S rRNA during ribosome assembly or in the peptidyltransferase reaction. Images Fig. 1 Fig. 2 PMID:8524828

  6. Use of whole-genome sequencing data to analyze 23S rRNA-mediated azithromycin resistance.

    PubMed

    Johnson, Steven R; Grad, Yonatan; Abrams, A Jeanine; Pettus, Kevin; Trees, David L

    2017-02-01

    The whole-genome sequences of 24 isolates of Neisseria gonorrhoeae with elevated minimum inhibitory concentrations (MICs) to azithromycin (≥2.0 µg/mL) were analyzed against a modified sequence derived from the whole-genome sequence of N. gonorrhoeae FA1090 to determine, by signal ratio, the number of mutant copies of the 23S rRNA gene and the copy number effect on 50S ribosome-mediated azithromycin resistance. Isolates that were predicted to contain four mutated copies were accurately identified compared with the results of direct sequencing. Fewer than four mutated copies gave less accurate results but were consistent with elevated MICs.

  7. Complete nucleotide sequence of the 23S rRNA gene of the Cyanobacterium, Anacystis nidulans.

    PubMed Central

    Douglas, S E; Doolittle, W F

    1984-01-01

    The nucleotide sequence of the Anacystis nidulans 23S rRNA gene, including the 5'- and 3'-flanking regions has been determined. The gene is 2876 nucleotides long and shows higher primary sequence homology to the 23S rRNAs of plastids (84.5%) than to that of E. coli (79%). The predicted rRNA transcript also shares many secondary structural features with those of plastids, reinforcing the endosymbiont hypothesis for the origin of these organelles. PMID:6326060

  8. Transition mutations in the 23S rRNA of erythromycin-resistant isolates of Mycoplasma pneumoniae.

    PubMed Central

    Lucier, T S; Heitzman, K; Liu, S K; Hu, P C

    1995-01-01

    Erythromycin is the drug of choice for treatment of Mycoplasma pneumoniae infections due to its susceptibility to low levels of this antibiotic. After exposure of susceptible strains to erythromycin in vitro and in vivo, mutants resistant to erythromycin and other macrolides were isolated. Their phenotypes have been characterized, but the genetic basis for resistance has never been determined. We isolated two resistant mutants (M129-ER1 and M129-ER2) by growing M. pneumoniae M129 on agar containing different amounts of erythromycin. In broth dilution tests both strains displayed resistance to high levels of several macrolide-lincosamide-streptogramin B (MLS) antibiotics. In binding studies, ribosomes isolated from the resistant strains exhibited significantly lower affinity for [14C]erythromycin than did ribosomes from the M129 parent strain. Sequencing of DNA amplified from the region of the 2S rRNA gene encoding domain V revealed an A-to-G transition in the central loop at position 2063 of M129-ER1 and a similar A-to-G transition at position 2064 in M129-ER2. Transitions at homologous locations in the 23S rRNA from other organisms have been shown to result in resistance to MLS antibiotics. Thus, MLS-like resistance can occur in M. pneumoniae as the result of point mutations in the 23S rRNA gene which reduce the affinity of these antibiotics for the ribosome. Since they involve only single-base changes, development of resistance to erythromycin in vivo by these mechanisms could be relatively frequent event. PMID:8593017

  9. Single-base mutations at position 2661 of Escherichia coli 23S rRNA increase efficiency of translational proofreading.

    PubMed Central

    Melançon, P; Tapprich, W E; Brakier-Gingras, L

    1992-01-01

    Two single-base substitutions were constructed in the 2660 loop of Escherichia coli 23S rRNA (G2661-->C or U) and were introduced into the rrnB operon cloned in plasmid pKK3535. Ribosomes were isolated from bacteria transformed with the mutated plasmids and assayed in vitro in a poly(U)-directed system for their response to the misreading effect of streptomycin, neomycin, and gentamicin, three aminoglycoside antibiotics known to impair the proofreading control of translational accuracy. Both mutations decreased the stimulation of misreading by these drugs, but neither interfered with their binding to the ribosome. The response of the mutant ribosomes to these drugs suggests that the 2660 loop, which belongs to the elongation factor Tu binding site, is involved in the proofreading step of the accuracy control. In vivo, both mutations reduced read-through of nonsense codons and frameshifting, which can also be related to the increased efficiency in proofreading control which they confer to ribosomes. PMID:1281147

  10. Lessons from an evolving rRNA: 16S and 23S rRNA structures from a comparative perspective.

    PubMed Central

    Gutell, R R; Larsen, N; Woese, C R

    1994-01-01

    The 16S and 23S rRNA higher-order structures inferred from comparative analysis are now quite refined. The models presented here differ from their immediate predecessors only in minor detail. Thus, it is safe to assert that all of the standard secondary-structure elements in (prokaryotic) rRNAs have been identified, with approximately 90% of the individual base pairs in each molecule having independent comparative support, and that at least some of the tertiary interactions have been revealed. It is interesting to compare the rRNAs in this respect with tRNA, whose higher-order structure is known in detail from its crystal structure (36) (Table 2). It can be seen that rRNAs have as great a fraction of their sequence in established secondary-structure elements as does tRNA. However, the fact that the former show a much lower fraction of identified tertiary interactions and a greater fraction of unpaired nucleotides than the latter implies that many of the rRNA tertiary interactions remain to be located. (Alternatively, the ribosome might involve protein-rRNA rather than intramolecular rRNA interactions to stabilize three-dimensional structure.) Experimental studies on rRNA are consistent to a first approximation with the structures proposed here, confirming the basic assumption of comparative analysis, i.e., that bases whose compositions strictly covary are physically interacting. In the exhaustive study of Moazed et al. (45) on protection of the bases in the small-subunit rRNA against chemical modification, the vast majority of bases inferred to pair by covariation are found to be protected from chemical modification, both in isolated small-subunit rRNA and in the 30S subunit. The majority of the tertiary interactions are reflected in the chemical protection data as well (45). On the other hand, many of the bases not shown as paired in Fig. 1 are accessible to chemical attack (45). However, in this case a sizeable fraction of them are also protected against chemical

  11. Lessons from an evolving rRNA: 16S and 23S rRNA structures from a comparative perspective

    NASA Technical Reports Server (NTRS)

    Gutell, R. R.; Larsen, N.; Woese, C. R.

    1994-01-01

    The 16S and 23S rRNA higher-order structures inferred from comparative analysis are now quite refined. The models presented here differ from their immediate predecessors only in minor detail. Thus, it is safe to assert that all of the standard secondary-structure elements in (prokaryotic) rRNAs have been identified, with approximately 90% of the individual base pairs in each molecule having independent comparative support, and that at least some of the tertiary interactions have been revealed. It is interesting to compare the rRNAs in this respect with tRNA, whose higher-order structure is known in detail from its crystal structure (36) (Table 2). It can be seen that rRNAs have as great a fraction of their sequence in established secondary-structure elements as does tRNA. However, the fact that the former show a much lower fraction of identified tertiary interactions and a greater fraction of unpaired nucleotides than the latter implies that many of the rRNA tertiary interactions remain to be located. (Alternatively, the ribosome might involve protein-rRNA rather than intramolecular rRNA interactions to stabilize three-dimensional structure.) Experimental studies on rRNA are consistent to a first approximation with the structures proposed here, confirming the basic assumption of comparative analysis, i.e., that bases whose compositions strictly covary are physically interacting. In the exhaustive study of Moazed et al. (45) on protection of the bases in the small-subunit rRNA against chemical modification, the vast majority of bases inferred to pair by covariation are found to be protected from chemical modification, both in isolated small-subunit rRNA and in the 30S subunit. The majority of the tertiary interactions are reflected in the chemical protection data as well (45). On the other hand, many of the bases not shown as paired in Fig. 1 are accessible to chemical attack (45). However, in this case a sizeable fraction of them are also protected against chemical

  12. Infective Arthritis: Bacterial 23S rRNA Gene Sequencing as a Supplementary Diagnostic Method

    PubMed Central

    Moser, Claus; Andresen, Keld; Kjerulf, Anne; Salamon, Suheil; Kemp, Michael; Christensen, Jens Jørgen

    2008-01-01

    Consecutively collected synovial fluids were examined for presence of bacterial DNA (a 700-bp fragment of the bacterial 23S rRNA gene) followed by DNA sequencing of amplicons, and by conventional bacteriological methods. One or more microorganisms were identified in 22 of the 227 synovial fluids (9,7%) originating from 17 patients. Sixteen of the patients had clinical signs of arthritis. For 11 patients molecular and conventional bacterial examinations were in agreement. Staphylococcus aureus, Streptococcus dysgalactiae subspecies equisimilis and Streptococcus pneumoniae, were detected in synovial fluids from 6, 2 and 2 patients, respectively. In 3 patients only 23S rRNA analysis was positive; 2 synovial fluids contained S. dysgalactiae subspecies equisimilis and 1 S. pneumoniae). The present study indicates a significant contribution by PCR with subsequent DNA sequencing of the 23S rRNA gene analysis in recognizing and identification of microorganisms from synovial fluids. PMID:19088916

  13. Reconstitution of functional eukaryotic ribosomes from Dictyostelium discoideum ribosomal proteins and RNA.

    PubMed

    Mangiarotti, G; Chiaberge, S

    1997-08-08

    40 and 60 S ribosomal subunits have been reconstituted in vitro from purified ribosomal RNA and ribosomal proteins of Dictyostelium discoideum. The functionality of the reconstituted ribosomes was demonstrated in in vitro mRNA-directed protein synthesis. The reassembly proceeded well with immature precursors of ribosomal RNA but poorly if at all with mature cytoplasmic RNA species. Reassembly also required a preparation of small nuclear RNA(s), acting as morphopoietic factor(s).

  14. Radical SAM-Mediated Methylation of Ribosomal RNA

    PubMed Central

    Stojkovic, Vanja; Fujimori, Danica Galonić

    2015-01-01

    Post-transcriptional modifications of RNA play an important role in a wide range of biological processes. In ribosomal RNA (rRNA), methylation of nucleotide bases is the predominant modification. In recent years, methylation of adenosine 2503 (A2503) in bacterial 23S rRNA has attracted significant attention due to both the unusual regioselectivity of the methyl group incorporation, as well as the pathophysiological roles of the resultant methylations. Specifically, A2503 is methylated at the C2 and C8 positions of the adenine ring, and the introduced modifications have a profound impact on translational fidelity and antibiotic resistance, respectively. These modifications are performed by RlmN and Cfr, two members, of the recently discovered class of radical S-adenosylmethionine (radical SAM) methylsynthases. Here, we present several methods that can be used to evaluate the activity of these enzymes, under both in vivo and in vitro conditions. PMID:26253978

  15. DbpA: a DEAD box protein specifically activated by 23s rRNA.

    PubMed Central

    Fuller-Pace, F V; Nicol, S M; Reid, A D; Lane, D P

    1993-01-01

    The Escherichia coli protein DbpA is a member of the 'DEAD box' family of putative RNA-dependent ATPases and RNA helicases, so called because they share the highly conserved motif Asp-Glu-Ala-Asp, together with several other conserved elements. We have investigated DbpA expression under conditions where an endogenous promoter is used. In this context, translation initiation does not occur at the previously identified AUG, but at an upstream, in-frame GUG. Mutation of the GUG initiation codon to AUG virtually abolishes DbpA expression, suggesting an unusual translation initiation mechanism. Using an inducible overexpression plasmid, we have purified milligram quantities of DbpA to homogeneity and shown that the purified protein hydrolyses ATP in an RNA-dependent manner. This ATPase activity is interesting in that, unlike that of other DEAD box proteins investigated to date, it absolutely requires a specific bacterial RNA, which we have identified as 23S rRNA. This observation is particularly significant since DbpA will bind other RNAs and DNA, but will only hydrolyse ATP in the presence of 23S rRNA. Images PMID:8253085

  16. Different sensitivity of H69 modification enzymes RluD and RlmH to mutations in Escherichia coli 23S rRNA.

    PubMed

    Leppik, Margus; Ero, Rya; Liiv, Aivar; Kipper, Kalle; Remme, Jaanus

    2012-05-01

    Nucleoside modifications are introduced into the ribosomal RNA during the assembly of the ribosome. The number and the localization of the modified nucleosides in rRNAs are known for several organisms. In bacteria, rRNA modified nucleosides are synthesized by a set of specific enzymes, the majority of which have been identified in Escherichia coli. Each rRNA modification enzyme recognizes its substrate nucleoside(s) at a specific stage of ribosome assembly. Not much is known about the specificity determinants involved in the substrate recognition of the modification enzymes. In order to shed light on the substrate specificity of RluD and RlmH, the enzymes responsible for the introduction of modifications into the stem-loop 69 (H69), we monitored the formation of H69 pseudouridines (Ψ) and methylated pseudouridine (m3Ψ) in vitro on ribosomes with alterations in 23S rRNA. While the synthesis of Ψs in H69 by RluD is relatively insensitive to the point mutations at neighboring positions, methylation of one of the Ψs by RlmH exhibited a much stronger sensitivity. Apparently, in spite of synthesizing modifications in the same region or even at the same position of rRNA, the two enzymes employ different substrate recognition mechanisms.

  17. The ribosome challenge to the RNA world.

    PubMed

    Bowman, Jessica C; Hud, Nicholas V; Williams, Loren Dean

    2015-04-01

    An RNA World that predated the modern world of polypeptide and polynucleotide is one of the most widely accepted models in origin of life research. In this model, the translation system shepherded the RNA World into the extant biology of DNA, RNA, and protein. Here, we examine the RNA World Hypothesis in the context of increasingly detailed information available about the origins, evolution, functions, and mechanisms of the translation system. We conclude that the translation system presents critical challenges to RNA World Hypotheses. Firstly, a timeline of the RNA World is problematic when the ribosome is incorporated. The mechanism of peptidyl transfer of the ribosome appears distinct from evolved enzymes, signaling origins in a chemical rather than biological milieu. Secondly, we have no evidence that the basic biochemical toolset of life is subject to substantive change by Darwinian evolution, as required for the transition from the RNA world to extant biology. Thirdly, we do not see specific evidence for biological takeover of ribozyme function by protein enzymes. Finally, we can find no basis for preservation of the ribosome as ribozyme or the universality of translation, if it were the case that other information transducing ribozymes, such as ribozyme polymerases, were replaced by protein analogs and erased from the phylogenetic record. We suggest that an updated model of the RNA World should address the current state of knowledge of the translation system.

  18. 23S rRNA domain V, a fragment that can be specifically methylated in vitro by the ErmSF (TlrA) methyltransferase.

    PubMed Central

    Kovalic, D; Giannattasio, R B; Jin, H J; Weisblum, B

    1994-01-01

    The DNA sequence that encodes 23S rRNA domain V of Bacillus subtilis, nucleotides 2036 to 2672 (C. J. Green, G. C. Stewart, M. A. Hollis, B. S. Vold, and K. F. Bott, Gene 37:261-266, 1985), was cloned and used as a template from which to transcribe defined domain V RNA in vitro. The RNA transcripts served as a substrate in vitro for specific methylation of B. subtilis adenine 2085 (adenine 2058 in Escherichia coli 23S rRNA) by the ErmSF methyltransferase, an enzyme that confers resistance to the macrolide-lincosamide-streptogramin B group of antibiotics on Streptomyces fradiae NRRL 2702, the host from which it was cloned. Thus, neither RNA sequences belonging to domains other than V nor the association of 23S rRNA with ribosomal proteins is needed for the specific methylation of adenine that confers resistance to the macrolide-lincosamide-streptogramin B group of antibiotics. Images PMID:7961463

  19. Networks of interactions in the secondary and tertiary structure of ribosomal RNA

    NASA Astrophysics Data System (ADS)

    Lee, Chang-Yong; Lee, Jung C.; Gutell, Robin R.

    2007-12-01

    We construct four different structural networks for both the secondary and tertiary structures of the 16S and 23S ribosomal RNAs (rRNAs) in the high-resolution crystal structures of the Thermus thermophilus 30S and Haloarcula marismortui 50S ribosomal subunits, and investigate topological characteristics of the rRNA structures by determining relevant measures, such as the characteristic path length, the clustering coefficient, and the helix betweenness. This study reveals that the 23S rRNA network is more compact than the 16S rRNA networks, reflecting the more globular overall structure of the 23S rRNA relative to the 16S rRNA. In particular, the large number of tertiary interactions in the 23S rRNA tends to cluster, accounting for its small-world network properties. In addition, although the rRNA networks are not the scale-free network, their helix betweenness has a power-law distribution and is correlated with the phylogenetic conservation of helices. The higher the helix betweenness, the more conserved the helix. These results suggest a potential role of the rRNA network as a new quantitative approach in rRNA research.

  20. Cloning and sequence analysis of two copies of a 23S rRNA gene from Helicobacter pylori and association of clarithromycin resistance with 23S rRNA mutations.

    PubMed Central

    Taylor, D E; Ge, Z; Purych, D; Lo, T; Hiratsuka, K

    1997-01-01

    In this study, two identical copies of a 23S-5S gene cluster, which are separately situated within the Helicobacter pylori UA802 chromosome, were cloned and sequenced. Comparison of the DNA sequence of the H. pylori 23S rRNA gene with known sequences of other bacterial 23S rRNA genes indicated that the H. pylori UA802 23S rRNA genes are closely related to those of Campylobacter spp. and therefore belong in the proposed Proteobacteria subdivision. The 5'-terminal nucleotide T or A of the 23S rRNA is close to a Pribnow box which could be a -10 region of the transcription promoter for the 23S rRNA gene, suggesting that a posttranscriptional process is likely not involved in the maturation of the H. pylori 23S rRNA. Clinical isolates of H. pylori resistant to clarithromycin were examined by using natural transformation and pulsed-field gel electrophoresis. Cross-resistance to clarithromycin and erythromycin, which was transferred by natural transformation from the Cla(r) Ery(r) donor strain H. pylori E to the Cla(s) Ery(s) recipient strain H. pylori UA802, was associated with an single A-to-G transition mutation at position 2142 of both copies of the 23S rRNA in UA802 Cla(r) Ery(r) mutants. The transformation frequency for Cla(r) and Ery(r) was found to be approximately 2 x 10(-6) transformants per viable cell, and the MICs of both clarithromycin and erythromycin for the Cla(r) Ery(r) mutants were equal to those for the donor isolate. Our results confirmed the previous findings that mutations at positions 2142 and 2143 of the H. pylori 23S rRNA gene are responsible for clarithromycin resistance and suggest that acquisition of clarithromycin resistance in H. pylori could also result from horizontal transfer. PMID:9420030

  1. Chloroplast RNA-Binding Protein RBD1 Promotes Chilling Tolerance through 23S rRNA Processing in Arabidopsis

    PubMed Central

    Yang, Leiyun; Yang, Fen; Wang, Yi; Zhu, Jian-Kang; Hua, Jian

    2016-01-01

    Plants have varying abilities to tolerate chilling (low but not freezing temperatures), and it is largely unknown how plants such as Arabidopsis thaliana achieve chilling tolerance. Here, we describe a genome-wide screen for genes important for chilling tolerance by their putative knockout mutants in Arabidopsis thaliana. Out of 11,000 T-DNA insertion mutant lines representing half of the genome, 54 lines associated with disruption of 49 genes had a drastic chilling sensitive phenotype. Sixteen of these genes encode proteins with chloroplast localization, suggesting a critical role of chloroplast function in chilling tolerance. Study of one of these proteins RBD1 with an RNA binding domain further reveals the importance of chloroplast translation in chilling tolerance. RBD1 is expressed in the green tissues and is localized in the chloroplast nucleoid. It binds directly to 23S rRNA and the binding is stronger under chilling than at normal growth temperatures. The rbd1 mutants are defective in generating mature 23S rRNAs and deficient in chloroplast protein synthesis especially under chilling conditions. Together, our study identifies RBD1 as a regulator of 23S rRNA processing and reveals the importance of chloroplast function especially protein translation in chilling tolerance. PMID:27138552

  2. Large Variations in Bacterial Ribosomal RNA Genes

    PubMed Central

    Lim, Kyungtaek; Furuta, Yoshikazu; Kobayashi, Ichizo

    2012-01-01

    Ribosomal RNA (rRNA) genes, essential to all forms of life, have been viewed as highly conserved and evolutionarily stable, partly because very little is known about their natural variations. Here, we explored large-scale variations of rRNA genes through bioinformatic analyses of available complete bacterial genomic sequences with an emphasis on formation mechanisms and biological significance. Interestingly, we found bacterial genomes in which no 16S rRNA genes harbor the conserved core of the anti–Shine-Dalgarno sequence (5′-CCTCC-3′). This loss was accompanied by elimination of Shine-Dalgarno–like sequences upstream of their protein-coding genes. Those genomes belong to 1 or 2 of the following categories: primary symbionts, hemotropic Mycoplasma, and Flavobacteria. We also found many rearranged rRNA genes and reconstructed their history. Conjecturing the underlying mechanisms, such as inversion, partial duplication, transposon insertion, deletion, and substitution, we were able to infer their biological significance, such as co-orientation of rRNA transcription and chromosomal replication, lateral transfer of rRNA gene segments, and spread of rRNA genes with an apparent structural defect through gene conversion. These results open the way to understanding dynamic evolutionary changes of rRNA genes and the translational machinery. PMID:22446745

  3. RlmCD-mediated U747 methylation promotes efficient G748 methylation by methyltransferase RlmAII in 23S rRNA in Streptococcus pneumoniae; interplay between two rRNA methylations responsible for telithromycin susceptibility

    PubMed Central

    Shoji, Tatsuma; Takaya, Akiko; Sato, Yoshiharu; Kimura, Satoshi; Suzuki, Tsutomu; Yamamoto, Tomoko

    2015-01-01

    Adenine at position 752 in a loop of helix 35 from positions 745 to 752 in domain II of 23S rRNA is involved in binding to the ribosome of telithromycin (TEL), a member of ketolides. Methylation of guanine at position 748 by the intrinsic methyltransferase RlmAII enhances binding of telithromycin (TEL) to A752 in Streptococcus pneumoniae. We have found that another intrinsic methylation of the adjacent uridine at position 747 enhances G748 methylation by RlmAII, rendering TEL susceptibility. U747 and another nucleotide, U1939, were methylated by the dual-specific methyltransferase RlmCD encoded by SP_1029 in S. pneumoniae. Inactivation of RlmCD reduced N1-methylated level of G748 by RlmAII in vivo, leading to TEL resistance when the nucleotide A2058, located in domain V of 23S rRNA, was dimethylated by the dimethyltransferase Erm(B). In vitro methylation of rRNA showed that RlmAII activity was significantly enhanced by RlmCD-mediated pre-methylation of 23S rRNA. These results suggest that RlmCD-mediated U747 methylation promotes efficient G748 methylation by RlmAII, thereby facilitating TEL binding to the ribosome. PMID:26365244

  4. Identification of a Novel G2073A Mutation in 23S rRNA in Amphenicol-Selected Mutants of Campylobacter jejuni

    PubMed Central

    Naren, Gaowa; Li, Hui; Xia, Xi; Wu, Congming; Shen, Jianzhong; Zhang, Qijing; Wang, Yang

    2014-01-01

    Objectives This study was conducted to examine the development and molecular mechanisms of amphenicol resistance in Campylobacter jejuni by using in vitro selection with chloramphenicol and florfenicol. The impact of the resistance development on growth rates was also determined using in vitro culture. Methods Chloramphenicol and florfenicol were used as selection agents to perform in vitro stepwise selection. Mutants resistant to the selective agents were obtained from the selection process. The mutant strains were compared with the parent strain for changes in MICs and growth rates. The 23S rRNA gene and the L4 and L22 ribosomal protein genes in the mutant strains and the parent strain were amplified and sequenced to identify potential resistance-associated mutations. Results C. jejuni strains that were highly resistant to chloramphenicol and florfenicol were obtained from in vitro selection. A novel G2073A mutation in all three copies of the 23S rRNA gene was identified in all the resistant mutants examined, which showed resistance to both chloramphenicol and florfenicol. In addition, all the mutants selected by chloramphenicol also exhibited the G74D modification in ribosomal protein L4, which was previously shown to confer a low-level erythromycin resistance in Campylobacter species. The mutants selected by florfenicol did not have the G74D mutation in L4. Notably, the amphenicol-resistant mutants also exhibited reduced susceptibility to erythromycin, suggesting that the selection resulted in cross resistance to macrolides. Conclusions This study identifies a novel point mutation (G2073A) in 23S rRNA in amphenicol-selected mutants of C. jejuni. Development of amphenicol resistance in Campylobacter likely incurs a fitness cost as the mutant strains showed slower growth rates in antibiotic-free media. PMID:24728007

  5. Nonenzymatic microorganism identification based on ribosomal RNA

    NASA Astrophysics Data System (ADS)

    Ives, Jeffrey T.; Pierini, Alicia M.; Stokes, Jeffrey A.; Wahlund, Thomas M.; Read, Betsy; Bechtel, James H.; Bronk, Burt V.

    1999-11-01

    Effective defense against biological warfare (BW) agents requires rapid, fieldable and accurate systems. For micro- organisms like bacteria and viruses, ribosomal RNA (rRNA) provides a valuable target with multiple advantages of species specificity and intrinsic target amplification. Vegetative and spore forms of bacteria contain approximately 104 copies of rRNA. Direct detection of rRNA copies can eliminate some of the interference and preparation difficulties involved in enzymatic amplification methods. In order to apply the advantages of rRNA to BW defense, we are developing a fieldable system based on 16S rRNA, physical disruption of the micro-organism, solid phase hybridization, and fluorescence detection. Our goals include species-specific identification, complete operation from raw sample to identification in 15 minutes or less, and compact, fieldable instrumentation. Initial work on this project has investigated the lysis and hybridization steps, the species-specificity of oligonucleotides probes, and the development of a novel electromagnetic method to physically disrupt the micro- organisms. Target bacteria have been Escherichia coli (E. coli) and Bacillus subtilis (B. subtilis). Continuing work includes further development of methods to rapidly disrupt the micro-organisms and release the rRNA, improved integration and processing, and extension to bacterial and mammalian viruses like MS2 and vesicular stomatitis virus.

  6. A detailed view of a ribosomal active site: the structure of the L11-RNA complex.

    PubMed

    Wimberly, B T; Guymon, R; McCutcheon, J P; White, S W; Ramakrishnan, V

    1999-05-14

    We report the crystal structure of a 58 nucleotide fragment of 23S ribosomal RNA bound to ribosomal protein L11. This highly conserved ribonucleoprotein domain is the target for the thiostrepton family of antibiotics that disrupt elongation factor function. The highly compact RNA has both familiar and novel structural motifs. While the C-terminal domain of L11 binds RNA tightly, the N-terminal domain makes only limited contacts with RNA and is proposed to function as a switch that reversibly associates with an adjacent region of RNA. The sites of mutations conferring resistance to thiostrepton and micrococcin line a narrow cleft between the RNA and the N-terminal domain. These antibiotics are proposed to bind in this cleft, locking the putative switch and interfering with the function of elongation factors.

  7. Regulating the Ribosome: A Spotlight on RNA Dark Matter

    PubMed Central

    Lintner, Nathanael G.; Cate, Jamie H.D.

    2014-01-01

    In this issue Pircher et al.(2014) show that an abundant ribosome-associated 18-nt noncoding RNA (ncRNA),derived from the open reading frame of an mRNA,acts directly on the ribosome and regulates global translation levels in response to hypertonic shock. PMID:24725592

  8. Structural features of the tmRNA-ribosome interaction.

    PubMed

    Bugaeva, Elizaveta Y; Surkov, Serhiy; Golovin, Andrey V; Ofverstedt, Lars-Göran; Skoglund, Ulf; Isaksson, Leif A; Bogdanov, Alexey A; Shpanchenko, Olga V; Dontsova, Olga A

    2009-12-01

    Trans-translation is a process which switches the synthesis of a polypeptide chain encoded by a nonstop messenger RNA to the mRNA-like domain of a transfer-messenger RNA (tmRNA). It is used in bacterial cells for rescuing the ribosomes arrested during translation of damaged mRNA and directing this mRNA and the product polypeptide for degradation. The molecular basis of this process is not well understood. Earlier, we developed an approach that allowed isolation of tmRNA-ribosomal complexes arrested at a desired step of tmRNA passage through the ribosome. We have here exploited it to examine the tmRNA structure using chemical probing and cryo-electron microscopy tomography. Computer modeling has been used to develop a model for spatial organization of the tmRNA inside the ribosome at different stages of trans-translation.

  9. Mutations in the 23S rRNA gene of Helicobacter pylori associated with clarithromycin resistance.

    PubMed Central

    Kim, Kyung Suk; Kang, Jung Oak; Eun, Chang Soo; Han, Dong Soo; Choi, Tae Yeal

    2002-01-01

    Among 12 clarithromycin-resistant Helicobacter pylori strains isolated in Guri, Korea, 8 showed an adenine to guanine mutation at position 2143 (formerly A2144G or E. coli 2059) in the 23S rRNA gene by the PCR-restriction fragment length polymorphism (RFLP) method. The remaining 4 strains, digested by neither BsaI nor BbsI, showed a thymine to cytosine mutation at position 2182 (T2182C) by direct sequencing of the PCR products. The T2182C mutants showed a tendency of higher levels of minimum inhibitory concentration to clarithromycin than the A2143G mutants. In conclusion, either the A2143G or the T2182C mutation was present in 100% of clarithromycin-resistant H. pylori isolates examined. The PCR-RFLP technique with restriction enzymes BbsI and BsaI was a rapid and relatively simple method to detect the clarithromycin resistance. But undigested isolates were quite frequent among our isolates (33.3%), the PCR-RFLP method with restriction enzymes BbsI and BsaI should not be used alone, and development of other rapid detection method for clarithromycin resistance is mandatory. PMID:12378008

  10. Occurrence of 20S RNA and 23S RNA replicons in industrial yeast strains and their variation under nutritional stress conditions.

    PubMed

    López, Victoria; Gil, Rosario; Vicente Carbonell, José; Navarro, Alfonso

    2002-04-01

    We have characterized industrial yeast strains used in the brewing, baking, and winemaking industries for the presence or absence of cytoplasmic single-stranded 20S and 23S RNAs. Furthermore, the variation of intracellular concentrations of these replicons in brewing and laboratory strains under nutritional stress conditions was determined. Our results show a correlation between the relative abundance of these replicons and exposure of yeast to nutritionally stressful conditions, indicating that these RNAs could be employed as molecular probes to evaluate the exposure of 20S(+) and/or 23S(+) yeast strains to stress situations during industrial manipulation. During this study, several 20S(-)23S(+) Saccharomyces cerevisiae strains were isolated and identified. This is the first time that a yeast strain containing only 23S RNA has been reported, demonstrating that 20S RNA is not required for 23S RNA replication.

  11. Compilation of small ribosomal subunit RNA structures.

    PubMed Central

    Neefs, J M; Van de Peer, Y; De Rijk, P; Chapelle, S; De Wachter, R

    1993-01-01

    The database on small ribosomal subunit RNA structure contained 1804 nucleotide sequences on April 23, 1993. This number comprises 365 eukaryotic, 65 archaeal, 1260 bacterial, 30 plastidial, and 84 mitochondrial sequences. These are stored in the form of an alignment in order to facilitate the use of the database as input for comparative studies on higher-order structure and for reconstruction of phylogenetic trees. The elements of the postulated secondary structure for each molecule are indicated by special symbols. The database is available on-line directly from the authors by ftp and can also be obtained from the EMBL nucleotide sequence library by electronic mail, ftp, and on CD ROM disk. PMID:8332525

  12. Mapping the interaction of SmpB with ribosomes by footprinting of ribosomal RNA

    PubMed Central

    Ivanova, Natalia; Pavlov, Michael Y.; Bouakaz, Elli; Ehrenberg, Måns; Schiavone, Lovisa Holmberg

    2005-01-01

    In trans-translation transfer messenger RNA (tmRNA) and small protein B (SmpB) rescue ribosomes stalled on truncated or in other ways problematic mRNAs. SmpB promotes the binding of tmRNA to the ribosome but there is uncertainty about the number of participating SmpB molecules as well as their ribosomal location. Here, the interaction of SmpB with ribosomal subunits and ribosomes was studied by isolation of SmpB containing complexes followed by chemical modification of ribosomal RNA with dimethyl sulfate, kethoxal and hydroxyl radicals. The results show that SmpB binds 30S and 50S subunits with 1:1 molar ratios and the 70S ribosome with 2:1 molar ratio. SmpB-footprints are similar on subunits and the ribosome. In the 30S subunit, SmpB footprints nucleotides that are in the vicinity of the P-site facing the E-site, and in the 50S subunit SmpB footprints nucleotides that are located below the L7/L12 stalk in the 3D structure of the ribosome. Based on these results, we suggest a mechanism where two molecules of SmpB interact with tmRNA and the ribosome during trans-translation. The first SmpB molecule binds near the factor-binding site on the 50S subunit helping tmRNA accommodation on the ribosome, whereas the second SmpB molecule may functionally substitute for a missing anticodon stem–loop in tmRNA during later steps of trans-translation. PMID:15972795

  13. 23S rRNA gene mutations contributing to macrolide resistance in Campylobacter jejuni and Campylobacter coli

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Operon specific 23S rRNA mutations affecting minimum inhibitory concentrations (MICs) of macrolides (erythromycin [ERY], azithromycin [AZM], tylosin [TYL]) and a lincosamide (clindamycin [CLI]) were examined in a collection of Campylobacter jejuni and C. coli isolates. The three copies of the Campy...

  14. The Expression of Antibiotic Resistance Methyltransferase Correlates with mRNA Stability Independently of Ribosome Stalling

    PubMed Central

    Dzyubak, Ekaterina

    2016-01-01

    Members of the Erm methyltransferase family modify 23S rRNA of the bacterial ribosome and render cross-resistance to macrolides and multiple distantly related antibiotics. Previous studies have shown that the expression of erm is activated when a macrolide-bound ribosome stalls the translation of the leader peptide preceding the cotranscribed erm. Ribosome stalling is thought to destabilize the inhibitory stem-loop mRNA structure and exposes the erm Shine-Dalgarno (SD) sequence for translational initiation. Paradoxically, mutations that abolish ribosome stalling are routinely found in hyper-resistant clinical isolates; however, the significance of the stalling-dead leader sequence is largely unknown. Here, we show that nonsense mutations in the Staphylococcus aureus ErmB leader peptide (ErmBL) lead to high basal and induced expression of downstream ErmB in the absence or presence of macrolide concomitantly with elevated ribosome methylation and resistance. The overexpression of ErmB is associated with the reduced turnover of the ermBL-ermB transcript, and the macrolide appears to mitigate mRNA cleavage at a site immediately downstream of the ermBL SD sequence. The stabilizing effect of antibiotics on mRNA is not limited to ermBL-ermB; cationic antibiotics representing a ribosome-stalling inducer and a noninducer increase the half-life of specific transcripts. These data unveil a new layer of ermB regulation and imply that ErmBL translation or ribosome stalling serves as a “tuner” to suppress aberrant production of ErmB because methylated ribosome may impose a fitness cost on the bacterium as a result of misregulated translation. PMID:27645242

  15. DExD/H-box RNA helicases in ribosome biogenesis

    PubMed Central

    Martin, Roman; Straub, Annika U.; Doebele, Carmen; Bohnsack, Markus T.

    2013-01-01

    Ribosome synthesis requires a multitude of cofactors, among them DExD/H-box RNA helicases. Bacterial RNA helicases involved in ribosome assembly are not essential, while eukaryotes strictly require multiple DExD/H-box proteins that are involved in the much more complex ribosome biogenesis pathway. Here, RNA helicases are thought to act in structural remodeling of the RNPs including the modulation of protein binding, and they are required for allowing access or the release of specific snoRNPs from pre-ribosomes. Interestingly, helicase action is modulated by specific cofactors that can regulate recruitment and enzymatic activity. This review summarizes the current knowledge and focuses on recent findings and open questions on RNA helicase function and regulation in ribosome synthesis. PMID:22922795

  16. Ribosomal RNA sequence suggest microsporidia are extremely ancient eukaryotes

    NASA Technical Reports Server (NTRS)

    Vossbrinck, C. R.; Maddox, J. V.; Friedman, S.; Debrunner-Vossbrinck, B. A.; Woese, C. R.

    1987-01-01

    A comparative sequence analysis of the 18S small subunit ribosomal RNA (rRNA) of the microsporidium Vairimorpha necatrix is presented. The results show that this rRNA sequence is more unlike those of other eukaryotes than any known eukaryote rRNA sequence. It is concluded that the lineage leading to microsporidia branched very early from that leading to other eukaryotes.

  17. Binding of tRNA to the ribosomal A and P sites protects two distinct sets of nucleotides in 16 S rRNA.

    PubMed

    Moazed, D; Noller, H F

    1990-01-05

    Transfer RNA protects a characteristic set of bases in 16 S rRNA from chemical probes when it binds to ribosomes. We used several criteria, based on construction of well-characterized in vitro ribosome-tRNA complexes, to assign these proteins to A or P-site binding. All of these approaches lead to similar conclusions. In the A site, tRNA caused protection of G529, G530, A1492 and A1493 (strongly), and A1408 and G1494 (weakly). In the P site, the protected bases are G693, A794, C795, G926 and G1401 (strong), and A532, G966, G1338 and G1339 (weak). In contrast to what is observed for 23 S rRNA, blocking the release of EF-Tu.GDP from the ribosome by kirromycin has no detectable effect on the protection of bases in 16 S rRNA.

  18. The three-dimensional structure of the RNA-binding domain of ribosomal protein L2; a protein at the peptidyl transferase center of the ribosome.

    PubMed Central

    Nakagawa, A; Nakashima, T; Taniguchi, M; Hosaka, H; Kimura, M; Tanaka, I

    1999-01-01

    Ribosomal protein L2 is the largest protein component in the ribosome. It is located at or near the peptidyl transferase center and has been a prime candidate for the peptidyl transferase activity. It binds directly to 23S rRNA and plays a crucial role in its assembly. The three-dimensional structure of the RNA-binding domain of L2 from Bacillus stearothermophilus has been determined at 2.3 A resolution by X-ray crystallography using the selenomethionyl MAD method. The RNA-binding domain of L2 consists of two recurring motifs of approximately 70 residues each. The N-terminal domain (positions 60-130) is homologous to the OB-fold, and the C-terminal domain (positions 131-201) is homologous to the SH3-like barrel. Residues Arg86 and Arg155, which have been identified by mutation experiments to be involved in the 23S rRNA binding, are located at the gate of the interface region between the two domains. The molecular architecture suggests how this important protein has evolved from the ancient nucleic acid-binding proteins to create a 23S rRNA-binding domain in the very remote past. PMID:10075918

  19. Impact of P-Site tRNA and Antibiotics on Ribosome Mediated Protein Folding: Studies Using the Escherichia coli Ribosome

    PubMed Central

    Mondal, Surojit; Pathak, Bani Kumar; Ray, Sutapa; Barat, Chandana

    2014-01-01

    Background The ribosome, which acts as a platform for mRNA encoded polypeptide synthesis, is also capable of assisting in folding of polypeptide chains. The peptidyl transferase center (PTC) that catalyzes peptide bond formation resides in the domain V of the 23S rRNA of the bacterial ribosome. Proper positioning of the 3′ –CCA ends of the A- and P-site tRNAs via specific interactions with the nucleotides of the PTC are crucial for peptidyl transferase activity. This RNA domain is also the center for ribosomal chaperoning activity. The unfolded polypeptide chains interact with the specific nucleotides of the PTC and are released in a folding competent form. In vitro transcribed RNA corresponding to this domain (bDV RNA) also displays chaperoning activity. Results The present study explores the effects of tRNAs, antibiotics that are A- and P-site PTC substrate analogs (puromycin and blasticidin) and macrolide antibiotics (erythromycin and josamycin) on the chaperoning ability of the E. coli ribosome and bDV RNA. Our studies using mRNA programmed ribosomes show that a tRNA positioned at the P-site effectively inhibits the ribosome's chaperoning function. We also show that the antibiotic blasticidin (that mimics the interaction between 3′–CCA end of P/P-site tRNA with the PTC) is more effective in inhibiting ribosome and bDV RNA chaperoning ability than either puromycin or the macrolide antibiotics. Mutational studies of the bDV RNA could identify the nucleotides U2585 and G2252 (both of which interact with P-site tRNA) to be important for its chaperoning ability. Conclusion Both protein synthesis and their proper folding are crucial for maintenance of a functional cellular proteome. The PTC of the ribosome is attributed with both these abilities. The silencing of the chaperoning ability of the ribosome in the presence of P-site bound tRNA might be a way to segregate these two important functions. PMID:25000563

  20. Helicobacter pylori specific nested PCR assay for the detection of 23S rRNA mutation associated with clarithromycin resistance

    PubMed Central

    Maeda, S; Yoshida, H; Ogura, K; Kanai, F; Shiratori, Y; Omata, M

    1998-01-01

    Background—Clarithromycin is one of the most important antibiotics for Helicobacter pylori eradication. However, 5-10% of strains are reported to be resistant. It has been shown that one point mutation in the 23S rRNA gene is associated with resistance to clarithromycin. 
Aims—To establish a polymerase chain reaction (PCR) system which amplifies a segment of the 23S rRNA gene containing the mutation points with primers specific for H pylori, so that H pylori infection and the mutation associated with clarithromycin resistance can be examined simultaneously. 
Methods—To detect H pylori infection and the mutation simultaneously, primers specific for the H pylori 23S rRNA gene were designed based on sequence conservation among H pylori strains and sequence specificity as compared with other bacteria. DNA from 57 cultured strains and from 39 gastric juice samples was amplified in the seminested 23S rRNA PCR. Clinical applicability was evaluated in 85patients. 
Results—DNA samples from 57 cultured strains were all amplified. The novel assay and the urease A PCR agreed in 37/39 gastric juice samples with no false positives. The assay did not amplify the DNA of bacteria other than H pylori. Eight of 85 samples had the mutation before treatment. In clarithromycin based treatment, eradication was achieved in 2/5 (40%) with the mutation and 29/34 (85%) without the mutation. 
Conclusion—The assay using gastric juice is quick (within 12 hours) and non-invasive (endoscopy not required), enabling rapid initiation of appropriate antibiotic treatment. 

 Keywords: Helicobacter pylori; eradication; clarithromycin; resistance; point mutation PMID:9863474

  1. RNA tertiary interactions in the large ribosomal subunit: The A-minor motif

    SciTech Connect

    Nissen, Poul; Ippolito, Joseph A.; Ban, Nenad; Moore, Peter B.; Steitz, Thomas A.

    2009-10-07

    Analysis of the 2.4-{angstrom} resolution crystal structure of the large ribosomal subunit from Haloarcula marismortui reveals the existence of an abundant and ubiquitous structural motif that stabilizes RNA tertiary and quaternary structures. This motif is termed the A-minor motif, because it involves the insertion of the smooth, minor groove edges of adenines into the minor groove of neighboring helices, preferentially at C-G base pairs, where they form hydrogen bonds with one or both of the 2' OHs of those pairs. A-minor motifs stabilize contacts between RNA helices, interactions between loops and helices, and the conformations of junctions and tight turns. The interactions between the 3' terminal adenine of tRNAs bound in either the A site or the P site with 23S rRNA are examples of functionally significant A-minor interactions. The A-minor motif is by far the most abundant tertiary structure interaction in the large ribosomal subunit; 186 adenines in 23S and 5S rRNA participate, 68 of which are conserved. It may prove to be the universally most important long-range interaction in large RNA structures.

  2. Model of ribosome translation and mRNA unwinding.

    PubMed

    Xie, Ping

    2013-05-01

    A ribosome is an enzyme that catalyzes translation of the genetic information encoded in messenger RNA (mRNA) into proteins. Besides translation through the single-stranded mRNA, the ribosome is also able to translate through the duplex region of mRNA via unwinding the duplex. Here, based on our proposed ribosome translation model, we study analytically the dynamics of Escherichia coli ribosome translation through the duplex region of mRNA, and compare with the available single molecule experimental data. It is shown that the ribosome uses only one active mechanism (mechanical unwinding), rather than two active mechanisms (open-state stabilization and mechanical unwinding), as proposed before, to unwind the duplex. The reduced rate of translation through the duplex region is due to the occurrence of futile transitions, which are induced by the energy barrier from the duplex unwinding to the forward translocation along the single-stranded mRNA. Moreover, we also present predicted results of the average translation rate versus the external force acting on the ribosome translating through the duplex region and through the single-stranded region of mRNA, which can be easily tested by future experiments.

  3. Translating Ribosomes Inhibit Poliovirus Negative-Strand RNA Synthesis

    PubMed Central

    Barton, David J.; Morasco, B. Joan; Flanegan, James B.

    1999-01-01

    Poliovirus has a single-stranded RNA genome of positive polarity that serves two essential functions at the start of the viral replication cycle in infected cells. First, it is translated to synthesize viral proteins and, second, it is copied by the viral polymerase to synthesize negative-strand RNA. We investigated these two reactions by using HeLa S10 in vitro translation-RNA replication reactions. Preinitiation RNA replication complexes were isolated from these reactions and then used to measure the sequential synthesis of negative- and positive-strand RNAs in the presence of different protein synthesis inhibitors. Puromycin was found to stimulate RNA replication overall. In contrast, RNA replication was inhibited by diphtheria toxin, cycloheximide, anisomycin, and ricin A chain. Dose-response experiments showed that precisely the same concentration of a specific drug was required to inhibit protein synthesis and to either stimulate or inhibit RNA replication. This suggested that the ability of these drugs to affect RNA replication was linked to their ability to alter the normal clearance of translating ribosomes from the input viral RNA. Consistent with this idea was the finding that the protein synthesis inhibitors had no measurable effect on positive-strand synthesis in normal RNA replication complexes. In marked contrast, negative-strand synthesis was stimulated by puromycin and was inhibited by cycloheximide. Puromycin causes polypeptide chain termination and induces the dissociation of polyribosomes from mRNA. Cycloheximide and other inhibitors of polypeptide chain elongation “freeze” ribosomes on mRNA and prevent the normal clearance of ribosomes from viral RNA templates. Therefore, it appears that the poliovirus polymerase was not able to dislodge translating ribosomes from viral RNA templates and mediate the switch from translation to negative-strand synthesis. Instead, the initiation of negative-strand synthesis appears to be coordinately regulated

  4. Database on the structure of large ribosomal subunit RNA.

    PubMed Central

    De Rijk, P; Van de Peer, Y; Chapelle, S; De Wachter, R

    1994-01-01

    A database on large ribosomal subunit RNA is made available. It contains 258 sequences. It provides sequence, alignment and secondary structure information in computer-readable formats. Files can be obtained using ftp. PMID:7524023

  5. Epigenetic engineering of ribosomal RNA genes enhances protein production.

    PubMed

    Santoro, Raffaella; Lienemann, Philipp; Fussenegger, Martin

    2009-08-14

    Selection of mammalian high-producer cell lines remains a major challenge for the biopharmaceutical manufacturing industry. Ribosomal RNA (rRNA) genes encode the major component of the ribosome but many rRNA gene copies are not transcribed due to epigenetic silencing by the nucleolar remodelling complex (NoRC) [6], which may limit the cell's full production capacity. Here we show that the knockdown of TIP5, a subunit of NoRC, decreases the number of silent rRNA genes, upregulates rRNA transcription, enhances ribosome synthesis and increases production of recombinant proteins. However, general enhancement of rRNA transcription rate did not stimulate protein synthesis. Our data demonstrates that the number of transcriptionally competent rRNA genes limits efficient ribosome synthesis. Epigenetic engineering of ribosomal RNA genes offers new possibilities for improving biopharmaceutical manufacturing and provides novel insights into the complex regulatory network which governs the translation machinery in normal cellular processes as well as in pathological conditions like cancer.

  6. Methylation of 23S rRNA Nucleotide G748 by RlmAII Methyltransferase Renders Streptococcus pneumoniae Telithromycin Susceptible

    PubMed Central

    Sato, Yoshiharu; Shoji, Tatsuma; Yamamoto, Tomoko

    2013-01-01

    Several posttranscriptional modifications of bacterial rRNAs are important in determining antibiotic resistance or sensitivity. In all Gram-positive bacteria, dimethylation of nucleotide A2058, located in domain V of 23S rRNA, by the dimethyltransferase Erm(B) results in low susceptibility and resistance to telithromycin (TEL). However, this is insufficient to produce high-level resistance to TEL in Streptococcus pneumoniae. Inactivation of the methyltransferase RlmAII, which methylates the N-1 position of nucleotide G748, located in hairpin 35 of domain II of 23S rRNA, results in increased resistance to TEL in erm(B)-carrying S. pneumoniae. Sixteen TEL-resistant mutants (MICs, 16 to 32 μg/ml) were obtained from a clinically isolated S. pneumoniae strain showing low TEL susceptibility (MIC, 2 μg/ml), with mutation resulting in constitutive dimethylation of A2058 because of nucleotide differences in the regulatory region of erm(B) mRNA. Primer extension analysis showed that the degree of methylation at G748 in all TEL-resistant mutants was significantly reduced by a mutation in the gene encoding RlmAII to create a stop codon or change an amino acid residue. Furthermore, RNA footprinting with dimethyl sulfate and a molecular modeling study suggested that methylation of G748 may contribute to the stable interaction of TEL with domain II of 23S rRNA, even after dimethylation of A2058 by Erm(B). This novel finding shows that methylation of G748 by RlmAII renders S. pneumoniae TEL susceptible. PMID:23716046

  7. Mutational Evidence for a Functional Connection between Two Domains of 23S rRNA in Translation Termination

    PubMed Central

    Arkov, Alexey L.; Hedenstierna, Klas O. F.; Murgola, Emanuel J.

    2002-01-01

    Nucleotide 1093 in domain II of Escherichia coli 23S rRNA is part of a highly conserved structure historically referred to as the GTPase center. The mutation G1093A was previously shown to cause readthrough of nonsense codons and high temperature-conditional lethality. Defects in translation termination caused by this mutation have also been demonstrated in vitro. To identify sites in 23S rRNA that may be functionally associated with the G1093 region during termination, we selected for secondary mutations in 23S rRNA that would compensate for the temperature-conditional lethality caused by G1093A. Here we report the isolation and characterization of such a secondary mutation. The mutation is a deletion of two consecutive nucleotides from helix 73 in domain V, close to the peptidyltransferase center. The deletion results in a shortening of the CGCG sequence between positions 2045 and 2048 by two nucleotides to CG. In addition to restoring viability in the presence of G1093A, this deletion dramatically decreased readthrough of UGA nonsense mutations caused by G1093A. An analysis of the amount of mutant rRNA in polysomes revealed that this decrease cannot be explained by an inability of G1093A-containing rRNA to be incorporated into polysomes. Furthermore, the deletion was found to cause UGA readthrough on its own, thereby implicating helix 73 in termination for the first time. These results also indicate the existence of a functional connection between the G1093 region and helix 73 during translation termination. PMID:12193621

  8. Sequence diversity in the 16S-23S intergenic spacer region (ISR) of the rRNA operons in representatives of the Escherichia coli ECOR collection.

    PubMed

    Antón, A I; Martínez-Murcia, A J; Rodríguez-Valera, F

    1998-07-01

    The ribosomal RNA multigene family in Escherichia coli comprises seven rrn operons of similar, but not identical, sequence. Four operons (rrnC, B, G, and E) contain genes in the 16S-23S intergenic spacer region (ISR) for tRNA(Glu-2) and three (rrnA, D, and H) contain genes for tRNA(Ile-1) and tRNA(Ala-1B). To increase our understanding of their molecular evolution, we have determined the ISR sequence of the seven operons in a set of 12 strains from the ECOR collection. Each operon was specifically amplified using polymerase chain reaction primers designed from genes or open reading frames located upstream of the 16S rRNA genes in E. coli K12. With a single exception (ECOR 40), ISRs containing one or two tRNA genes were found at the same respective loci as those of strain K12. Intercistronic heterogeneity already found in K12 was representative of most variation among the strains studied and the location of polymorphic sites was the same. Dispersed nucleotide substitutions were very few but 21 variable sites were found grouped in a stem-loop, although the secondary structure was conserved. Some regions were found in which a stretch of nucleotides was substituted in block by one alternative, apparently unrelated, sequence (as illustrated by the known putative insertion of rsl in K12). Except for substitutions of different sizes and insertions/deletions found in the ISR, the pattern of nucleotide variation is very similar to that found for the 16S rRNA gene in E. coli. Strains K12 and ECOR 40 showed the highest intercistronic heterogeneity. Most strains showed a strong tendency to homogenization. Concerted evolution could explain the notorious conservation of this region that is supposed to have low functional restrictions.

  9. Designation of Streptomycete 16S and 23S rRNA-based target regions for oligonucleotide probes.

    PubMed

    Stackebrandt, E; Witt, D; Kemmerling, C; Kroppenstedt, R; Liesack, W

    1991-05-01

    The 16S and 23S rRNA of various Streptomyces species were partially sequenced and screened for the presence of stretches that could define all members of the genus, groups of species, or individual species. Nucleotide 929 (Streptomyces ambofaciens nomenclature [J.L. Pernodet, M.T. Alegre, F. Boccard, and M. Guerineau, Gene 79:33-46, 1989]) is a nucleotide highly unique to Streptomyces species which, in combination with flanking regions, allowed the designation of a genus-specific probe. Regions 158 through 203 of the 16S rRNA and 1518 through 1645 of the 23S rRNA (helix 54 [Pernodet et al., Gene 79:33-46, 1989]) have a high potential to define species, whereas the degree of variation in regions 982 through 998 and 1102 through 1122 of the 16S rRNA is less pronounced but characteristic for at least certain species. Alone or in combination with each other, these regions may serve as target sites for synthetic oligonucleotide probes and primers to be used in the determination of pure cultures and in the characterization of community structures. The specificity of several probes is demonstrated by dot blot hybridization.

  10. Ribosome-messenger recognition: mRNA target sites for ribosomal protein S1.

    PubMed Central

    Boni, I V; Isaeva, D M; Musychenko, M L; Tzareva, N V

    1991-01-01

    Ribosomal protein S1 is known to play an important role in translational initiation, being directly involved in recognition and binding of mRNAs by 30S ribosomal particles. Using a specially developed procedure based on efficient crosslinking of S1 to mRNA induced by UV irradiation, we have identified S1 binding sites on several phage RNAs in preinitiation complexes. Targets for S1 on Q beta and fr RNAs are localized upstream from the coat protein gene and contain oligo(U)-sequences. In the case of Q beta RNA, this S1 binding site overlaps the S-site for Q beta replicase and the site for S1 binding within a binary complex. It is reasonable that similar U-rich sequences represent S1 binding sites on bacterial mRNAs. To test this idea we have used E. coli ssb mRNA prepared in vitro with the T7 promoter/RNA polymerase system. By the methods of toeprinting, enzymatic footprinting, and UV crosslinking we have shown that binding of the ssb mRNA to 30S ribosomes is S1-dependent. The oligo(U)-sequence preceding the SD domain was found to be the target for S1. We propose that S1 binding sites, represented by pyrimidine-rich sequences upstream from the SD region, serve as determinants involved in recognition of mRNA by the ribosome. Images PMID:2011495

  11. Bayesian prediction of RNA translation from ribosome profiling.

    PubMed

    Malone, Brandon; Atanassov, Ilian; Aeschimann, Florian; Li, Xinping; Großhans, Helge; Dieterich, Christoph

    2017-01-26

    Ribosome profiling via high-throughput sequencing (ribo-seq) is a promising new technique for characterizing the occupancy of ribosomes on messenger RNA (mRNA) at base-pair resolution. The ribosome is responsible for translating mRNA into proteins, so information about its occupancy offers a detailed view of ribosome density and position which could be used to discover new translated open reading frames (ORFs), among other things. In this work, we propose Rp-Bp, an unsupervised Bayesian approach to predict translated ORFs from ribosome profiles. We use state-of-the-art Markov chain Monte Carlo techniques to estimate posterior distributions of the likelihood of translation of each ORF. Hence, an important feature of Rp-Bp is its ability to incorporate and propagate uncertainty in the prediction process. A second novel contribution is automatic Bayesian selection of read lengths and ribosome P-site offsets (BPPS). We empirically demonstrate that our read length selection technique modestly improves sensitivity by identifying more canonical and non-canonical ORFs. Proteomics- and quantitative translation initiation sequencing-based validation verifies the high quality of all of the predictions. Experimental comparison shows that Rp-Bp results in more peptide identifications and proteomics-validated ORF predictions compared to another recent tool for translation prediction.

  12. Protein-guided RNA dynamics during early ribosome assembly.

    PubMed

    Kim, Hajin; Abeysirigunawarden, Sanjaya C; Chen, Ke; Mayerle, Megan; Ragunathan, Kaushik; Luthey-Schulten, Zaida; Ha, Taekjip; Woodson, Sarah A

    2014-02-20

    The assembly of 30S ribosomes requires the precise addition of 20 proteins to the 16S ribosomal RNA. How early binding proteins change the ribosomal RNA structure so that later proteins may join the complex is poorly understood. Here we use single-molecule fluorescence resonance energy transfer (FRET) to observe real-time encounters between Escherichia coli ribosomal protein S4 and the 16S 5' domain RNA at an early stage of 30S assembly. Dynamic initial S4-RNA complexes pass through a stable non-native intermediate before converting to the native complex, showing that non-native structures can offer a low free-energy path to protein-RNA recognition. Three-colour FRET and molecular dynamics simulations reveal how S4 changes the frequency and direction of RNA helix motions, guiding a conformational switch that enforces the hierarchy of protein addition. These protein-guided dynamics offer an alternative explanation for induced fit in RNA-protein complexes.

  13. Protein-guided RNA dynamics during early ribosome assembly

    NASA Astrophysics Data System (ADS)

    Kim, Hajin; Abeysirigunawarden, Sanjaya C.; Chen, Ke; Mayerle, Megan; Ragunathan, Kaushik; Luthey-Schulten, Zaida; Ha, Taekjip; Woodson, Sarah A.

    2014-02-01

    The assembly of 30S ribosomes requires the precise addition of 20 proteins to the 16S ribosomal RNA. How early binding proteins change the ribosomal RNA structure so that later proteins may join the complex is poorly understood. Here we use single-molecule fluorescence resonance energy transfer (FRET) to observe real-time encounters between Escherichia coli ribosomal protein S4 and the 16S 5' domain RNA at an early stage of 30S assembly. Dynamic initial S4-RNA complexes pass through a stable non-native intermediate before converting to the native complex, showing that non-native structures can offer a low free-energy path to protein-RNA recognition. Three-colour FRET and molecular dynamics simulations reveal how S4 changes the frequency and direction of RNA helix motions, guiding a conformational switch that enforces the hierarchy of protein addition. These protein-guided dynamics offer an alternative explanation for induced fit in RNA-protein complexes.

  14. Sequence variation of the 16S to 23S rRNA spacer region in Salmonella enterica.

    PubMed

    Christensen, H; Møller, P L; Vogensen, F K; Olsen, J E

    2000-01-01

    The possibility for identification of Salmonella enterica serotypes by sequence analysis of the 16S to 23S rRNA internal transcribed spacer was investigated by direct sequencing of polymerase chain reaction-amplified DNA from all operons simultaneously in a collection of 25 strains of 18 different serotypes of S. enterica, and by sequencing individual cloned operons from a single strain. It was only possible to determine the first 117 bases upstream from the 23S rRNA gene by direct sequencing because of variation between the rrn operons. Comparison of sequences from this region allowed separation of only 15 out of the 18 serotypes investigated and was not specific even at the subspecies level of S. enterica. To determine the differences between internal transcribed spacers in more detail, the individual rrn operons of strain JEO 197, serotype IV 43:z4,z23:-, were cloned and sequenced. The strain contained four short internal transcribed spacer fragments of 382-384 bases in length, which were 98.4-99.7% similar to each other and three long fragments of 505 bases with 98.0-99.8% similarity. The study demonstrated a higher degree of interbacterial variation than intrabacterial variation between operons for serotypes of S. enterica.

  15. Identification to the species level of Lactobacillus isolated in probiotic prospecting studies of human, animal or food origin by 16S-23S rRNA restriction profiling

    PubMed Central

    Moreira, João Luiz S; Mota, Rodrigo M; Horta, Maria F; Teixeira, Santuza MR; Neumann, Elisabeth; Nicoli, Jacques R; Nunes, Álvaro C

    2005-01-01

    Background The accurate identification of Lactobacillus and other co-isolated bacteria during microbial ecological studies of ecosystems such as the human or animal intestinal tracts and food products is a hard task by phenotypic methods requiring additional tests such as protein and/or lipids profiling. Results Bacteria isolated in different probiotic prospecting studies, using de Man, Rogosa and Sharpe medium (MRS), were typed at species level by PCR amplification of 16S-23S rRNA intergenic spacers using universal primers that anneal within 16S and 23S genes, followed by restriction digestion analyses of PCR products. The set of enzymes chosen differentiates most species of Lactobacillus genus and also co-isolated bacteria such as Enterococcus, Streptococcus, Weissella, Staphylococcus, and Escherichia species. The in silico predictions of restriction patterns generated by the Lactobacillus shorter spacers digested with 11 restriction enzymes with 6 bp specificities allowed us to distinguish almost all isolates at the species level but not at the subspecies one. Simultaneous theoretical digestions of the three spacers (long, medium and short) with the same set of enzymes provided more complex patterns and allowed us to distinguish the species without purifying and cloning of PCR products. Conclusion Lactobacillus isolates and several other strains of bacteria co-isolated on MRS medium from gastrointestinal ecosystem and fermented food products could be identified using DNA fingerprints generated by restriction endonucleases. The methodology based on amplified ribosomal DNA restriction analysis (ARDRA) is easier, faster and more accurate than the current methodologies based on fermentation profiles, used in most laboratories for the purpose of identification of these bacteria in different prospecting studies. PMID:15788104

  16. Evaluation of ribosomal RNA removal protocols for Salmonella RNA-Seq projects

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Next generation sequencing is a powerful technology and its application to sequencing entire RNA populations of food-borne pathogens will provide valuable insights. A problem unique to prokaryotic RNA-Seq is the massive abundance of ribosomal RNA. Unlike eukaryotic messenger RNA (mRNA), bacterial ...

  17. A minimal ribosomal RNA: sequence and secondary structure of the 9S kinetoplast ribosomal RNA from Leishmania tarentolae.

    PubMed Central

    de la Cruz, V F; Lake, J A; Simpson, A M; Simpson, L

    1985-01-01

    The portion of the Leishmania tarentolae kinetoplast maxicircle DNA encoding the 9S RNA gene was sequenced, and the 5' and 3' ends of the transcript were determined. A secondary structure for the 9S RNA was determined based on the Escherichia coli 16S model. The 610-nucleotide 9S RNA exhibits a minimal secondary structure in which all four domains of the E. coli 16S structure are preserved. Within domains, however, some stems and loops have been greatly reduced or eliminated entirely. It is presumed that these reduced domains represent the minimal essential small ribosomal RNA secondary structures necessary for a functional ribosome. Alignment of the L. tarentolae 9S rRNA sequence with the published Trypanosoma brucei 9S rRNA sequence shows a nucleotide similarity of 84% and a transversion/transition ratio of 1.66. Images PMID:3856267

  18. Ribosomal RNA: a key to phylogeny

    NASA Technical Reports Server (NTRS)

    Olsen, G. J.; Woese, C. R.

    1993-01-01

    As molecular phylogeny increasingly shapes our understanding of organismal relationships, no molecule has been applied to more questions than have ribosomal RNAs. We review this role of the rRNAs and some of the insights that have been gained from them. We also offer some of the practical considerations in extracting the phylogenetic information from the sequences. Finally, we stress the importance of comparing results from multiple molecules, both as a method for testing the overall reliability of the organismal phylogeny and as a method for more broadly exploring the history of the genome.

  19. Spontaneous Erythromycin Resistance Mutation in a 23S rRNA Gene, rrlA, of the Extreme Thermophile Thermus thermophilus IB-21

    PubMed Central

    Gregory, Steven T.; Cate, Jamie H. D.; Dahlberg, Albert E.

    2001-01-01

    Spontaneous, erythromycin-resistant mutants of Thermus thermophilus IB-21 were isolated and found to carry the mutation A2058G in one of two 23S rRNA operons. The heterozygosity of these mutants indicates that A2058G confers a dominant or codominant phenotype in this organism. This mutation provides a valuable tool for the genetic manipulation of the 23S rRNA genes of Thermus. PMID:11418580

  20. Insights into the structure, function and evolution of the radical-SAM 23S rRNA methyltransferase Cfr that confers antibiotic resistance in bacteria

    PubMed Central

    Kaminska, Katarzyna H.; Purta, Elzbieta; Hansen, Lykke H.; Bujnicki, Janusz M.; Vester, Birte; Long, Katherine S.

    2010-01-01

    The Cfr methyltransferase confers combined resistance to five classes of antibiotics that bind to the peptidyl tranferase center of bacterial ribosomes by catalyzing methylation of the C-8 position of 23S rRNA nucleotide A2503. The same nucleotide is targeted by the housekeeping methyltransferase RlmN that methylates the C-2 position. Database searches with the Cfr sequence have revealed a large group of closely related sequences from all domains of life that contain the conserved CX3CX2C motif characteristic of radical S-adenosyl-l-methionine (SAM) enzymes. Phylogenetic analysis of the Cfr/RlmN family suggests that the RlmN subfamily is likely the ancestral form, whereas the Cfr subfamily arose via duplication and horizontal gene transfer. A structural model of Cfr has been calculated and used as a guide for alanine mutagenesis studies that corroborate the model-based predictions of a 4Fe–4S cluster, a SAM molecule coordinated to the iron–sulfur cluster (SAM1) and a SAM molecule that is the putative methyl group donor (SAM2). All mutations at predicted functional sites affect Cfr activity significantly as assayed by antibiotic susceptibility testing and primer extension analysis. The investigation has identified essential amino acids and Cfr variants with altered reaction mechanisms and represents a first step towards understanding the structural basis of Cfr activity. PMID:20007606

  1. Resistance to the macrolide antibiotic tylosin is conferred by single methylations at 23S rRNA nucleotides G748 and A2058 acting in synergy

    PubMed Central

    Liu, Mingfu; Douthwaite, Stephen

    2002-01-01

    The macrolide antibiotic tylosin has been used extensively in veterinary medicine and exerts potent antimicrobial activity against Gram-positive bacteria. Tylosin-synthesizing strains of the Gram-positive bacterium Streptomyces fradiae protect themselves from their own product by differential expression of four resistance determinants, tlrA, tlrB, tlrC, and tlrD. The tlrB and tlrD genes encode methyltransferases that add single methyl groups at 23S rRNA nucleotides G748 and A2058, respectively. Here we show that methylation by neither TlrB nor TlrD is sufficient on its own to give tylosin resistance, and resistance is conferred by the G748 and A2058 methylations acting together in synergy. This synergistic mechanism of resistance is specific for the macrolides tylosin and mycinamycin that possess sugars extending from the 5- and 14-positions of the macrolactone ring and is not observed for macrolides, such as carbomycin, spiramycin, and erythromycin, that have different constellations of sugars. The manner in which the G748 and A2058 methylations coincide with the glycosylation patterns of tylosin and mycinamycin reflects unambiguously how these macrolides fit into their binding site within the bacterial 50S ribosomal subunit. PMID:12417742

  2. Resistance to the macrolide antibiotic tylosin is conferred by single methylations at 23S rRNA nucleotides G748 and A2058 acting in synergy.

    PubMed

    Liu, Mingfu; Douthwaite, Stephen

    2002-11-12

    The macrolide antibiotic tylosin has been used extensively in veterinary medicine and exerts potent antimicrobial activity against Gram-positive bacteria. Tylosin-synthesizing strains of the Gram-positive bacterium Streptomyces fradiae protect themselves from their own product by differential expression of four resistance determinants, tlrA, tlrB, tlrC, and tlrD. The tlrB and tlrD genes encode methyltransferases that add single methyl groups at 23S rRNA nucleotides G748 and A2058, respectively. Here we show that methylation by neither TlrB nor TlrD is sufficient on its own to give tylosin resistance, and resistance is conferred by the G748 and A2058 methylations acting together in synergy. This synergistic mechanism of resistance is specific for the macrolides tylosin and mycinamycin that possess sugars extending from the 5- and 14-positions of the macrolactone ring and is not observed for macrolides, such as carbomycin, spiramycin, and erythromycin, that have different constellations of sugars. The manner in which the G748 and A2058 methylations coincide with the glycosylation patterns of tylosin and mycinamycin reflects unambiguously how these macrolides fit into their binding site within the bacterial 50S ribosomal subunit.

  3. A new RNA-RNA crosslinking reagent and its application to ribosomal 5S RNA.

    PubMed Central

    Wagner, R; Garrett, R A

    1978-01-01

    The synthesis of a new RNA specific bifunctional crosslinking reagent, 1.4-phenyl-diglyoxal, is described which reacts exclusively with guanosines. The properties of the crosslinked products enabled us to develop a straightforward method for identifying the reacted nucleotides. Results obtained with ribosomal 5S RNA of Escherichia coli demonstrate the formation of an intramolecular crosslink between guanosine-2 and guanosine-112 in the stem region. Images PMID:724507

  4. Protein-guided RNA dynamics during early ribosome assembly

    PubMed Central

    Kim, Hajin; Abeysirigunawardena, Sanjaya C.; Chen, Ke; Mayerle, Megan; Ragunathan, Kaushik; Luthey-Schulten, Zaida; Ha, Taekjip; Woodson, Sarah A.

    2014-01-01

    The assembly of 30S ribosomes requires the precise addition of 20 proteins to the 16S ribosomal RNA. How early binding proteins change the rRNA structure so that later proteins may join the complex is poorly understood. Here we use single molecule fluorescence resonance energy transfer (smFRET) to observe real-time encounters between ribosomal protein S4 and the 16S 5′ domain RNA at an early stage of 30S assembly. Dynamic initial S4-RNA complexes pass through a stable non-native intermediate before converting to the native complex, showing that non-native structures can offer a low free energy path to protein-RNA recognition. Three-color FRET and molecular dynamics (MD) simulations reveal how S4 changes the frequency and direction of RNA helix motions, guiding a conformational switch that enforces the hierarchy of protein addition. This protein-guided dynamics offers an alternative explanation for induced fit in RNA-protein complexes. PMID:24522531

  5. Positive modulation of RNA polymerase III transcription by ribosomal proteins

    SciTech Connect

    Dieci, Giorgio; Carpentieri, Andrea; Amoresano, Angela; Ottonello, Simone

    2009-02-06

    A yeast nuclear fraction of unknown composition, named TFIIIE, was reported previously to enhance transcription of tRNA and 5S rRNA genes in vitro. We show that TFIIIE activity co-purifies with a specific subset of ribosomal proteins (RPs) which, as revealed by chromatin immunoprecipitation analysis, generally interact with tRNA and 5S rRNA genes, but not with a Pol II-specific promoter. Only Rpl6Ap and Rpl6Bp, among the tested RPs, were found associated to a TATA-containing tRNA{sup Ile}(TAT) gene. The RPL6A gene also emerged as a strong multicopy suppressor of a conditional mutation in the basal transcription factor TFIIIC, while RPL26A and RPL14A behaved as weak suppressors. The data delineate a novel extra-ribosomal role for one or a few RPs which, by influencing 5S rRNA and tRNA synthesis, could play a key role in the coordinate regulation of the different sub-pathways required for ribosome biogenesis and functionality.

  6. Differentiation of Acinetobacter baumannii biotypes by amplification of 16S-23S rRNA intergenic spacer sequences.

    PubMed

    Garcia, A; Montoya, R; Bello, H; Gonzalez, G; Dominguez, M; Zemelman, R

    1996-01-01

    Isolates of Acinetobacter baumannii (32 strains) from blood samples obtained from patients in five Chilean hospitals were identified and biotyped according to their phenotypic properties. They were also submitted to random amplified polymorphic DNA (RAPD) using eight randomly designed 10-mers and the core sequence of M13 phage (15-mers) as well as amplification of the spacer regions between 16S and 23S genes in the prokaryotic rRNA genetic loci. With some primers, RAPD discriminated between biotypes, whereas with others each isolate showed a particular profile. When amplification of spacer regions was performed, a clear correlation between patterns and biotypes was found. This last technique allowed correct biotyping of clinical isolates. Both genetic methods might be used for the identification of A. baumannii biotypes.

  7. Rapid differentiation of Francisella species and subspecies by fluorescent in situ hybridization targeting the 23S rRNA

    PubMed Central

    2010-01-01

    Background Francisella (F.) tularensis is the causative agent of tularemia. Due to its low infectious dose, ease of dissemination and high case fatality rate, F. tularensis was the subject in diverse biological weapons programs and is among the top six agents with high potential if misused in bioterrorism. Microbiological diagnosis is cumbersome and time-consuming. Methods for the direct detection of the pathogen (immunofluorescence, PCR) have been developed but are restricted to reference laboratories. Results The complete 23S rRNA genes of representative strains of F. philomiragia and all subspecies of F. tularensis were sequenced. Single nucleotide polymorphisms on species and subspecies level were confirmed by partial amplification and sequencing of 24 additional strains. Fluorescent In Situ Hybridization (FISH) assays were established using species- and subspecies-specific probes. Different FISH protocols allowed the positive identification of all 4 F. philomiragia strains, and more than 40 F. tularensis strains tested. By combination of different probes, it was possible to differentiate the F. tularensis subspecies holarctica, tularensis, mediasiatica and novicida. No cross reactivity with strains of 71 clinically relevant bacterial species was observed. FISH was also successfully applied to detect different F. tularensis strains in infected cells or tissue samples. In blood culture systems spiked with F. tularensis, bacterial cells of different subspecies could be separated within single samples. Conclusion We could show that FISH targeting the 23S rRNA gene is a rapid and versatile method for the identification and differentiation of F. tularensis isolates from both laboratory cultures and clinical samples. PMID:20205957

  8. Database on the structure of large ribosomal subunit RNA.

    PubMed Central

    De Rijk, P; Caers, A; Van de Peer, Y; De Wachter, R

    1998-01-01

    The rRNA WWW Server at URL http://rrna.uia.ac.be/ now provides a database of 496 large subunit ribosomal RNA sequences. All these sequences are aligned, incorporate secondary structure information, and can be obtained in a number of formats. Other information about the sequences, such as literature references, accession numbers and taxonomic information is also available and searchable. If necessary, the data on the server can also be obtained by anonymous ftp. PMID:9399830

  9. Cis-regulatory RNA elements that regulate specialized ribosome activity

    PubMed Central

    Xue, Shifeng; Barna, Maria

    2015-01-01

    Recent evidence has shown that the ribosome itself can play a highly regulatory role in the specialized translation of specific subpools of mRNAs, in particular at the level of ribosomal proteins (RP). However, the mechanism(s) by which this selection takes place has remained poorly understood. In our recent study, we discovered a combination of unique RNA elements in the 5′UTRs of mRNAs that allows for such control by the ribosome. These mRNAs contain a Translation Inhibitory Element (TIE) that inhibits general cap-dependent translation, and an Internal Ribosome Entry Site (IRES) that relies on a specific RP for activation. The unique combination of an inhibitor of general translation and an activator of specialized translation is key to ribosome-mediated control of gene expression. Here we discuss how these RNA regulatory elements provide a new level of control to protein expression and their implications for gene expression, organismal development and evolution. PMID:26327194

  10. Effect of ribosome shielding on mRNA stability

    NASA Astrophysics Data System (ADS)

    Deneke, Carlus; Lipowsky, Reinhard; Valleriani, Angelo

    2013-08-01

    Based on the experimental evidence that translating ribosomes stabilize the mRNAs, we introduce and study a theoretical model for the dynamic shielding of mRNA by ribosomes. We present an improved fitting of published decay assay data in E. coli and show that only one third of the decay patterns are exponential. Our new transcriptome-wide estimate of the average lifetimes and mRNA half-lives shows that these timescales are considerably shorter than previous estimates. We also explain why there is a negative correlation between mRNA length and average lifetime when the mRNAs are subdivided in classes sharing the same degradation parameters. As a by-product, our model indicates that co-transcriptional translation in E. coli may be less common than previously believed.

  11. Fasciola hepatica - where is 28S ribosomal RNA?

    PubMed

    Haçarız, Orçun; Sayers, Gearóid

    2013-10-01

    Advanced molecular biology techniques are currently used to develop new effective strategies against fasciolosis. Assessment of the quality of extracted total RNA is an important step prior to commencing many molecular biology methods such as transcriptomics. However, RNA quality assessment is complicated for some organisms, including Fasciola hepatica, by the absence of a 28S rRNA peak/band, when assessed with modern protocols. In this study, electrophoretic profiles of F. hepatica ribosomal RNAs were evaluated using microfluidics capillary based and conventional non-denaturing gel electrophoresis methods. An important modification to recommended protocols, the exclusion of heat-denaturation step, in the microfluidics capillary based electrophoresis is critical to visualise the expected 28S rRNA and obtain an RNA integrity number (RIN). The intensity of the 28S rRNA band is reduced by the effect of non-denaturing gel electrophoresis.

  12. Ribosomal 18S rRNA base pairs with mRNA during eukaryotic translation initiation

    PubMed Central

    Martin, Franck; Ménétret, Jean-François; Simonetti, Angelita; Myasnikov, Alexander G.; Vicens, Quentin; Prongidi-Fix, Lydia; Natchiar, S. Kundhavai; Klaholz, Bruno P.; Eriani, Gilbert

    2016-01-01

    Eukaryotic mRNAs often contain a Kozak sequence that helps tether the ribosome to the AUG start codon. The mRNA of histone H4 (h4) does not undergo classical ribosome scanning but has evolved a specific tethering mechanism. The cryo-EM structure of the rabbit ribosome complex with mouse h4 shows that the mRNA forms a folded, repressive structure at the mRNA entry site on the 40S subunit next to the tip of helix 16 of 18S ribosomal RNA (rRNA). Toe-printing and mutational assays reveal that an interaction exists between a purine-rich sequence in h4 mRNA and a complementary UUUC sequence of helix h16. Together the present data establish that the h4 mRNA harbours a sequence complementary to an 18S rRNA sequence which tethers the mRNA to the ribosome to promote proper start codon positioning, complementing the interactions of the 40S subunit with the Kozak sequence that flanks the AUG start codon. PMID:27554013

  13. Site-Specific Cleavage of Ribosomal RNA in Escherichia coli-Based Cell-Free Protein Synthesis Systems

    PubMed Central

    Failmezger, Jurek; Nitschel, Robert; Sánchez-Kopper, Andrés; Kraml, Michael; Siemann-Herzberg, Martin

    2016-01-01

    Cell-free protein synthesis, which mimics the biological protein production system, allows rapid expression of proteins without the need to maintain a viable cell. Nevertheless, cell-free protein expression relies on active in vivo translation machinery including ribosomes and translation factors. Here, we examined the integrity of the protein synthesis machinery, namely the functionality of ribosomes, during (i) the cell-free extract preparation and (ii) the performance of in vitro protein synthesis by analyzing crucial components involved in translation. Monitoring the 16S rRNA, 23S rRNA, elongation factors and ribosomal protein S1, we show that processing of a cell-free extract results in no substantial alteration of the translation machinery. Moreover, we reveal that the 16S rRNA is specifically cleaved at helix 44 during in vitro translation reactions, resulting in the removal of the anti-Shine-Dalgarno sequence. These defective ribosomes accumulate in the cell-free system. We demonstrate that the specific cleavage of the 16S rRNA is triggered by the decreased concentrations of Mg2+. In addition, we provide evidence that helix 44 of the 30S ribosomal subunit serves as a point-of-entry for ribosome degradation in Escherichia coli. Our results suggest that Mg2+ homeostasis is fundamental to preserving functional ribosomes in cell-free protein synthesis systems, which is of major importance for cell-free protein synthesis at preparative scale, in order to create highly efficient technical in vitro systems. PMID:27992588

  14. Antibiotic resistance evolved via inactivation of a ribosomal RNA methylating enzyme

    PubMed Central

    Stojković, Vanja; Noda-Garcia, Lianet; Tawfik, Dan S.; Fujimori, Danica Galonić

    2016-01-01

    Modifications of the bacterial ribosome regulate the function of the ribosome and modulate its susceptibility to antibiotics. By modifying a highly conserved adenosine A2503 in 23S rRNA, methylating enzyme Cfr confers resistance to a range of ribosome-targeting antibiotics. The same adenosine is also methylated by RlmN, an enzyme widely distributed among bacteria. While RlmN modifies C2, Cfr modifies the C8 position of A2503. Shared nucleotide substrate and phylogenetic relationship between RlmN and Cfr prompted us to investigate evolutionary origin of antibiotic resistance in this enzyme family. Using directed evolution of RlmN under antibiotic selection, we obtained RlmN variants that mediate low-level resistance. Surprisingly, these variants confer resistance not through the Cfr-like C8 methylation, but via inhibition of the endogenous RlmN C2 methylation of A2503. Detection of RlmN inactivating mutations in clinical resistance isolates suggests that the mechanism used by the in vitro evolved variants is also relevant in a clinical setting. Additionally, as indicated by a phylogenetic analysis, it appears that Cfr did not diverge from the RlmN family but from another distinct family of predicted radical SAM methylating enzymes whose function remains unknown. PMID:27496281

  15. Deciphering Poxvirus Gene Expression by RNA Sequencing and Ribosome Profiling

    PubMed Central

    Cao, Shuai; Martens, Craig A.; Porcella, Stephen F.; Xie, Zhi; Ma, Ming; Shen, Ben

    2015-01-01

    ABSTRACT The more than 200 closely spaced annotated open reading frames, extensive transcriptional read-through, and numerous unpredicted RNA start sites have made the analysis of vaccinia virus gene expression challenging. Genome-wide ribosome profiling provided an unprecedented assessment of poxvirus gene expression. By 4 h after infection, approximately 80% of the ribosome-associated mRNA was viral. Ribosome-associated mRNAs were detected for most annotated early genes at 2 h and for most intermediate and late genes at 4 and 8 h. Cluster analysis identified a subset of early mRNAs that continued to be translated at the later times. At 2 h, there was excellent correlation between the abundance of individual mRNAs and the numbers of associated ribosomes, indicating that expression was primarily transcriptionally regulated. However, extensive transcriptional read-through invalidated similar correlations at later times. The mRNAs with the highest density of ribosomes had host response, DNA replication, and transcription roles at early times and were virion components at late times. Translation inhibitors were used to map initiation sites at single-nucleotide resolution at the start of most annotated open reading frames although in some cases a downstream methionine was used instead. Additional putative translational initiation sites with AUG or alternative codons occurred mostly within open reading frames, and fewer occurred in untranslated leader sequences, antisense strands, and intergenic regions. However, most open reading frames associated with these additional translation initiation sites were short, raising questions regarding their biological roles. The data were used to construct a high-resolution genome-wide map of the vaccinia virus translatome. IMPORTANCE This report contains the first genome-wide, high-resolution analysis of poxvirus gene expression at both transcriptional and translational levels. The study was made possible by recent methodological

  16. c-Myc co-ordinates mRNA cap methylation and ribosomal RNA production

    PubMed Central

    Dunn, Sianadh; Lombardi, Olivia; Cowling, Victoria H.

    2017-01-01

    The mRNA cap is a structure added to RNA pol II transcripts in eukaryotes, which recruits factors involved in RNA processing, nuclear export and translation initiation. RNA guanine-7 methyltransferase (RNMT)–RNA-activating miniprotein (RAM), the mRNA cap methyltransferase complex, completes the basic functional mRNA cap structure, cap 0, by methylating the cap guanosine. Here, we report that RNMT–RAM co-ordinates mRNA processing with ribosome production. Suppression of RNMT–RAM reduces synthesis of the 45S ribosomal RNA (rRNA) precursor. RNMT–RAM is required for c-Myc expression, a major regulator of RNA pol I, which synthesises 45S rRNA. Constitutive expression of c-Myc restores rRNA synthesis when RNMT–RAM is suppressed, indicating that RNMT–RAM controls rRNA production predominantly by controlling c-Myc expression. We report that RNMT–RAM is recruited to the ribosomal DNA locus, which may contribute to rRNA synthesis in certain contexts. PMID:27934633

  17. c-Myc co-ordinates mRNA cap methylation and ribosomal RNA production.

    PubMed

    Dunn, Sianadh; Lombardi, Olivia; Cowling, Victoria H

    2017-02-01

    The mRNA cap is a structure added to RNA pol II transcripts in eukaryotes, which recruits factors involved in RNA processing, nuclear export and translation initiation. RNA guanine-7 methyltransferase (RNMT)-RNA-activating miniprotein (RAM), the mRNA cap methyltransferase complex, completes the basic functional mRNA cap structure, cap 0, by methylating the cap guanosine. Here, we report that RNMT-RAM co-ordinates mRNA processing with ribosome production. Suppression of RNMT-RAM reduces synthesis of the 45S ribosomal RNA (rRNA) precursor. RNMT-RAM is required for c-Myc expression, a major regulator of RNA pol I, which synthesises 45S rRNA. Constitutive expression of c-Myc restores rRNA synthesis when RNMT-RAM is suppressed, indicating that RNMT-RAM controls rRNA production predominantly by controlling c-Myc expression. We report that RNMT-RAM is recruited to the ribosomal DNA locus, which may contribute to rRNA synthesis in certain contexts.

  18. Database on the structure of large ribosomal subunit RNA.

    PubMed Central

    De Rijk, P; Van de Peer, Y; De Wachter, R

    1996-01-01

    Our database on large ribosomal subunit RNA contained 334 sequences in July, 1995. All sequences in the database are aligned, taking into account secondary structure. The aligned sequences are provided, together with incorporated secondary structure information, in several computer-readable formats. These data can easily be obtained through the World Wide Web. The files in the database are also available via anonymous ftp. PMID:8594610

  19. Database on the structure of small ribosomal subunit RNA.

    PubMed Central

    Van de Peer, Y; Caers, A; De Rijk, P; De Wachter, R

    1998-01-01

    About 8600 complete or nearly complete sequences are now available from the Antwerp database on small ribosomal subunit RNA. All these sequences are aligned with one another on the basis of the adopted secondary structure model, which is corroborated by the observation of compensating substitutions in the alignment. Literature references, accession numbers and detailed taxonomic information are also compiled. The database can be consulted via the World Wide Web at URL http://rrna.uia.ac.be/ssu/ PMID:9399829

  20. Use of 16S rRNA, 23S rRNA, and gyrB Gene Sequence Analysis To Determine Phylogenetic Relationships of Bacillus cereus Group Microorganisms

    PubMed Central

    Bavykin, Sergei G.; Lysov, Yuri P.; Zakhariev, Vladimir; Kelly, John J.; Jackman, Joany; Stahl, David A.; Cherni, Alexey

    2004-01-01

    In order to determine if variations in rRNA sequence could be used for discrimination of the members of the Bacillus cereus group, we analyzed 183 16S rRNA and 74 23S rRNA sequences for all species in the B. cereus group. We also analyzed 30 gyrB sequences for B. cereus group strains with published 16S rRNA sequences. Our findings indicated that the three most common species of the B. cereus group, B. cereus, Bacillus thuringiensis, and Bacillus mycoides, were each heterogeneous in all three gene sequences, while all analyzed strains of Bacillus anthracis were found to be homogeneous. Based on analysis of 16S and 23S rRNA sequence variations, the microorganisms within the B. cereus group were divided into seven subgroups, Anthracis, Cereus A and B, Thuringiensis A and B, and Mycoides A and B, and these seven subgroups were further organized into two distinct clusters. This classification of the B. cereus group conflicts with current taxonomic groupings, which are based on phenotypic traits. The presence of B. cereus strains in six of the seven subgroups and the presence of B. thuringiensis strains in three of the subgroups do not support the proposed unification of B. cereus and B. thuringiensis into one species. Analysis of the available phenotypic data for the strains included in this study revealed phenotypic traits that may be characteristic of several of the subgroups. Finally, our results demonstrated that rRNA and gyrB sequences may be used for discriminating B. anthracis from other microorganisms in the B. cereus group. PMID:15297521

  1. Use of 16S rRNA, 23S rRNA, and gyrB gene sequence analysis to determine phylogenetic relationships of Bacillus cereus group.

    SciTech Connect

    Bayvkin, S. G.; Lysov, Y. P.; Zakhariev, V.; Kelly, J. J.; Jackman, J.; Stahl, D. A.; Cherni, A.; Engelhardt Inst. of Molecular Biology; Loyola Univ.; Johns Hopkins Univ.; Univ. of Washington

    2004-08-01

    In order to determine if variations in rRNA sequence could be used for discrimination of the members of the Bacillus cereus group, we analyzed 183 16S rRNA and 74 23S rRNA sequences for all species in the B. cereus group. We also analyzed 30 gyrB sequences for B. cereus group strains with published 16S rRNA sequences. Our findings indicated that the three most common species of the B. cereus group, B. cereus, Bacillus thuringiensis, and Bacillus mycoides, were each heterogeneous in all three gene sequences, while all analyzed strains of Bacillus anthracis were found to be homogeneous. Based on analysis of 16S and 23S rRNA sequence variations, the microorganisms within the B. cereus group were divided into seven subgroups, Anthracis, Cereus A and B, Thuringiensis A and B, and Mycoides A and B, and these seven subgroups were further organized into two distinct clusters. This classification of the B. cereus group conflicts with current taxonomic groupings, which are based on phenotypic traits. The presence of B. cereus strains in six of the seven subgroups and the presence of B. thuringiensis strains in three of the subgroups do not support the proposed unification of B. cereus and B. thuringiensis into one species. Analysis of the available phenotypic data for the strains included in this study revealed phenotypic traits that may be characteristic of several of the subgroups. Finally, our results demonstrated that rRNA and gyrB sequences may be used for discriminating B. anthracis from other microorganisms in the B. cereus group.

  2. Transformation of Chloroplast Ribosomal RNA Genes in Chlamydomonas: Molecular and Genetic Characterization of Integration Events

    PubMed Central

    Newman, S. M.; Boynton, J. E.; Gillham, N. W.; Randolph-Anderson, B. L.; Johnson, A. M.; Harris, E. H.

    1990-01-01

    Transformation of chloroplast ribosomal RNA (rRNA) genes in Chlamydomonas has been achieved by the biolistic process using cloned chloroplast DNA fragments carrying mutations that confer antibiotic resistance. The sites of exchange employed during the integration of the donor DNA into the recipient genome have been localized using a combination of antibiotic resistance mutations in the 16S and 23S rRNA genes and restriction fragment length polymorphisms that flank these genes. Complete or nearly complete replacement of a region of the chloroplast genome in the recipient cell by the corresponding sequence from the donor plasmid was the most common integration event. Exchange events between the homologous donor and recipient sequences occurred preferentially near the vector:insert junctions. Insertion of the donor rRNA genes and flanking sequences into one inverted repeat of the recipient genome was followed by intramolecular copy correction so that both copies of the inverted repeat acquired identical sequences. Increased frequencies of rRNA gene transformants were achieved by reducing the copy number of the chloroplast genome in the recipient cells and by decreasing the heterology between donor and recipient DNA sequences flanking the selectable markers. In addition to producing bona fide chloroplast rRNA transformants, the biolistic process induced mutants resistant to low levels of streptomycin, typical of nuclear mutations in Chlamydomonas. PMID:1981764

  3. Length-dependent translation of messenger RNA by ribosomes

    NASA Astrophysics Data System (ADS)

    Valleriani, Angelo; Zhang, Gong; Nagar, Apoorva; Ignatova, Zoya; Lipowsky, Reinhard

    2011-04-01

    A simple measure for the efficiency of protein synthesis by ribosomes is provided by the steady state amount of protein per messenger RNA (mRNA), the so-called translational ratio, which is proportional to the translation rate. Taking the degradation of mRNA into account, we show theoretically that both the translation rate and the translational ratio decrease with increasing mRNA length, in agreement with available experimental data for the prokaryote Escherichia coli. We also show that, compared to prokaryotes, mRNA degradation in eukaryotes leads to a less rapid decrease of the translational ratio. This finding is consistent with the fact that, compared to prokaryotes, eukaryotes tend to have longer proteins.

  4. Clinical and Microbiological Aspects of Linezolid Resistance Mediated by the cfr Gene Encoding a 23S rRNA Methyltransferase▿

    PubMed Central

    Arias, Cesar A.; Vallejo, Martha; Reyes, Jinnethe; Panesso, Diana; Moreno, Jaime; Castañeda, Elizabeth; Villegas, Maria V.; Murray, Barbara E.; Quinn, John P.

    2008-01-01

    The cfr (chloramphenicol-florfenicol resistance) gene encodes a 23S rRNA methyltransferase that confers resistance to linezolid. Detection of linezolid resistance was evaluated in the first cfr-carrying human hospital isolate of linezolid and methicillin-resistant Staphylococcus aureus (designated MRSA CM-05) by dilution and diffusion methods (including Etest). The presence of cfr was investigated in isolates of staphylococci colonizing the patient's household contacts and clinical isolates recovered from patients in the same unit where MRSA CM-05 was isolated. Additionally, 68 chloramphenicol-resistant Colombian MRSA isolates recovered from hospitals between 2001 and 2004 were screened for the presence of the cfr gene. In addition to erm(B), the erm(A) gene was also detected in CM-05. The isolate belonged to sequence type 5 and carried staphylococcal chromosomal cassette mec type I. We were unable to detect the cfr gene in any of the human staphylococci screened (either clinical or colonizing isolates). Agar and broth dilution methods detected linezolid resistance in CM-05. However, the Etest and disk diffusion methods failed to detect resistance after 24 h of incubation. Oxazolidinone resistance mediated by the cfr gene is rare, and acquisition by a human isolate appears to be a recent event in Colombia. The detection of cfr-mediated linezolid resistance might be compromised by the use of the disk diffusion or Etest method. PMID:18174304

  5. PCR-based method for targeting 16S-23S rRNA intergenic spacer regions among Vibrio species

    PubMed Central

    2010-01-01

    Background The genus Vibrio is a diverse group of Gram-negative bacteria comprised of 74 species. Furthermore, the genus has and is expected to continue expanding with the addition of several new species annually. Consequently, it is of paramount importance to have a method which is able to reliably and efficiently differentiate the numerous Vibrio species. Results In this study, a novel and rapid polymerase chain reaction (PCR)-based intergenic spacer (IGS)-typing system for vibrios was developed that is based on the well-known IGS regions located between the 16S and 23S rRNA genes on the bacterial chromosome. The system was optimized to resolve heteroduplex formation as well as to take advantage of capillary gel electrophoresis technology such that reproducible analyses could be achieved in a rapid manner. System validation was achieved through testing of 69 archetypal Vibrio strains, representing 48 Vibrio species, from which an 'IGS-type' profile database was generated. These data, presented here in several cluster analyses, demonstrated successful differentiation of the 69 type strains showing that this PCR-based fingerprinting method easily discriminates bacterial strains at the species level among Vibrio. Furthermore, testing 36 strains each of V. parahaemolyticus and V. vulnificus, important food borne pathogens, isolated from a variety of geographical locations with the IGS-typing method demonstrated distinct IGS-typing patterns indicative of subspecies divergence in both populations making this technique equally useful for intraspecies differentiation, as well. Conclusion This rapid, reliable and efficient IGS-typing system, especially in combination with 16S rRNA gene sequencing, has the capacity to not only discern and identify vibrios at the species level but, in some cases, at the sub-species level, as well. This procedure is particularly well-suited for preliminary species identification and, lends itself nicely to epidemiological investigations

  6. Database on the structure of large ribosomal subunit RNA.

    PubMed Central

    De Rijk, P; Van de Peer, Y; De Wachter, R

    1997-01-01

    The latest release of the large ribosomal subunit RNA database contains 429 sequences. All these sequences are aligned, and incorporate secondary structure information. The rRNA WWW Server at URL http://rrna.uia.ac.be/ provides researchers with an easily accessible resource to obtain the data in this database in a number of computer-readable formats. A new query interface has been added to the server. If necessary, the data can also be obtained by anonymous ftp from the same site. PMID:9016517

  7. Antitermination of transcription from an Escherichia coli ribosomal RNA promoter.

    PubMed

    Holben, W E; Morgan, E A

    1984-11-01

    The Escherichia coli lac and ara promoters and rrnC ribosomal RNA promoter-leader region were fused to lacZYA. Transcription termination signals were introduced into the lac genes of these fusions by Tn9 and IS1 insertions. Measurement of lac enzymes from upstream and downstream of the insertions showed that termination signals resulting from these insertions are very efficient when transcription begins at lac or ara promoters but are very inefficient when transcription begins at the rrnC promoter-leader region. The rrnC promoter-leader region must, therefore, modify RNA polymerase to enable it to read through transcription termination signals.

  8. Depletion of Ribosomal RNA Sequences from Single-Cell RNA-Sequencing Library.

    PubMed

    Fang, Nan; Akinci-Tolun, Rumeysa

    2016-07-01

    Recent advances in single-cell RNA sequencing technologies have revealed high heterogeneity of gene expression profiles in individual cells. However, most current single-cell RNA-seq methods use oligo-dT priming in the reverse transcription steps and detect only polyA-positive for more accuracy, since there are also polyA-positive non-coding RNAs transcripts, not other important RNA species, such as polyA-negative noncoding RNA. Reverse transcription using random oligos enables detection of not only the noncoding RNA species without polyA tails, but also ribosomal RNA (rRNA). rRNA comprises more than 90% of the total RNA and should be depleted from the RNA-seq library to ensure efficient usage of the sequencing capacity. Commonly used hybridization-based rRNA depletion methods can preserve noncoding RNA in the standard RNA-seq library. However, such rRNA depletion methods require high input amounts of total RNA and do not work at the single-cell level or with limited input DNA. This unit describes a novel procedure for RNA-seq library construction from single cells or a minimal amount of RNA. A thermostable duplex-specific nuclease is used in this method to effectively remove ribosomal RNA sequences following whole-transcriptome amplification and sequencing library construction. © 2016 by John Wiley & Sons, Inc.

  9. Mechanisms for ribotoxin-induced ribosomal RNA cleavage

    SciTech Connect

    He, Kaiyu; Zhou, Hui-Ren; Pestka, James J.

    2012-11-15

    The Type B trichothecene deoxynivalenol (DON), a ribotoxic mycotoxin known to contaminate cereal-based foods, induces ribosomal RNA (rRNA) cleavage in the macrophage via p38-directed activation of caspases. Here we employed the RAW 264.7 murine macrophage model to test the hypothesis that this rRNA cleavage pathway is similarly induced by other ribotoxins. Capillary electrophoresis confirmed that the antibiotic anisomycin (≥ 25 ng/ml), the macrocylic trichothecene satratoxin G (SG) (≥ 10 ng/ml) and ribosome-inactivating protein ricin (≥ 300 ng/ml) induced 18s and 28s rRNA fragmentation patterns identical to that observed for DON. Also, as found for DON, inhibition of p38, double-stranded RNA-activated kinase (PKR) and hematopoietic cell kinase (Hck) suppressed MAPK anisomycin-induced rRNA cleavage, while, in contrast, their inhibition did not affect SG- and ricin-induced rRNA fragmentation. The p53 inhibitor pifithrin-μ and pan caspase inhibitor Z-VAD-FMK suppressed rRNA cleavage induced by anisomycin, SG and ricin, indicating that these ribotoxins shared with DON a conserved downstream pathway. Activation of caspases 8, 9 and 3 concurrently with apoptosis further suggested that rRNA cleavage occurred in parallel with both extrinsic and intrinsic pathways of programmed cell death. When specific inhibitors of cathepsins L and B (lysosomal cysteine cathepsins active at cytosolic neutral pH) were tested, only the former impaired anisomycin-, SG-, ricin- and DON-induced rRNA cleavage. Taken together, the data suggest that (1) all four ribotoxins induced p53-dependent rRNA cleavage via activation of cathepsin L and caspase 3, and (2) activation of p53 by DON and anisomycin involved p38 whereas SG and ricin activated p53 by an alternative mechanism. Highlights: ► Deoxynivalenol (DON) anisomycin, satratoxin G (SG) and ricin are ribotoxins. ► Ribotoxins induce 18s and 28s rRNA cleavage in the RAW 264.7 macrophage model. ► Ribotoxins induce rRNA cleavage via

  10. The RNA-binding domain of ribosomal protein L11 recognizes an rRNA tertiary structure stabilized by both thiostrepton and magnesium ion

    PubMed Central

    Blyn, Lawrence B.; Risen, Lisa M.; Griffey, Richard H.; Draper, David E.

    2000-01-01

    Antibiotics that inhibit ribosomal function may do so by one of several mechanisms, including the induction of incorrect RNA folding or prevention of protein and/or RNA conformational transitions. Thiostrepton, which binds to the ‘GTPase center’ of the large subunit, has been postulated to prevent conformational changes in either the L11 protein or rRNA to which it binds. Scintillation proximity assays designed to look at the binding of the L11 C-terminal RNA-binding domain to a 23S ribosomal RNA (rRNA) fragment, as well as the ability of thiostrepton to induce that binding, were used to demonstrate the role of Mg2+, L11 and thiostrepton in the formation and maintenance of the rRNA fragment tertiary structure. Experiments using these assays with both an Escherichia coli rRNA fragment and a thermostable variant of that RNA show that Mg2+, L11 and thiostrepton all induce the RNA to fold to an essentially identical tertiary structure. PMID:10734197

  11. Crosslinking of Ribosomal Proteins to RNA in Maize Ribosomes by UV-B and Its Effects on Translation1[w

    PubMed Central

    Casati, Paula; Walbot, Virginia

    2004-01-01

    Ultraviolet-B (UV-B) photons can cause substantial cellular damage in biomolecules, as is well established for DNA. Because RNA has the same absorption spectrum for UV as DNA, we have investigated damage to this cellular constituent. In maize (Zea mays) leaves, UV-B radiation damages ribosomes by crosslinking cytosolic ribosomal proteins S14, L23a, and L32, and chloroplast ribosomal protein L29 to RNA. Ribosomal damage accumulated during a day of UV-B exposure correlated with a progressive decrease in new protein production; however, de novo synthesis of some ribosomal proteins is increased after 6 h of UV-B exposure. After 16 h without UV-B, damaged ribosomes were eliminated and translation was restored to normal levels. Ribosomal protein S6 and an S6 kinase are phosphorylated during UV-B exposure; these modifications are associated with selective translation of some ribosomal proteins after ribosome damage in mammalian fibroblast cells and may be an adaptation in maize. Neither photosynthesis nor pigment levels were affected significantly by UV-B, demonstrating that the treatment applied is not lethal and that maize leaf physiology readily recovers. PMID:15466230

  12. Transcriptional activation of ribosomal RNA genes during compensatory renal hypertrophy

    SciTech Connect

    Ouellette, A.J.; Moonka, R.; Zelenetz, A.; Malt, R.A.

    1986-05-01

    The overall rate of rDNA transcription increases by 50% during the first 24 hours of compensatory renal hypertrophy in the mouse. To study mechanisms of ribosome accumulation after uninephrectomy, transcription rates were measured in isolated kidneys by transcriptional runoff. /sup 32/P-labeled nascent transcripts were hybridized to blots containing linearized, denatured cloned rDNA, and hybridization was quantitated autoradiographically and by direct counting. Overall transcriptional activity of rDNA was increased by 30% above control levels at 6 hrs after nephrectomy and by 50% at 12, 18, and 24 hrs after operation. Hybridizing RNA was insensitive to inhibiby alpha-amanitin, and no hybridization was detected to vector DNA. Thus, accelerated rDNA transcription is one regulatory element in the accretion of ribosomes in renal growth, and the regulatory event is an early event. Mechanisms of activation may include enhanced transcription of active genes or induction of inactive DNA.

  13. Transfer RNA-mediated regulation of ribosome dynamics during protein synthesis.

    PubMed

    Fei, Jingyi; Richard, Arianne C; Bronson, Jonathan E; Gonzalez, Ruben L

    2011-08-21

    Translocation of tRNAs through the ribosome during protein synthesis involves large-scale structural rearrangement of the ribosome and ribosome-bound tRNAs that is accompanied by extensive and dynamic remodeling of tRNA-ribosome interactions. How the rearrangement of individual tRNA-ribosome interactions influences tRNA movement during translocation, however, remains largely unknown. To address this question, we used single-molecule FRET to characterize the dynamics of ribosomal pretranslocation (PRE) complex analogs carrying either wild-type or systematically mutagenized tRNAs. Our data reveal how specific tRNA-ribosome interactions regulate the rate of PRE complex rearrangement into a critical, on-pathway translocation intermediate and how these interactions control the stability of the resulting configuration. Notably, our results suggest that the conformational flexibility of the tRNA molecule has a crucial role in directing the structural dynamics of the PRE complex during translocation.

  14. RNA-protein distance patterns in ribosomes reveal the mechanism of translational attenuation.

    PubMed

    Yu, DongMei; Zhang, Chao; Qin, PeiWu; Cornish, Peter V; Xu, Dong

    2014-11-01

    Elucidating protein translational regulation is crucial for understanding cellular function and drug development. A key molecule in protein translation is ribosome, which is a super-molecular complex extensively studied for more than a half century. The structure and dynamics of ribosome complexes were resolved recently thanks to the development of X-ray crystallography, Cryo-EM, and single molecule biophysics. Current studies of the ribosome have shown multiple functional states, each with a unique conformation. In this study, we analyzed the RNA-protein distances of ribosome (2.5 MDa) complexes and compared these changes among different ribosome complexes. We found that the RNA-protein distance is significantly correlated with the ribosomal functional state. Thus, the analysis of RNA-protein binding distances at important functional sites can distinguish ribosomal functional states and help understand ribosome functions. In particular, the mechanism of translational attenuation by nascent peptides and antibiotics was revealed by the conformational changes of local functional sites.

  15. Translational feedback regulation of the gene for L35 in Escherichia coli requires binding of ribosomal protein L20 to two sites in its leader mRNA: a possible case of ribosomal RNA-messenger RNA molecular mimicry.

    PubMed Central

    Guillier, Maude; Allemand, Frédéric; Raibaud, Sophie; Dardel, Frédéric; Springer, Mathias; Chiaruttini, Claude

    2002-01-01

    In addition to being a component of the large ribosomal subunit, ribosomal protein L20 of Escherichia coli also acts as a translational repressor. L20 is synthesized from the IF3 operon that contains three cistrons coding for IF3, and ribosomal proteins L35 and L20. L20 directly represses the expression of the gene encoding L35 and the expression of its own gene by translational coupling. All of the cis-acting sequences required for repression by L20, called the operator, are found on an mRNA segment extending from the middle of the IF3 gene to the start of the L35 gene. L20-mediated repression requires a long-range base-pairing interaction between nucleotide residues within the IF3 gene and residues just upstream of the L35 gene. This interaction results in the formation of a pseudoknot. Here we show that L20 causes protection of nucleotide residues in two regions of the operator in vitro. The first region is the pseudoknot itself and the second lies in an irregular stem located upstream of the L35 gene. By primer extension analysis, we show that L20 specifically induces reverse transcriptase stops in both regions. Therefore, these two regions define two L20-binding sites in the operator. Using mutations and deletions of rpml'-'lacZ fusions, we show that both sites are essential for repression in vivo. However L20 can bind to each site independently in vitro. One site is similar to the L20-binding site on 23S rRNA. Here we propose that L20 recognizes its mRNA and its rRNA in similar way. PMID:12166643

  16. Database on the structure of small ribosomal subunit RNA.

    PubMed Central

    Van de Peer, Y; Nicolaï, S; De Rijk, P; De Wachter, R

    1996-01-01

    The Antwerp database on small ribosomal subunit RNA offers over 4300 nucleotide sequences (August 1995). All these sequences are stored in the form of an alignment based on the adopted secondary structure model, which in turn is corroborated by the observation of compensating substitutions in the alignment. Besides the primary and secondary structure information, literature references, accession numbers and detailed taxonomic information are also compiled. The complete database is made available to the scientific community through anonymous ftp and World Wide Web(WWW). PMID:8594609

  17. Database on the structure of small ribosomal subunit RNA.

    PubMed Central

    Van de Peer, Y; Van den Broeck, I; De Rijk, P; De Wachter, R

    1994-01-01

    The database on small ribosomal subunit RNA structure contains (June 1994) 2824 nucleotide sequences. All these sequences are stored in the form of an alignment based on the adopted secondary structure model, which in turn is corroborated by the observation of compensating substitutions in the alignment. The complete database is made available to the scientific community through anonymous ftp on our server in Antwerp. A special effort was made to improve electronic retrieval and a program is supplied that allows to create different file formats. The database can also be obtained from the EMBL nucleotide sequence library. PMID:7524022

  18. Database on the structure of small ribosomal subunit RNA.

    PubMed Central

    Van de Peer, Y; Jansen, J; De Rijk, P; De Wachter, R

    1997-01-01

    The Antwerp database on small ribosomal subunit RNA now offers more than 6000 nucleotide sequences (August 1996). All these sequences are stored in the form of an alignment based on the adopted secondary structure model, which is corroborated by the observation of compensating substitutions in the alignment. Besides the primary and secondary structure information, literature references, accession numbers and detailed taxonomic information are also compiled. For ease of use, the complete database is made available to the scientific community via World Wide Web at URL http://rrna.uia.ac.be/ssu/ . PMID:9016516

  19. Structures of the Bacterial Ribosome in Classical and Hybrid States of tRNA Binding

    SciTech Connect

    Dunkle, Jack A.; Wang, Leyi; Feldman, Michael B.; Pulk, Arto; Chen, Vincent B.; Kapral, Gary J.; Noeske, Jonas; Richardson, Jane S.; Blanchard, Scott C.; Cate, Jamie H. Doudna

    2011-09-06

    During protein synthesis, the ribosome controls the movement of tRNA and mRNA by means of large-scale structural rearrangements. We describe structures of the intact bacterial ribosome from Escherichia coli that reveal how the ribosome binds tRNA in two functionally distinct states, determined to a resolution of {approx}3.2 angstroms by means of x-ray crystallography. One state positions tRNA in the peptidyl-tRNA binding site. The second, a fully rotated state, is stabilized by ribosome recycling factor and binds tRNA in a highly bent conformation in a hybrid peptidyl/exit site. The structures help to explain how the ratchet-like motion of the two ribosomal subunits contributes to the mechanisms of translocation, termination, and ribosome recycling.

  20. Comparison of ribosomal RNA removal methods for transcriptome sequencing workflows in teleost fish

    Technology Transfer Automated Retrieval System (TEKTRAN)

    RNA sequencing (RNA-Seq) is becoming the standard for transcriptome analysis. Removal of contaminating ribosomal RNA (rRNA) is a priority in the preparation of libraries suitable for sequencing. rRNAs are commonly removed from total RNA via either mRNA selection or rRNA depletion. These methods have...

  1. Discrimination of Bacillus anthracis from closely related microorganisms by analysis of 16S and 23S rRNA with oligonucleotide microchips

    DOEpatents

    Bavykin, Sergei G.; Mirzabekova, legal representative, Natalia V.; Mirzabekov, deceased, Andrei D.

    2007-12-04

    The present invention relates to methods and compositions for using nucleotide sequence variations of 16S and 23S rRNA within the B. cereus group to discriminate a highly infectious bacterium B. anthracis from closely related microorganisms. Sequence variations in the 16S and 23S rRNA of the B. cereus subgroup including B. anthracis are utilized to construct an array that can detect these sequence variations through selective hybridizations and discriminate B. cereus group that includes B. anthracis. Discrimination of single base differences in rRNA was achieved with a microchip during analysis of B. cereus group isolates from both single and in mixed samples, as well as identification of polymorphic sites. Successful use of a microchip to determine the appropriate subgroup classification using eight reference microorganisms from the B. cereus group as a study set, was demonstrated.

  2. The sequence of Methanospirillum hungatei 23S rRNA confirms the specific relationship between the extreme halophiles and the Methanomicrobiales

    NASA Technical Reports Server (NTRS)

    Burggraf, S.; Ching, A.; Stetter, K. O.; Woese, C. R.

    1991-01-01

    We have determined the sequence of the 23S rRNA from the methanogenic archaeon Methanospirillum hungatei. This is the first such sequence from a member of the Methanomicrobiales. Moreover, it brings additional evidence to bear on the possible specific relationship between this particular group of methanogens and the extreme halophiles. Such evidence is critical in that several new (and relatively untested) methods of phylogenetic inference have lead to the controversial conclusion that the extreme halophiles are either not related to the archaea, or are only peripherally so. Analysis of the Methanospirillum hungatei 23S rRNA sequence shows the Methanomicrobiales are indeed a sister group of the extreme halophiles, further strengthening the conclusions reached from analysis of 16S rRNA sequences.

  3. UtpA and UtpB chaperone nascent pre-ribosomal RNA and U3 snoRNA to initiate eukaryotic ribosome assembly

    NASA Astrophysics Data System (ADS)

    Hunziker, Mirjam; Barandun, Jonas; Petfalski, Elisabeth; Tan, Dongyan; Delan-Forino, Clémentine; Molloy, Kelly R.; Kim, Kelly H.; Dunn-Davies, Hywel; Shi, Yi; Chaker-Margot, Malik; Chait, Brian T.; Walz, Thomas; Tollervey, David; Klinge, Sebastian

    2016-06-01

    Early eukaryotic ribosome biogenesis involves large multi-protein complexes, which co-transcriptionally associate with pre-ribosomal RNA to form the small subunit processome. The precise mechanisms by which two of the largest multi-protein complexes--UtpA and UtpB--interact with nascent pre-ribosomal RNA are poorly understood. Here, we combined biochemical and structural biology approaches with ensembles of RNA-protein cross-linking data to elucidate the essential functions of both complexes. We show that UtpA contains a large composite RNA-binding site and captures the 5' end of pre-ribosomal RNA. UtpB forms an extended structure that binds early pre-ribosomal intermediates in close proximity to architectural sites such as an RNA duplex formed by the 5' ETS and U3 snoRNA as well as the 3' boundary of the 18S rRNA. Both complexes therefore act as vital RNA chaperones to initiate eukaryotic ribosome assembly.

  4. Novel Diagnostic Algorithm for Identification of Mycobacteria Using Genus-Specific Amplification of the 16S-23S rRNA Gene Spacer and Restriction Endonucleases

    PubMed Central

    Roth, Andreas; Reischl, Udo; Streubel, Anna; Naumann, Ludmila; Kroppenstedt, Reiner M.; Habicht, Marion; Fischer, Marga; Mauch, Harald

    2000-01-01

    A novel genus-specific PCR for mycobacteria with simple identification to the species level by restriction fragment length polymorphism (RFLP) was established using the 16S-23S ribosomal RNA gene (rDNA) spacer as a target. Panspecificity of primers was demonstrated on the genus level by testing 811 bacterial strains (122 species in 37 genera from 286 reference strains and 525 clinical isolates). All mycobacterial isolates (678 strains among 48 defined species and 5 indeterminate taxons) were amplified by the new primers. Among nonmycobacterial isolates, only Gordonia terrae was amplified. The RFLP scheme devised involves estimation of variable PCR product sizes together with HaeIII and CfoI restriction analysis. It yielded 58 HaeIII patterns, of which 49 (84%) were unique on the species level. Hence, HaeIII digestion together with CfoI results was sufficient for correct identification of 39 of 54 mycobacterial taxons and one of three or four of seven RFLP genotypes found in Mycobacterium intracellulare and Mycobacterium kansasii, respectively. Following a clearly laid out diagnostic algorithm, the remaining unidentified organisms fell into five clusters of closely related species (i.e., the Mycobacterium avium complex or Mycobacterium chelonae-Mycobacterium abscessus) that were successfully separated using additional enzymes (TaqI, MspI, DdeI, or AvaII). Thus, next to slowly growing mycobacteria, all rapidly growing species studied, including M. abscessus, M. chelonae, Mycobacterium farcinogenes, Mycobacterium fortuitum, Mycobacterium peregrinum, and Mycobacterium senegalense (with a very high 16S rDNA sequence similarity) were correctly identified. A high intraspecies sequence stability and the good discriminative power of patterns indicate that this method is very suitable for rapid and cost-effective identification of a wide variety of mycobacterial species without the need for sequencing. Phylogenetically, spacer sequence data stand in good agreement with 16S r

  5. A conserved heptamer motif for ribosomal RNA transcription termination in animal mitochondria.

    PubMed Central

    Valverde, J R; Marco, R; Garesse, R

    1994-01-01

    A search of sequence data bases for a tridecamer transcription termination signal, previously described in human mtDNA as being responsible for the accumulation of mitochondrial ribosomal RNAs (rRNAs) in excess over the rest of mitochondrial genes, has revealed that this termination signal occurs in equivalent positions in a wide variety of organisms from protozoa to mammals. Due to the compact organization of the mtDNA, the tridecamer motif usually appears as part of the 3' adjacent gene sequence. Because in phylogenetically widely separated organisms the mitochondrial genome has experienced many rearrangements, it is interesting that its occurrence near the 3' end of the large rRNA is independent of the adjacent gene. The tridecamer sequence has diverged in phylogenetically widely separated organisms. Nevertheless, a well-conserved heptamer--TGGCAGA, the mitochondrial rRNA termination box--can be defined. Although extending the experimental evidence of its role as a transcription termination signal in humans will be of great interest, its evolutionary conservation strongly suggests that mitochondrial rRNA transcription termination could be a widely conserved mechanism in animals. Furthermore, the conservation of a homologous tridecamer motif in one of the last 3' secondary loops of nonmitochondrial 23S-like rRNAs suggests that the role of the sequence has changed during mitochondrial evolution. PMID:7515499

  6. Panel of 23S rRNA Gene-Based Real-Time PCR Assays for Improved Universal and Group-Specific Detection of Phytoplasmas▿ †

    PubMed Central

    Hodgetts, Jennifer; Boonham, Neil; Mumford, Rick; Dickinson, Matthew

    2009-01-01

    Primers and probes based on the 23S rRNA gene have been utilized to design a range of real-time PCR assays for routine phytoplasma diagnostics. These assays have been authenticated as phytoplasma specific and shown to be at least as sensitive as nested PCR. A universal assay to detect all phytoplasmas has been developed, along with a multiplex assay to discriminate 16SrI group phytoplasmas from members of all of the other 16Sr groups. Assays for the 16SrII, 16SrIV, and 16SrXII groups have also been developed to confirm that the 23S rRNA gene can be used to design group-specific assays. PMID:19270148

  7. In vitro transcription of a cloned mouse ribosomal RNA gene.

    PubMed Central

    Mishima, Y; Yamamoto, O; Kominami, R; Muramatsu, M

    1981-01-01

    An in vitro transcription system which utilizes cloned mouse ribosomal RNA gene (rDNA) fragments and a mouse cell extract has been developed. RNA polymerases I is apparently responsible for this transcription as evidenced by the complete resistance to a high concentration (200 micrograms/ml) of alpha-amanitin. Run-off products obtained with three different truncated rDNA fragments indicated that RNA was transcribed from a unique site of rDNA. The S1 nuclease protection mapping of the in vitro product and of in vivo 45S RNA confirmed this site, indicating that, in this in vitro system, transcription of rDNA started from the same site as in vivo. This site is located at several hundred nucleotides upstream from the putative initiation site reported by us (1) and by others (2). Some sequence homology surrounding this region was noted among mouse, Xenopus laevis and Drosophila melanogaster. The data also suggest that some processing of the primary transcript occurs in this in vitro system. Images PMID:6278446

  8. A model for the study of ligand binding to the ribosomal RNA helix h44

    SciTech Connect

    Dibrov, Sergey M.; Parsons, Jerod; Hermann, Thomas

    2010-09-02

    Oligonucleotide models of ribosomal RNA domains are powerful tools to study the binding and molecular recognition of antibiotics that interfere with bacterial translation. Techniques such as selective chemical modification, fluorescence labeling and mutations are cumbersome for the whole ribosome but readily applicable to model RNAs, which are readily crystallized and often give rise to higher resolution crystal structures suitable for detailed analysis of ligand-RNA interactions. Here, we have investigated the HX RNA construct which contains two adjacent ligand binding regions of helix h44 in 16S ribosomal RNA. High-resolution crystal structure analysis confirmed that the HX RNA is a faithful structural model of the ribosomal target. Solution studies showed that HX RNA carrying a fluorescent 2-aminopurine modification provides a model system that can be used to monitor ligand binding to both the ribosomal decoding site and, through an indirect effect, the hygromycin B interaction region.

  9. A putative precursor for the small ribosomal RNA from mitochondria of Saccharomyces cerevisiae.

    PubMed Central

    Osinga, K A; Evers, R F; Van der Laan, J C; Tabak, H F

    1981-01-01

    We have characterized a putative precursor RNA (15.5S) for the 15S ribosomal RNA in mitochondria of Saccharomyces cerevisiae. Hybrids were formed with mitochondrial RNA and mtDNA fragments terminally labelled at restriction sites located within the gene coding for 15S ribosomal RNA and treated with S1 nuclease (Berk, A.J. and Sharp, J.A. (1977) 12, 721-732). Sites of resistant hybrids were measured by agarose gel electrophoresis and end points of RNAs determined. The 15.5S RNA is approximately 80 nucleotides longer than the 15S ribosomal RNA, with the extra sequences being located at the 5'-end. Both 15S ribosomal RNA and 15.5S RNA are fully localised within a 2000 base pair HapII fragment. This putative precursor and the mature 15S ribosomal RNA are also found in petite mutants which retain the 15S ribosomal RNA gene. The petite mutant with the smallest genetic complexity has its end point of deletion (junction) just outside the HapII site located in the 5' flank of the 15S ribosomal RNA genes as determined by S1 nuclease analysis. This leaves a DNA stretch approximately 300 base pairs long where an initiation signal for mitochondrial transcription may be present. Images PMID:6262728

  10. Identification of dairy lactic acid bacteria by tRNAAla-23S rDNA-RFLP.

    PubMed

    Mancini, Andrea; Lazzi, Camilla; Bernini, Valentina; Neviani, Erasmo; Gatti, Monica

    2012-12-01

    The aim of this study was to evaluate the potential of target tRNA(Ala)-23S ribosomal DNA for identification of lactic acid bacteria strains associated with dairy ecosystem. For this purpose tRNA(Ala)-23S ribosomal DNA Restriction Fragment Length Polymorphism (tRNA(Ala)-23S rDNA-RFLP) was compared with two widely used DNA fingerprinting methods - P1 Random Amplified Polymorphic DNA (RAPD), (GTG)5 repetitive extragenic palindromic PCR (rep-PCR) - for their ability to identify different species on a set of 10 type and 34 reference strains. Moreover, 75 unknown isolates collected during different stages of Grana Padano cheese production and ripening were identified using tRNA(Ala)-23S rDNA-RFLP and compared to the RFLP profiles of the strains in the reference database. This study demonstrated that the target tRNA(Ala)-23S rDNA has high potential in bacterial identification and tRNA(Ala)-23S rDNA-RFLP is a promising method for reliable species-level identification of lactic acid bacteria (LAB) in dairy products.

  11. Inhibition by Elongation Factor EF G of Aminoacyl-tRNA Binding to Ribosomes

    PubMed Central

    Cabrer, Bartolomé; Vázquez, David; Modolell, Juan

    1972-01-01

    Elongation factor G (EF G), bound to ribosomes either with GMPPCP or with fusidic acid and GDP, inhibits elongation factor Tu (EF Tu)-dependent binding of Phe-tRNA on the ribosome-poly(U) complex and binding of Ala-tRNA on the initiation complex formed with RNA from bacteriophage R17; GTP hydrolysis associated with Phe-tRNA binding is also inhibited. Moreover, nonenzymic binding of Phe-tRNA at high Mg++ concentration is completely blocked by EF G. Thus, EF G appears to bind at a site that overlaps or interacts with the ribosomal A-site. PMID:4551985

  12. Inversions between ribosomal RNA genes of Escherichia coli.

    PubMed Central

    Hill, C W; Harnish, B W

    1981-01-01

    It might be anticipated that the presence of redundant but oppositely oriented sequences in a chromosome could allow inversion of the intervening material through homologous recombination. For example, the ribosomal RNA gene rrnD of Escherichia coli has the opposite orientation fro rrnB and rrnE and is separated from these genes by roughly 20% of the chromosome. Starting with a derivative of Cavalli Hfr, we have constructed mutants that have an inversion of the segment between rrnD and either rrnB or rrnE. These mutants are generally quite viable but do exhibit a slight reduction in growth rate relative to the parental strain. A major line of laboratory E. coli, W3110 and its derivatives, also has an inversion between rrnD and rrnE, probably created directly by a recombinational event between these highly homologous genes. Images PMID:6273909

  13. The Ribosomal RNA is a Useful Marker to Visualize Rhizobia Interacting with Legume Plants

    ERIC Educational Resources Information Center

    Rinaudi, Luciana; Isola, Maria C.; Giordano, Walter

    2004-01-01

    Symbiosis between rhizobia and leguminous plants leads to the formation of nitrogen-fixing root nodules. In the present article, we recommend the use of the ribosomal RNA (rRNA) isolated from legume nodules in an experimental class with the purpose of introducing students to the structure of eukaryotic and prokaryotic ribosomes and of…

  14. Superresolution imaging of ribosomes and RNA polymerase in live Escherichia coli cells.

    PubMed

    Bakshi, Somenath; Siryaporn, Albert; Goulian, Mark; Weisshaar, James C

    2012-07-01

    Quantitative spatial distributions of ribosomes (S2-YFP) and RNA polymerase (RNAP; β'-yGFP) in live Escherichia coli are measured by superresolution fluorescence microscopy. In moderate growth conditions, nucleoid-ribosome segregation is strong, and RNAP localizes to the nucleoid lobes. The mean copy numbers per cell are 4600 RNAPs and 55,000 ribosomes. Only 10-15% of the ribosomes lie within the densest part of the nucleoid lobes, and at most 4% of the RNAPs lie in the two ribosome-rich endcaps. The predominant observed diffusion coefficient of ribosomes is D(ribo) = 0.04 µm(2) s(-1), attributed to free mRNA being translated by one or more 70S ribosomes. We find no clear evidence of subdiffusion, as would arise from tethering of ribosomes to the DNA. The degree of DNA-ribosome segregation strongly suggests that in E. coli most translation occurs on free mRNA transcripts that have diffused into the ribosome-rich regions. Both RNAP and ribosome radial distributions extend to the cytoplasmic membrane, consistent with the transertion hypothesis. However, few if any RNAP copies lie near the membrane of the endcaps. This suggests that if transertion occurs, it exerts a direct radially expanding force on the nucleoid, but not a direct axially expanding force.

  15. Proteomic Analysis of Ribosomes: Translational Control of mRNA populations by Glycogen Synthase GYS1

    PubMed Central

    Fuchs, Gabriele; Diges, Camille; Kohlstaedt, Lori A.; Wehner, Karen A.; Sarnow, Peter

    2011-01-01

    Ribosomes exist as a heterogenous pool of macromolecular complexes composed of ribosomal RNA molecules, ribosomal proteins and numerous associated, “non-ribosomal” proteins. To identify non-ribosomal proteins that may modulate ribosome activity, we examined the composition of translationally active and inactive ribosomes using a proteomic multidimensional protein identification technology. Notably, the phosphorylated isoform of glycogen synthase, GYS1, was preferentially associated with elongating ribosomes. Depletion of GYS1 affected the translation of a subset of cellular mRNAs, some of which encode proteins that modulate protein biosynthesis. These findings argue that GYS1 abundance, and by virtue of its ribosomal association, provide a feedback loop between the energy state of the cells and the translation machinery. PMID:21570405

  16. Molecular analysis of the 16S-23S rDNA internal spacer region (ISR) and truncated tRNA(Ala) gene segments in Campylobacter lari.

    PubMed

    Hayashi, K; Tazumi, A; Nakanishi, S; Nakajima, T; Matsubara, K; Ueno, H; Moore, J E; Millar, B C; Matsuda, M

    2012-06-01

    Following PCR amplification and sequencing, nucleotide sequence alignment analyses demonstrated the presence of two kinds of 16S-23S rDNA internal spacer regions (ISRs), namely, long length ISRs of 837-844 base pair (bp) [n = six for urease-negative (UN) Campylobacter lari isolates, UN C. lari JCM2530(T), RM2100, 176, 293, 299 and 448] and short length ISRs of 679-725 bp [n = six for UN C. lari: n = 14 for urease-positive thermophilic Campylobacter (UPTC) isolates]. The analyses also indicated that the short length ISRs mainly lacked the 156 bp sequence from the nucleotide positions 122-277 bp in long length ISRs for UN C. lari JCM2530(T). The 156 bp sequences shared 94.9-96.8 % sequence similarity among six isolates. Surprisingly, atypical tRNA(Ala) gene segment (5' end 35 bp), which was extremely truncated, occurred within the 156 bp sequences in the long length ISRs, as an unexpected tRNA(Ala) pseudogene. An order of the intercistronic tRNA genes within the short nucleotide spacer of 5'-16S rDNA-tRNA(Ala)-tRNA(Ile)-23S rDNA-3' occurred in all the C. lari isolates examined.

  17. Distinct tmRNA sequence elements facilitate RNase R engagement on rescued ribosomes for selective nonstop mRNA decay.

    PubMed

    Venkataraman, Krithika; Zafar, Hina; Karzai, A Wali

    2014-01-01

    trans-Translation, orchestrated by SmpB and tmRNA, is the principal eubacterial pathway for resolving stalled translation complexes. RNase R, the leading nonstop mRNA surveillance factor, is recruited to stalled ribosomes in a trans-translation dependent process. To elucidate the contributions of SmpB and tmRNA to RNase R recruitment, we evaluated Escherichia coli-Francisella tularensis chimeric variants of tmRNA and SmpB. This evaluation showed that while the hybrid tmRNA supported nascent polypeptide tagging and ribosome rescue, it suffered defects in facilitating RNase R recruitment to stalled ribosomes. To gain further insights, we used established tmRNA and SmpB variants that impact distinct stages of the trans-translation process. Analysis of select tmRNA variants revealed that the sequence composition and positioning of the ultimate and penultimate codons of the tmRNA ORF play a crucial role in recruiting RNase R to rescued ribosomes. Evaluation of defined SmpB C-terminal tail variants highlighted the importance of establishing the tmRNA reading frame, and provided valuable clues into the timing of RNase R recruitment to rescued ribosomes. Taken together, these studies demonstrate that productive RNase R-ribosomes engagement requires active trans-translation, and suggest that RNase R captures the emerging nonstop mRNA at an early stage after establishment of the tmRNA ORF as the surrogate mRNA template.

  18. Homoduplex and Heteroduplex Polymorphisms of the Amplified Ribosomal 16S-23S Internal Transcribed Spacers Describe Genetic Relationships in the “Bacillus cereus Group”

    PubMed Central

    Daffonchio, Daniele; Cherif, Ameur; Borin, Sara

    2000-01-01

    Bacillus anthracis, Bacillus cereus, Bacillus mycoides, Bacillus pseudomycoides, Bacillus thuringiensis, and Bacillus weihenstephanensis are closely related in phenotype and genotype, and their genetic relationship is still open to debate. The present work uses amplified 16S-23S internal transcribed spacers (ITS) to discriminate between the strains and species and to describe the genetic relationships within the “B. cereus group,” advantage being taken of homoduplex-heteroduplex polymorphisms (HHP) resolved by polyacrylamide gel electrophoresis and silver staining. One hundred forty-one strains belonging to the six species were investigated, and 73 ITS-HHP pattern types were distinguished by MDE, a polyacrylamide matrix specifically designed to resolve heteroduplex and single-strand conformation polymorphisms. The discriminating bands were confirmed as ITS by Southern hybridization, and the homoduplex or heteroduplex nature was identified by single-stranded DNA mung bean nuclease digestion. Several of the ITS-HHP types corresponded to specific phenotypes such as B. anthracis or serotypes of B. thuringiensis. Unweighted pair group method arithmetic average cluster analysis revealed two main groups. One included B. mycoides, B. weihenstephanensis, and B. pseudomycoides. The second included B. cereus and B. thuringiensis, B. anthracis appeared as a lineage of B. cereus. PMID:11097928

  19. Differentiation of acetic acid bacteria based on sequence analysis of 16S-23S rRNA gene internal transcribed spacer sequences.

    PubMed

    González, Angel; Mas, Albert

    2011-06-30

    The 16S-23S gene internal transcribed spacer sequence of sixty-four strains belonging to different acetic acid bacteria genera were analyzed, and phylogenetic trees were generated for each genera. The topologies of the different trees were in accordance with the 16S rRNA gene trees, although the similarity percentages obtained between the species was shown to be much lower. These values suggest the usefulness of including the 16S-23S gene internal transcribed spacer region as a part of the polyphasic approach required for the further classification of acetic acid bacteria. Furthermore, the region could be a good target for primer and probe design. It has also been validated for use in the identification of unknown samples of this bacterial group from wine vinegar and fruit condiments.

  20. A novel RNA-binding motif in omnipotent suppressors of translation termination, ribosomal proteins and a ribosome modification enzyme?

    PubMed

    Koonin, E V; Bork, P; Sander, C

    1994-06-11

    Using computer methods for database search, multiple alignment, protein sequence motif analysis and secondary structure prediction, a putative new RNA-binding motif was identified. The novel motif is conserved in yeast omnipotent translation termination suppressor SUP1, the related DOM34 protein and its pseudogene homologue; three groups of eukaryotic and archaeal ribosomal proteins, namely L30e, L7Ae/S6e and S12e; an uncharacterized Bacillus subtilis protein related to the L7A/S6e group; and Escherichia coli ribosomal protein modification enzyme RimK. We hypothesize that a new type of RNA-binding domain may be utilized to deliver additional activities to the ribosome.

  1. The mRNA of human cytoplasmic arginyl-tRNA synthetase recruits prokaryotic ribosomes independently.

    PubMed

    Yang, Fang; Ji, Quan-Quan; Ruan, Liang-Liang; Ye, Qing; Wang, En-Duo

    2014-07-25

    There are two isoforms of cytoplasmic arginyl-tRNA synthetase (hcArgRS) in human cells. The long form is a component of the multiple aminoacyl-tRNA synthetase complex, and the other is an N-terminal truncated form (NhcArgRS), free in the cytoplasm. It has been shown that the two forms of ArgRS arise from alternative translational initiation in a single mRNA. The short form is produced from the initiation at a downstream, in-frame AUG start codon. Interestingly, our data suggest that the alternative translational initiation of hcArgRS mRNA also takes place in Escherichia coli transformants. When the gene encoding full-length hcArgRS was overexpressed in E. coli, two forms of hcArgRS were observed. The N-terminal sequencing experiment identified that the short form was identical to the NhcArgRS in human cytoplasm. By constructing a bicistronic system, our data support that the mRNA encoding the N-terminal extension of hcArgRS has the capacity of independently recruiting E. coli ribosomes. Furthermore, two critical elements for recruiting prokaryotic ribosomes were identified, the “AGGA” core of the Shine-Dalgarno sequence and the “A-rich” sequence located just proximal to the alternative in-frame initiation site. Although the mechanisms of prokaryotic and eukaryotic translational initiation are distinct, they share some common features. The ability of the hcArgRS mRNA to recruit the prokaryotic ribosome may provide clues for shedding light on the mechanism of alternative translational initiation of hcArgRS mRNA in eukaryotic cells.

  2. Ralstonia paucula (Formerly CDC group IV c-2): unsuccessful strain differentiation with PCR-based methods, study of the 16S-23S spacer of the rRNA operon, and comparison with other Ralstonia species (R. eutropha, R. pickettii, R. gilardii, and R. solanacearum).

    PubMed

    Moissenet, D; Bidet, P; Garbarg-Chenon, A; Arlet, G; Vu-Thien, H

    2001-01-01

    Ralstonia paucula (formerly CDC group IV c-2) can cause serious human infections. Confronted in 1995 with five cases of nosocomial bacteremia, we found that pulsed-field gel electrophoresis could not distinguish between the isolates and that randomly amplified polymorphic DNA analysis was poorly discriminatory. In this study, we used PCR-ribotyping and PCR-restriction fragment length polymorphism analysis of the spacer 16S-23S ribosomal DNA (rDNA); both methods were unable to differentiate R. paucula isolates. Eighteen strains belonging to other Ralstonia species (one R. eutropha strain, six R. pickettii strains, three R. solanacearum strains, and eight R. gilardii strains) were also tested by PCR-ribotyping, which failed to distinguish between the four species. The 16S-23S rDNA intergenic spacer of R. paucula contains the tRNA(Ile) and tRNA(Ala) genes, which are identical to genes described for R. pickettii and R. solanacearum.

  3. EttA regulates translation by binding to the ribosomal E site and restricting ribosome-tRNA dynamics

    PubMed Central

    Chen, Bo; Boël, Grégory; Hashem, Yaser; Ning, Wei; Fei, Jingyi; Wang, Chi; Gonzalez, Ruben L.; Hunt, John F.; Frank, Joachim

    2014-01-01

    Cells express many ribosome-interacting factors whose functions and molecular mechanisms remain unknown. Here, we elucidate the mechanism of a newly characterized regulatory translation factor, Energy-dependent Translational Throttle A (EttA), which is an Escherichia coli representative of the ATP-binding cassette F (ABC-F) protein family. Using cryo-EM, we demonstrate that the ATP-bound form of EttA binds to the ribosomal tRNA exit (E) site, where it forms bridging interactions between the ribosomal L1 stalk and the tRNA bound in the peptidyl-tRNA binding (P) site. Using single-molecule fluorescence resonance energy transfer (smFRET), we show that the ATP-bound form of EttA restricts ribosome and tRNA dynamics required for protein synthesis. This work represents the first example, to our knowledge, where the detailed molecular mechanism of any ABC-F family protein has been determined and establishes a framework for elucidating the mechanisms of other regulatory translation factors. PMID:24389465

  4. rRNA maturation as a "quality" control step in ribosomal subunit assembly in Dictyostelium discoideum.

    PubMed

    Mangiarotti, G; Chiaberge, S; Bulfone, S

    1997-10-31

    In Dictyostelium discoideum, newly assembled ribosomal subunits enter polyribosomes while they still contain immature rRNA. rRNA maturation requires the engagement of the subunits in protein synthesis and leads to stabilization of their structure. Maturation of pre-17 S rRNA occurs only after the newly formed 40 S ribosomal particle has entered an 80 S ribosome and participated at least in the formation of one peptide bond or in one translocation event; maturation of pre-26 S rRNA requires the presence on the 80 S particle of a peptidyl-tRNA containing at least 6 amino acids. Newly assembled particles that cannot fulfill these requirements for structural reasons are disassembled into free immature rRNA and ribosomal proteins.

  5. The pathway of hepatitis C virus mRNA recruitment to the human ribosome.

    PubMed

    Fraser, Christopher S; Hershey, John W B; Doudna, Jennifer A

    2009-04-01

    Eukaryotic protein synthesis begins with mRNA positioning in the ribosomal decoding channel in a process typically controlled by translation-initiation factors. Some viruses use an internal ribosome entry site (IRES) in their mRNA to harness ribosomes independently of initiation factors. We show here that a ribosome conformational change that is induced upon hepatitis C viral IRES binding is necessary but not sufficient for correct mRNA positioning. Using directed hydroxyl radical probing to monitor the assembly of IRES-containing translation-initiation complexes, we have defined a crucial step in which mRNA is stabilized upon initiator tRNA binding. Unexpectedly, however, this stabilization occurs independently of the AUG codon, underscoring the importance of initiation factor-mediated interactions that influence the configuration of the decoding channel. These results reveal how an IRES RNA supplants some, but not all, of the functions normally carried out by protein factors during initiation of protein synthesis.

  6. Global Analysis of Truncated RNA Ends Reveals New Insights into Ribosome Stalling in Plants[OPEN

    PubMed Central

    Lee, Wen‐Chi; Chou, Hsiao‐Chun; Chen, Ai‐Ping; Chou, Shu‐Jen

    2016-01-01

    High-throughput approaches for profiling the 5′ ends of RNA degradation intermediates on a genome-wide scale are frequently applied to analyze and validate cleavage sites guided by microRNAs (miRNAs). However, the complexity of the RNA degradome other than miRNA targets is currently largely uncharacterized, and this limits the application of RNA degradome studies. We conducted a global analysis of 5′-truncated mRNA ends that mapped to coding sequences (CDSs) of Arabidopsis thaliana, rice (Oryza sativa), and soybean (Glycine max). Based on this analysis, we provide multiple lines of evidence to show that the plant RNA degradome contains in vivo ribosome-protected mRNA fragments. We observed a 3-nucleotide periodicity in the position of free 5′ RNA ends and a bias toward the translational frame. By examining conserved peptide upstream open reading frames (uORFs) of Arabidopsis and rice, we found a predominance of 5′ termini of RNA degradation intermediates that were separated by a length equal to a ribosome-protected mRNA fragment. Through the analysis of RNA degradome data, we discovered uORFs and CDS regions potentially associated with stacked ribosomes in Arabidopsis. Furthermore, our analysis of RNA degradome data suggested that the binding of Arabidopsis ARGONAUTE7 to a noncleavable target site of miR390 might directly hinder ribosome movement. This work demonstrates an alternative use of RNA degradome data in the study of ribosome stalling. PMID:27742800

  7. Requirement of rRNA Methylation for 80S Ribosome Assembly on a Cohort of Cellular Internal Ribosome Entry Sites▿

    PubMed Central

    Basu, Abhijit; Das, Priyanka; Chaudhuri, Sujan; Bevilacqua, Elena; Andrews, Joel; Barik, Sailen; Hatzoglou, Maria; Komar, Anton A.; Mazumder, Barsanjit

    2011-01-01

    Protein syntheses mediated by cellular and viral internal ribosome entry sites (IRESs) are believed to have many features in common. Distinct mechanisms for ribosome recruitment and preinitiation complex assembly between the two processes have not been identified thus far. Here we show that the methylation status of rRNA differentially influenced the mechanism of 80S complex formation on IRES elements from the cellular sodium-coupled neutral amino acid transporter 2 (SNAT2) versus the hepatitis C virus mRNA. Translation initiation involves the assembly of the 48S preinitiation complex, followed by joining of the 60S ribosomal subunit and formation of the 80S complex. Abrogation of rRNA methylation did not affect the 48S complex but resulted in impairment of 80S complex assembly on the cellular, but not the viral, IRESs tested. Impairment of 80S complex assembly on the amino acid transporter SNAT2 IRES was rescued by purified 60S subunits containing fully methylated rRNA. We found that rRNA methylation did not affect the activity of any of the viral IRESs tested but affected the activity of numerous cellular IRESs. This work reveals a novel mechanism operating on a cohort of cellular IRESs that involves rRNA methylation for proper 80S complex assembly and efficient translation initiation. PMID:21930789

  8. Discrimination of bacillus anthracis and closely related microorganisms by analysis of 16S and 23S rRNA with oligonucleotide microarray.

    SciTech Connect

    Bavykin, S. G.; Mikhailovich, V. M.; Zakharyev, V. M.; Lysov, Y. P.; Kelly, J. J.; Alferov, O. S.; Jackman, J.; Stahl, D. A.; Mirzabekov, A. D.; Gavin, I. M.; Kukhtin, A. V.; Chandler, D.

    2008-01-30

    Analysis of 16S rRNA sequences is a commonly used method for the identification and discrimination of microorganisms. However, the high similarity of 16S and 23S rRNA sequences of Bacillus cereus group organisms (up to 99-100%) and repeatedly failed attempts to develop molecular typing systems that would use DNA sequences to discriminate between species within this group have resulted in several suggestions to consider B. cereus and B. thuringiensis, or these two species together with B. anthracis, as one species. Recently, we divided the B. cereus group into seven subgroups, Anthracis, Cereus A and B, Thuringiensis A and B, and Mycoides A and B, based on 16S rRNA, 23S rRNA and gyrB gene sequences and identified subgroup-specific makers in each of these three genes. Here we for the first time demonstrated discrimination of these seven subgroups, including subgroup Anthracis, with a 3D gel element microarray of oligonucleotide probes targeting 16S and 23S rRNA markers. This is the first microarray enabled identification of B. anthracis and discrimination of these seven subgroups in pure cell cultures and in environmental samples using rRNA sequences. The microarray bearing perfect match/mismatch (p/mm) probe pairs was specific enough to discriminate single nucleotide polymorphisms (SNPs) and was able to identify targeted organisms in 5 min. We also demonstrated the ability of the microarray to determine subgroup affiliations for B. cereus group isolates without rRNA sequencing. Correlation of these seven subgroups with groupings based on multilocus sequence typing (MLST), fluorescent amplified fragment length polymorphism analysis (AFLP) and multilocus enzyme electrophoresis (MME) analysis of a wide spectrum of different genes, and the demonstration of subgroup-specific differences in toxin profiles, psychrotolerance, and the ability to harbor some plasmids, suggest that these seven subgroups are not based solely on neutral genomic polymorphisms, but instead reflect

  9. Mutations in ribosomal proteins S4 and S12 influence the higher order structure of 16 S ribosomal RNA.

    PubMed

    Allen, P N; Noller, H F

    1989-08-05

    We have studied the effects of protein mutations on the higher order structure of 16 S rRNA in Escherichia coli ribosomes, using a set of structure-sensitive chemical probes. Ten mutant strains were studied, which contained alterations in ribosomal proteins S4 and S12, including double mutants containing both altered S4 and S12. Two ribosomal ambiguity (ram) S4 mutant strains, four streptomycin resistant (SmR) S12 mutant strains, one streptomycin pseudodependent (SmP) S12 mutant strain, one streptomycin dependent (SmD) S12 mutant strain and two streptomycin independent (Sm1) double mutants (containing both-SmD and ram mutations) were probed and compared to an isogenic wild-type strain. In ribosomes from strains containing S4 ram mutations, nucleotides A8 and A26 become more reactive to dimethyl sulfate (DMS) at their N-1 positions. In ribosomes from strains bearing the SmD allele, A908, A909, A1413 and G1487 are significantly less reactive to chemical probes. These same effects are observed when the S4 and S12 mutations are present simultaneously in the double mutants. An interesting correlation is found between the reactivity of A908 and the miscoding potential of SmR, SmD, SmP and wild-type ribosomes; the reactivity of A908 increases as the translational error frequency of the ribosomes increases. In the case of ram ribosomes, the reactivity of A908 resembles that of wild-type, unless tRNA is bound, in which case it becomes hyper-reactive. Similarly, streptomycin has little effect on A908 in wild-type ribosomes unless tRNA is bound, in which case its reactivity increases to resemble that of ram ribosomes with bound tRNA. Finally, interaction of streptomycin with SmP and SmD ribosomes causes the reactivity of A908 to increase to near-wild-type levels. A simple model is proposed, in which the reactivity of A908 reflects the position of an equilibrium between two conformational states of the 30 S subunit, one of which is DMS-reactive, and the other DMS

  10. Specialized yeast ribosomes: a customized tool for selective mRNA translation.

    PubMed

    Bauer, Johann W; Brandl, Clemens; Haubenreisser, Olaf; Wimmer, Bjoern; Weber, Manuela; Karl, Thomas; Klausegger, Alfred; Breitenbach, Michael; Hintner, Helmut; von der Haar, Tobias; Tuite, Mick F; Breitenbach-Koller, Lore

    2013-01-01

    Evidence is now accumulating that sub-populations of ribosomes - so-called specialized ribosomes - can favour the translation of subsets of mRNAs. Here we use a large collection of diploid yeast strains, each deficient in one or other copy of the set of ribosomal protein (RP) genes, to generate eukaryotic cells carrying distinct populations of altered 'specialized' ribosomes. We show by comparative protein synthesis assays that different heterologous mRNA reporters based on luciferase are preferentially translated by distinct populations of specialized ribosomes. These mRNAs include reporters carrying premature termination codons (PTC) thus allowing us to identify specialized ribosomes that alter the efficiency of translation termination leading to enhanced synthesis of the wild-type protein. This finding suggests that these strains can be used to identify novel therapeutic targets in the ribosome. To explore this further we examined the translation of the mRNA encoding the extracellular matrix protein laminin β3 (LAMB3) since a LAMB3-PTC mutant is implicated in the blistering skin disease Epidermolysis bullosa (EB). This screen identified specialized ribosomes with reduced levels of RP L35B as showing enhanced synthesis of full-length LAMB3 in cells expressing the LAMB3-PTC mutant. Importantly, the RP L35B sub-population of specialized ribosomes leave both translation of a reporter luciferase carrying a different PTC and bulk mRNA translation largely unaltered.

  11. Choreography of molecular movements during ribosome progression along mRNA.

    PubMed

    Belardinelli, Riccardo; Sharma, Heena; Caliskan, Neva; Cunha, Carlos E; Peske, Frank; Wintermeyer, Wolfgang; Rodnina, Marina V

    2016-04-01

    During translation elongation, ribosome translocation along an mRNA entails rotations of the ribosomal subunits, swiveling motions of the small subunit (SSU) head and stepwise movements of the tRNAs together with the mRNA. Here, we reconstructed the choreography of the collective motions of the Escherichia coli ribosome during translocation promoted by elongation factor EF-G, by recording the fluorescence signatures of nine different reporters placed on both ribosomal subunits, tRNA and mRNA. We captured an early forward swiveling of the SSU head taking place while the SSU body rotates in the opposite, clockwise direction. Backward swiveling of the SSU head starts upon tRNA translocation and continues until the post-translocation state is reached. This work places structures of translocation intermediates along a time axis and unravels principles of the motions of macromolecular machines.

  12. Methylation of ribosomal RNA by NSUN5 is a conserved mechanism modulating organismal lifespan

    PubMed Central

    Schosserer, Markus; Minois, Nadege; Angerer, Tina B.; Amring, Manuela; Dellago, Hanna; Harreither, Eva; Calle-Perez, Alfonso; Pircher, Andreas; Gerstl, Matthias Peter; Pfeifenberger, Sigrid; Brandl, Clemens; Sonntagbauer, Markus; Kriegner, Albert; Linder, Angela; Weinhäusel, Andreas; Mohr, Thomas; Steiger, Matthias; Mattanovich, Diethard; Rinnerthaler, Mark; Karl, Thomas; Sharma, Sunny; Entian, Karl-Dieter; Kos, Martin; Breitenbach, Michael; Wilson, Iain B.H.; Polacek, Norbert; Grillari-Voglauer, Regina; Breitenbach-Koller, Lore; Grillari, Johannes

    2015-01-01

    Several pathways modulating longevity and stress resistance converge on translation by targeting ribosomal proteins or initiation factors, but whether this involves modifications of ribosomal RNA is unclear. Here, we show that reduced levels of the conserved RNA methyltransferase NSUN5 increase the lifespan and stress resistance in yeast, worms and flies. Rcm1, the yeast homologue of NSUN5, methylates C2278 within a conserved region of 25S rRNA. Loss of Rcm1 alters the structural conformation of the ribosome in close proximity to C2278, as well as translational fidelity, and favours recruitment of a distinct subset of oxidative stress-responsive mRNAs into polysomes. Thus, rather than merely being a static molecular machine executing translation, the ribosome exhibits functional diversity by modification of just a single rRNA nucleotide, resulting in an alteration of organismal physiological behaviour, and linking rRNA-mediated translational regulation to modulation of lifespan, and differential stress response. PMID:25635753

  13. Generation of chemically engineered ribosomes for atomic mutagenesis studies on protein biosynthesis.

    PubMed

    Erlacher, Matthias D; Chirkova, Anna; Voegele, Paul; Polacek, Norbert

    2011-05-01

    The protocol describes the site-specific chemical modification of 23S rRNA of Thermus aquaticus ribosomes. The centerpiece of this 'atomic mutagenesis' approach is the site-specific incorporation of non-natural nucleoside analogs into 23S rRNA in the context of the entire 70S ribosome. This technique exhaustively makes use of the available crystallographic structures of the ribosome for designing detailed biochemical experiments aiming at unraveling molecular insights of ribosomal functions. The generation of chemically engineered ribosomes carrying a particular non-natural 23S rRNA residue at the site of interest, a procedure that typically takes less than 2 d, allows the study of translation at the molecular level and goes far beyond the limits of standard mutagenesis approaches. This methodology, in combination with the presented tests for ribosomal functions adapted to chemically engineered ribosomes, allows unprecedented molecular insight into the mechanisms of protein biosynthesis.

  14. Heritability and Variability in Ribosomal RNA Genes of Vicia faba

    PubMed Central

    Rogers, Scott O.; Bendich, Arnold J.

    1987-01-01

    We have compared the restriction patterns and copy numbers of ribosomal RNA genes (rDNA) between and within individuals of Vicia faba . While the EcoRI blot-hybridization patterns changed only after one to two generations, copy number changes were found among different tissues of the same plant. Copy number differences among individuals in the population were as great as 95-fold, whereas as much as a 12-fold variation was seen among tissues of the same plant. Among individual F1 progeny from genetic crosses, nearly an 8-fold variation was seen, and among individuals of the F2 generation a spread of 22-fold was measured. Among individual siblings of self-pollinated parents, up to 7-fold variation was observed. However, changes in copy number did not necessarily indicate changes in rDNA EcoRI blot-hybridization pattern, and vice versa. Furthermore, nearest neighbor analysis of R-loop experiments showed that the arrangement of members of the "nontranscribed" spacer (NTS) size classes along the chromosome was not random, but some clustering was indicated. The data are consistent with the hypothesis that sister chromatid exchange in somatic cells of V. faba is the primary mechanism for altering the rDNA copy number as well as causing the extreme variation observed in the NTS. Variation among individuals in rDNA blot-hybridization pattern was also observed for Vicia villosa, Vicia dasycarpa, Vicia benghalensis and Vicia pannonica. PMID:17246404

  15. Molecular breeding of Saccharomyces cerevisiae with high RNA content by harnessing essential ribosomal RNA transcription regulator.

    PubMed

    Sasano, Yu; Kariya, Takahiro; Usugi, Shogo; Sugiyama, Minetaka; Harashima, Satoshi

    2017-12-01

    As yeast is commonly used for RNA production, it is industrially important to breed strains with high RNA contents. The upstream activating factor (UAF) plays an important role in transcription of ribosomal RNA (rRNA), a major constituent of intracellular RNA species. Here, we targeted the essential rRNA transcription regulator Rrn5 of Saccharomyces cerevisiae, a component of the UAF complex, and disrupted the genomic RRN5 gene using a helper plasmid carrying an RRN5 gene. Then we isolated nine suppressor mutants (Sup mutants) of RRN5 gene disruption, causing deficiency in rRNA transcription. The Sup mutants had RNA contents of approximately 40% of the wild type level and expansion of rDNA repeats to ca. 400-700 copies. Reintroduction of a functional RRN5 gene into Sup mutants caused a reduction in the number of rDNA repeats to close to the wild type level but did not change RNA content. However, we found that reintroduction of RRN5 into the Sup16 mutant (in which the FOB1 gene encoding the rDNA replication fork barrier site binding protein was disrupted) resulted in a significant increase (17%) in RNA content compared with wild type, although the rDNA repeat copy number was almost identical to the wild type strain. In this case, upregulated transcription of non-transcribed spacers (NTS) occurred, especially in the NTS2 region; this was likely mediated by RNA polymerase II and accounted for the increased RNA content. Thus, we propose a novel breeding strategy for developing high RNA content yeast by harnessing the essential rRNA transcription regulator.

  16. Transcription of ribosomal RNA: the role of antitermination of RNA polymerase

    NASA Astrophysics Data System (ADS)

    Klumpp, Stefan; Hwa, Terry

    2007-03-01

    The genes encoding ribosomal RNA are transcribed at high rates of 1-2 transcripts per second. These high transcription rates are crucial to maintain the large concentration of ribosomes necessary in fast growing bacteria. To understand how transcription is regulated under these conditions, we developed a model for the traffic of transcribing RNA polymerases (RNAP). Our simulations show that the transcription rate is limited by the elongation stage of transcription rather than by transcript initiation. The maximal transcription rate is severly impaired by RNAP pausing with pause durations in the second range which is ubiquitous under single-molecule conditions. We propose that ribosomal antitermination reduces pauses and thereby increases the transcription rate. This idea is in quantitative agreement with the observed increase of the elongation rate due to antitermination and predicts a two-fold increase of the transcription rate. Antitermination must be highly efficient, since incomplete antitermination with only a few percent of non-antiterminated, i.e. slow, RNAPs completely abolishes its effect. This result suggests that rho-dependent termination may selectively terminate slow RNAPs.

  17. An RNA trapping mechanism in Alphavirus mRNA promotes ribosome stalling and translation initiation

    PubMed Central

    Toribio, René; Díaz-López, Irene; Boskovic, Jasminka; Ventoso, Iván

    2016-01-01

    During translation initiation, eukaryotic initiation factor 2 (eIF2) delivers the Met-tRNA to the 40S ribosomal subunit to locate the initiation codon (AUGi) of mRNA during the scanning process. Stress-induced eIF2 phosphorylation leads to a general blockade of translation initiation and represents a key antiviral pathway in mammals. However, some viral mRNAs can initiate translation in the presence of phosphorylated eIF2 via stable RNA stem-loop structures (DLP; Downstream LooP) located in their coding sequence (CDS), which promote 43S preinitiation complex stalling on the initiation codon. We show here that during the scanning process, DLPs of Alphavirus mRNA become trapped in ES6S region (680–914 nt) of 18S rRNA that are projected from the solvent side of 40S subunit. This trapping can lock the progress of the 40S subunit on the mRNA in a way that places the upstream initiator AUGi on the P site of 40S subunit, obviating the participation of eIF2. Notably, the DLP structure is released from 18S rRNA upon 60S ribosomal subunit joining, suggesting conformational changes in ES6Ss during the initiation process. These novel findings illustrate how viral mRNA is threaded into the 40S subunit during the scanning process, exploiting the topology of the 40S subunit solvent side to enhance its translation in vertebrate hosts. PMID:26984530

  18. 23S rRNA gene-based enterococci community signatures in Lake Pontchartrain, Louisiana, USA, following urban runoff inputs after Hurricane Katrina.

    PubMed

    Bae, Hee-Sung; Hou, Aixin

    2013-02-01

    Little is known about the impacts of fecal polluted urban runoff inputs on the structure of enterococci communities in estuarine waters. This study employed a 23S rRNA gene-based polymerase chain reaction (PCR) assay with newly designed genus-specific primers, Ent127F-Ent907R, to determine the possible impacts of Hurricane Katrina floodwaters via the 17th Street Canal discharge on the community structure of enterococci in Lake Pontchartrain. A total of 94 phylotypes were identified through the restriction fragment length polymorphism (RFLP) screening of 494 clones while only 8 phylotypes occurred among 88 cultivated isolates. Sequence analyses of representative phylotypes and their temporal and spatial distribution in the lake and the canal indicated the Katrina floodwater input introduced a large portion of Enterococcus flavescens, Enterococcus casseliflavus, and Enterococcus dispar into the lake; typical fecal groups Enterococcus faecium, Enterococcus durans, Enterococcus hirae, and Enterococcus mundtii were detected primarily in the floodwater-impacted waters. This study provides a global picture of enterococci in estuarine waters impacted by Hurricane Katrina-derived urban runoff. It also demonstrates the culture-independent PCR approach using 23S rRNA gene as a molecular marker could be a good alternative in ecological studies of enterococci in natural environments to overcome the limitation of conventional cultivation methods.

  19. Modeling of ribosome dynamics on a ds-mRNA under an external load

    NASA Astrophysics Data System (ADS)

    Shakiba, Bahareh; Dayeri, Maryam; Mohammad-Rafiee, Farshid

    2016-07-01

    Protein molecules in cells are synthesized by macromolecular machines called ribosomes. According to the recent experimental data, we reduce the complexity of the ribosome and propose a model to express its activity in six main states. Using our model, we study the translation rate in different biological relevant situations in the presence of external force and the translation through the RNA double stranded region in the absence or presence of the external force. In the present study, we give a quantitative theory for translation rate and show that the ribosome behaves more like a Brownian Ratchet motor. Our findings could shed some light on understanding behaviors of the ribosome in biological conditions.

  20. Discrimination of Bacillus anthracis from closely related microorganisms by analysis of 16S and 23S rRNA with oligonucleotide microchips

    DOEpatents

    Bavykin, Sergei G.; Mirzabekov, Andrei D.

    2007-10-30

    The present invention is directed to a novel method of discriminating a highly infectious bacterium Bacillus anthracis from a group of closely related microorganisms. Sequence variations in the 16S and 23S rRNA of the B. cereus subgroup including B. anthracis are utilized to construct an array that can detect these sequence variations through selective hybridizations. The identification and analysis of these sequence variations enables positive discrimination of isolates of the B. cereus group that includes B. anthracis. Discrimination of single base differences in rRNA was achieved with a microchip during analysis of B. cereus group isolates from both single and in mixed probes, as well as identification of polymorphic sites. Successful use of a microchip to determine the appropriate subgroup classification using eight reference microorganisms from the B. cereus group as a study set, was demonstrated.

  1. Transfer RNA-mediated regulation of ribosome dynamics during protein synthesis

    PubMed Central

    Fei, Jingyi; Richard, Arianne C.; Bronson, Jonathan E.; Gonzalez, & Ruben L.

    2011-01-01

    Translocation of transfer RNAs (tRNAs) through the ribosome during protein synthesis involves large-scale structural rearrangements of the ribosome and the ribosome-bound tRNAs that are accompanied by extensive and dynamic remodeling of tRNA-ribosome interactions. The contributions that rearranging individual tRNA-ribosome interactions make to directing tRNA movements during translocation, however, remain largely unknown. To address this question, we have used single-molecule fluorescence resonance energy transfer to characterize the dynamics of ribosomal pre-translocation (PRE) complex analogs carrying either wild-type or systematically mutagenized tRNAs. Our data reveal how specific tRNA-ribosome interactions regulate the rate with which the PRE complex rearranges into a critical, on-pathway translocation intermediate and how these interactions control the stability of the resulting configuration. More interestingly, our results suggest that the conformational flexibility of the tRNA molecule itself plays a crucial role in directing the structural dynamics of the PRE complex during translocation. PMID:21857664

  2. YaeJ is a novel ribosome-associated protein in Escherichia coli that can hydrolyze peptidyl-tRNA on stalled ribosomes.

    PubMed

    Handa, Yoshihiro; Inaho, Noriyuki; Nameki, Nobukazu

    2011-03-01

    In bacteria, ribosomes often become stalled and are released by a trans-translation process mediated by transfer-messenger RNA (tmRNA). In the absence of tmRNA, however, there is evidence that stalled ribosomes are released from non-stop mRNAs. Here, we show a novel ribosome rescue system mediated by a small basic protein, YaeJ, from Escherichia coli, which is similar in sequence and structure to the catalytic domain 3 of polypeptide chain release factor (RF). In vitro translation experiments using the E. coli-based reconstituted cell-free protein synthesis system revealed that YaeJ can hydrolyze peptidyl-tRNA on ribosomes stalled by both non-stop mRNAs and mRNAs containing rare codon clusters that extend downstream from the P-site and prevent Ala-tmRNA•SmpB from entering the empty A-site. In addition, YaeJ had no effect on translation of a normal mRNA with a stop codon. These results suggested a novel tmRNA-independent rescue system for stalled ribosomes in E. coli. YaeJ was almost exclusively found in the 70S ribosome and polysome fractions after sucrose density gradient sedimentation, but was virtually undetectable in soluble fractions. The C-terminal basic residue-rich extension was also found to be required for ribosome binding. These findings suggest that YaeJ functions as a ribosome-attached rescue device for stalled ribosomes.

  3. Recognition of the 70S ribosome and polysome by the RNA degradosome in Escherichia coli.

    PubMed

    Tsai, Yi-Chun; Du, Dijun; Domínguez-Malfavón, Lilianha; Dimastrogiovanni, Daniela; Cross, Jonathan; Callaghan, Anastasia J; García-Mena, Jaime; Luisi, Ben F

    2012-11-01

    The RNA degradosome is a multi-enzyme assembly that contributes to key processes of RNA metabolism, and it engages numerous partners in serving its varied functional roles. Small domains within the assembly recognize collectively a diverse range of macromolecules, including the core protein components, the cytoplasmic lipid membrane, mRNAs, non-coding regulatory RNAs and precursors of structured RNAs. We present evidence that the degradosome can form a stable complex with the 70S ribosome and polysomes, and we demonstrate the proximity in vivo of ribosomal proteins and the scaffold of the degradosome, RNase E. The principal interactions are mapped to two, independent, RNA-binding domains from RNase E. RhlB, the RNA helicase component of the degradosome, also contributes to ribosome binding, and this is favoured through an activating interaction with RNase E. The catalytic activity of RNase E for processing 9S RNA (the ribosomal 5S RNA precursor) is repressed in the presence of the ribosome, whereas there is little affect on the cleavage of single-stranded substrates mediated by non-coding RNA, suggestings that the enzyme retains capacity to cleave unstructured substrates when associated with the ribosome. We propose that polysomes may act as antennae that enhance the rates of capture of the limited number of degradosomes, so that they become recruited to sites of active translation to act on mRNAs as they become exposed or tagged for degradation.

  4. Nucleolin provides a link between RNA polymerase I transcription and pre-ribosome assembly.

    PubMed

    Roger, Benoit; Moisand, André; Amalric, François; Bouvet, Philippe

    2003-03-01

    Despite the identification of numerous factors involved in ribosomal RNA synthesis and maturation, the molecular mechanisms of ribosome biogenesis, and in particular the relationship between the different steps, are still largely unknown. We have investigated the consequences of an increased amount of a major nucleolar non-ribosomal protein, nucleolin, in Xenopus laevisstage VI oocytes on the production of ribosomal subunits. We show that a threefold increase in nucleolin leads to the complete absence of pre-rRNA maturation in addition to significant repression of RNA polymerase I transcription. Observation of "Christmas trees" by electron microscopy and analysis of the sedimentation properties of 40S pre-ribosomal particles suggest that an increased amount of nucleolin leads to incorrect packaging of the 40S particle. Interestingly, nucleolin affects the maturation of the 40S particle only when it is present at the time of transcription. These results indicate that nucleolin participates in the co-transcriptional packaging of the pre-rRNA, and that the quality of this packaging will determine whether the 40S precursor undergoes maturation or is degraded. The interaction of nucleolin with nascent pre-rRNA could help the co-transcriptional assembly on pre-rRNA of factors necessary for the subsequent maturation of the pre-ribosomal particle containing the 40S pre-rRNA.

  5. Identification of previously unrecognized common elements in eukaryotic promoters. A ribosomal RNA gene initiator element for RNA polymerase I.

    PubMed

    Radebaugh, C A; Gong, X; Bartholomew, B; Paule, M R

    1997-02-07

    A new ribosomal RNA promoter element with a functional role similar to the RNA polymerase II initiator (Inr) was identified. This sequence, which we dub the ribosomal Inr (rInr) is unusually conserved, even in normally divergent RNA polymerase I promoters. It functions in the recruitment of the fundamental, TATA-binding protein (TBP)-containing transcription factor, TIF-IB. All upstream elements of the exceptionally strong Acanthamoeba castellanii ribosomal RNA core promoter, to within 6 base pairs of the transcription initiation site (tis), can be deleted without loss of specific transcription initiation. Thus, the A. castellanii promoter can function in a manner similar to RNA polymerase II TATA-less promoters. Sequence-specific photo-cross-linking localizes a 96-kDa subunit of TIF-IB and the second largest RNA polymerase I subunit (A133) to the rInr sequence. A185 also photo-cross-links when polymerase is stalled at +7.

  6. Potential key bases of ribosomal RNA to kingdom-specific spectra of antibiotic susceptibility and the possible archaeal origin of eukaryotes.

    PubMed

    Xie, Qiang; Wang, Yanhui; Lin, Jinzhong; Qin, Yan; Wang, Ying; Bu, Wenjun

    2012-01-01

    In support of the hypothesis of the endosymbiotic origin of eukaryotes, much evidence has been found to support the idea that some organelles of eukaryotic cells originated from bacterial ancestors. Less attention has been paid to the identity of the host cell, although some biochemical and molecular genetic properties shared by archaea and eukaryotes have been documented. Through comparing 507 taxa of 16S-18S rDNA and 347 taxa of 23S-28S rDNA, we found that archaea and eukaryotes share twenty-six nucleotides signatures in ribosomal DNA. These signatures exist in all living eukaryotic organisms, whether protist, green plant, fungus, or animal. This evidence explicitly supports the archaeal origin of eukaryotes. In the ribosomal RNA, besides A2058 in Escherichia coli vs. G2400 in Saccharomyces cerevisiae, there still exist other twenties of sites, in which the bases are kingdom-specific. Some of these sites concentrate in the peptidyl transferase centre (PTC) of the 23S-28S rRNA. The results suggest potential key sites to explain the kingdom-specific spectra of drug resistance of ribosomes.

  7. Organization and nucleotide sequence analysis of a ribosomal RNA gene cluster from Streptomyces ambofaciens.

    PubMed

    Pernodet, J L; Boccard, F; Alegre, M T; Gagnat, J; Guérineau, M

    1989-06-30

    The Streptomyces ambofaciens genome contains four rRNA gene clusters. These copies are called rrnA, B, C and D. The complete nucleotide (nt) sequence of rrnD has been determined. These genes possess striking similarity with other eubacterial rRNA genes. Comparison with other rRNA sequences allowed the putative localization of the sequences encoding mature rRNAs. The structural genes are arranged in the order 16S-23S-5S and are tightly linked. The mature rRNAs are predicted to contain 1528, 3120 and 120 nt, for the 16S, 23S and 5S rRNAs, respectively. The 23S rRNA is, to our knowledge, the longest of all sequenced prokaryotic 23S rRNAs. When compared to other large rRNAs it shows insertions at positions where they are also present in archaebacterial and in eukaryotic large rRNAs. Secondary structure models of S. ambofaciens rRNAs are proposed, based upon those existing for other bacterial rRNAs. Positions of putative transcription start points and of a termination signal are suggested. The corresponding putative primary transcript, containing the 16S, 23S and 5S rRNAs plus flanking regions, was folded into a secondary structure, and sequences possibly involved in rRNA maturation are described. The G + C content of the rRNA gene cluster is low (57%) compared with the overall G + C content of Streptomyces DNA (73%).

  8. Ribosome release factor RF4 and termination factor RF3 are involved in dissociation of peptidyl-tRNA from the ribosome.

    PubMed Central

    Heurgué-Hamard, V; Karimi, R; Mora, L; MacDougall, J; Leboeuf, C; Grentzmann, G; Ehrenberg, M; Buckingham, R H

    1998-01-01

    Peptidyl-tRNA dissociation from ribosomes is an energetically costly but apparently inevitable process that accompanies normal protein synthesis. The drop-off products of these events are hydrolysed by peptidyl-tRNA hydrolase. Mutant selections have been made to identify genes involved in the drop-off of peptidyl-tRNA, using a thermosensitive peptidyl-tRNA hydrolase mutant in Escherichia coli. Transposon insertions upstream of the frr gene, which encodes RF4 (ribosome release or recycling factor), restored growth to this mutant. The insertions impaired expression of the frr gene. Mutations inactivating prfC, encoding RF3 (release factor 3), displayed a similar phenotype. Conversely, production of RF4 from a plasmid increased the thermosensitivity of the peptidyl-tRNA hydrolase mutant. In vitro measurements of peptidyl-tRNA release from ribosomes paused at stop signals or sense codons confirmed that RF3 and RF4 were able to stimulate peptidyl-tRNA release from ribosomes, and showed that this action of RF4 required the presence of translocation factor EF2, known to be needed for the function of RF4 in ribosome recycling. When present together, the three factors were able to stimulate release up to 12-fold. It is suggested that RF4 may displace peptidyl-tRNA from the ribosome in a manner related to its proposed function in removing deacylated tRNA during ribosome recycling. PMID:9451005

  9. Complex RNA maturation pathway for a chloroplast ribosomal protein operon with an internal tRNA cistron.

    PubMed Central

    Christopher, D A; Hallick, R B

    1990-01-01

    We have studied the expression of a large chloroplast ribosomal protein operon from Euglena gracilis that resembles the Escherichia coli S10 and spc ribosomal protein operons. We present evidence that 11 ribosomal protein genes, a tRNA gene, and a new locus, orf214/orf302, are expressed as a single transcription unit. The primary transcript also contains at least 15 group II and group III introns. Gene-specific probes for each ribosomal protein gene, orf214/orf302, and for trnl hybridized to a common pre-mRNA of an estimated size of 8.3 kilobases. This is the RNA size predicted for a full-length transcript of the entire operon after splicing of all 15 introns. Polycistronic ribosomal protein mRNAs accumulated primarily as spliced hepta-, hexa-, penta-, tetra-, tri-, and dicistronic mRNAs, which were presumed to arise by stepwise processing of the 8.3-kilobase pre-mRNA. A novel finding was the cotranscription of the trnl gene as an internal cistron within the ribosomal protein operon. Several combined mRNA/tRNA molecules, such as the pentacistronic rpl5-rps8-rpl36-trnl-rps14, were characterized. The occurrence of the orf214/orf302 is a unique feature of the Euglena operon, distinguishing it from all chloroplast and prokaryotic ribosomal protein operons characterized to date. The orf214/orf302 are not similar to any known genes but are cotranscribed with the ribosomal protein loci and encode stable RNA species of 2.4, 1.8, and 1.4 kilobases. PMID:2136640

  10. An unusual mechanism for EF-Tu activation during tmRNA-mediated ribosome rescue.

    PubMed

    Miller, Mickey R; Buskirk, Allen R

    2014-02-01

    In bacteria, ribosomes stalled on truncated mRNAs are rescued by transfer-messenger RNA (tmRNA) and its protein partner SmpB. Acting like tRNA, the aminoacyl-tmRNA/SmpB complex is delivered to the ribosomal A site by EF-Tu and accepts the transfer of the nascent polypeptide. Although SmpB binding within the decoding center is clearly critical for licensing tmRNA entry into the ribosome, it is not known how activation of EF-Tu occurs in the absence of a codon-anticodon interaction. A recent crystal structure revealed that SmpB residue His136 stacks on 16S rRNA nucleotide G530, a critical player in the canonical decoding mechanism. Here we use pre-steady-state kinetic methods to probe the role of this interaction in ribosome rescue. We find that although mutation of His136 does not reduce SmpB's affinity for the ribosomal A-site, it dramatically reduces the rate of GTP hydrolysis by EF-Tu. Surprisingly, the same mutation has little effect on the apparent rate of peptide-bond formation, suggesting that release of EF-Tu from the tmRNA/SmpB complex on the ribosome may occur prior to GTP hydrolysis. Consistent with this idea, we find that peptidyl transfer to tmRNA is relatively insensitive to the antibiotic kirromycin. Taken together, our studies provide a model for the initial stages of ribosomal rescue by tmRNA.

  11. An unusual mechanism for EF-Tu activation during tmRNA-mediated ribosome rescue

    PubMed Central

    Miller, Mickey R.; Buskirk, Allen R.

    2014-01-01

    In bacteria, ribosomes stalled on truncated mRNAs are rescued by transfer-messenger RNA (tmRNA) and its protein partner SmpB. Acting like tRNA, the aminoacyl-tmRNA/SmpB complex is delivered to the ribosomal A site by EF-Tu and accepts the transfer of the nascent polypeptide. Although SmpB binding within the decoding center is clearly critical for licensing tmRNA entry into the ribosome, it is not known how activation of EF-Tu occurs in the absence of a codon–anticodon interaction. A recent crystal structure revealed that SmpB residue His136 stacks on 16S rRNA nucleotide G530, a critical player in the canonical decoding mechanism. Here we use pre-steady-state kinetic methods to probe the role of this interaction in ribosome rescue. We find that although mutation of His136 does not reduce SmpB's affinity for the ribosomal A-site, it dramatically reduces the rate of GTP hydrolysis by EF-Tu. Surprisingly, the same mutation has little effect on the apparent rate of peptide-bond formation, suggesting that release of EF-Tu from the tmRNA/SmpB complex on the ribosome may occur prior to GTP hydrolysis. Consistent with this idea, we find that peptidyl transfer to tmRNA is relatively insensitive to the antibiotic kirromycin. Taken together, our studies provide a model for the initial stages of ribosomal rescue by tmRNA. PMID:24345396

  12. Clinical resistance to erythromycin and clindamycin in cutaneous propionibacteria isolated from acne patients is associated with mutations in 23S rRNA.

    PubMed Central

    Ross, J I; Eady, E A; Cove, J H; Jones, C E; Ratyal, A H; Miller, Y W; Vyakrnam, S; Cunliffe, W J

    1997-01-01

    The genetic basis of erythromycin resistance in cutaneous propionibacteria was determined by comparing the nucleotide sequences of the peptidyl transferase region in the 23S rRNAs from 9 susceptible and 26 resistant clinical isolates as well as 4 laboratory-selected erythromycin-resistant mutants of a susceptible strain. In 13 isolates and the 4 laboratory mutants, cross-resistance to macrolides, lincosamides, and B-type streptogramins was associated with an A-->G transition at a position cognate with Escherichia coli 23S rRNA base 2058. These strains were resistant to > or = 512 microg of erythromycin per ml. Two other mutations were identified, an A-->G transition at base 2059 in seven strains, associated with high-level resistance to all macrolides, and a G-->A transition at base 2057 in six strains, associated with low-level resistance to erythromycin. These mutations correspond to three of four phenotypic classes previously identified by using MIC determinations. PMID:9145890

  13. The ribosome profiling strategy for monitoring translation in vivo by deep sequencing of ribosome-protected mRNA fragments

    PubMed Central

    Ingolia, Nicholas T.; Brar, Gloria A.; Rouskin, Silvia; McGeachy, Anna M.; Weissman, Jonathan S.

    2012-01-01

    Recent studies highlight the importance of translational control in determining protein abundance, underscoring the value of measuring gene expression at the level of translation. We present a protocol for genome-wide, quantitative analysis of in vivo translation by deep sequencing. This ribosome profiling approach maps the exact positions of ribosomes on transcripts by nuclease footprinting. The nuclease-protected mRNA fragments are converted into a DNA library suitable for deep sequencing using a strategy that minimizes bias. The abundance of different footprint fragments in deep sequencing data reports on the amount of translation of a gene. Additionally, footprints reveal the exact regions of the transcriptome that are translated. To better define translated reading frames, we describe an adaptation that reveals the sites of translation initiation by pre-treating cells with harringtonine to immobilize initiating ribosomes. The protocol we describe requires 5–7 days to generate a completed ribosome profiling sequencing library. Sequencing and data analysis requires a further 4 – 5 days. PMID:22836135

  14. Dynamics of translation by single ribosomes through mRNA secondary structures

    PubMed Central

    Chen, Chunlai; Zhang, Haibo; Broitman, Steven L.; Reiche, Michael; Farrell, Ian; Cooperman, Barry S.; Goldman, Yale E.

    2013-01-01

    During protein synthesis, the ribosome translates nucleotide triplets in single-stranded mRNA into polypeptide sequences. Strong downstream mRNA secondary (2°) structures, which must be unfolded for translation, can slow or even halt protein synthesis. Here we employ single molecule fluorescence resonance energy transfer to determine reaction rates for specific steps within the elongation cycle as the Escherichia coli ribosome encounters stem loop or pseudoknot mRNA 2° structures. Downstream stem-loops containing 100% G-C base pairs decrease the rates of both tRNA translocation within the ribosome and deacylated tRNA dissociation from the ribosomal exit (E) site. Downstream stem-loops or pseudoknots containing both G-C and A-U pairs also decrease the rate of tRNA dissociation, but they have little effect on tRNA translocation rate. Thus, somewhat surprisingly, unfolding of mRNA 2° structures is more closely coupled to E-site tRNA dissociation than to tRNA translocation. PMID:23542154

  15. Dynamics of translation by single ribosomes through mRNA secondary structures.

    PubMed

    Chen, Chunlai; Zhang, Haibo; Broitman, Steven L; Reiche, Michael; Farrell, Ian; Cooperman, Barry S; Goldman, Yale E

    2013-05-01

    During protein synthesis, the ribosome translates nucleotide triplets in single-stranded mRNA into polypeptide sequences. Strong downstream mRNA secondary structures, which must be unfolded for translation, can slow or even halt protein synthesis. Here we used single-molecule fluorescence resonance energy transfer to determine reaction rates for specific steps within the elongation cycle as the Escherichia coli ribosome encounters stem-loop or pseudoknot mRNA secondary structures. Downstream stem-loops containing 100% GC base pairs decrease the rates of both tRNA translocation within the ribosome and deacylated tRNA dissociation from the ribosomal exit site (E site). Downstream stem-loops or pseudoknots containing both GC and AU pairs also decrease the rate of tRNA dissociation, but they have little effect on tRNA translocation rate. Thus, somewhat unexpectedly, unfolding of mRNA secondary structures is more closely coupled to E-site tRNA dissociation than to tRNA translocation.

  16. Translation by Ribosomes with mRNA Degradation: Exclusion Processes on Aging Tracks

    NASA Astrophysics Data System (ADS)

    Nagar, Apoorva; Valleriani, Angelo; Lipowsky, Reinhard

    2011-12-01

    We investigate the role of degradation of mRNA on protein synthesis using the totally asymmetric simple exclusion process (TASEP) as the underlying model for ribosome dynamics. mRNA degradation has a strong effect on the lifetime distribution of the mRNA, which in turn affects polysome statistics such as the number of ribosomes present on an mRNA strand of a given size. An average over mRNA of all ages is equivalent to an average over possible configurations of the corresponding TASEP—both before steady state and in steady state. To evaluate the relevant quantities for the translation problem, we first study the approach towards steady state of the TASEP, starting with an empty lattice representing an unloaded mRNA. When approaching the high density phase, the system shows two distinct phases with the entry and exit boundaries taking control of the density at their respective ends in the second phase. The approach towards the maximal current phase exhibits the surprising property that the ribosome entry flux can exceed the maximum possible steady state value. In all phases, the averaging over the mRNA age distribution shows a decrease in the average ribosome density profile as a function of distance from the entry boundary. For entry/exit parameters corresponding to the high density phase of TASEP, the average ribosome density profile also has a maximum near the exit end.

  17. A report of cat scratch disease in Korea confirmed by PCR amplification of the 16S-23S rRNA intergenic region of Bartonella henselae.

    PubMed

    Suh, Borum; Chun, Jin-Kyoung; Yong, Dongeun; Lee, Yang Soon; Jeong, Seok Hoon; Yang, Woo Ick; Kim, Dong Soo

    2010-02-01

    We report a case of cat scratch disease in an 8-yr-old girl who presented with fever and enlargement of both axillary lymph nodes. Both aerobic and anaerobic cultures of the lymph node aspirate were negative for microbial growth. Gram staining and Warthin-Starry silver staining did not reveal any organism. Purified DNA from the PCR-amplicon of the 16S-23S rRNA intergenic region was sequenced and showed 99.7% identity with the corresponding sequence of Bartonella henselae strain Houston-1. Our findings suggest that the internal transcribed spacer is a reliable region for PCR identification of Bartonella species. In patients with lymphadenitis, a history of contact with cats or dogs necessitates the use of diagnostic approaches that employ not only the conventional staining and culture but also molecular methods to detect B. henselae.

  18. Sequencing of the intergenic 16S-23S rRNA spacer (ITS) region of Mollicutes species and their identification using microarray-based assay and DNA sequencing.

    PubMed

    Volokhov, Dmitriy V; George, Joseph; Liu, Sue X; Ikonomi, Pranvera; Anderson, Christine; Chizhikov, Vladimir

    2006-08-01

    We have completed sequencing the 16S-23S rRNA intergenic transcribed spacer (ITS) region of most known Mycoplasma , Acholeplasma , Ureaplasma , Mesoplasma , and Spiroplasma species. Analysis of the sequence data revealed a significant interspecies variability and low intraspecies polymorphism of the ITS region among Mollicutes . This finding enabled the application of a combined polymerase chain reaction-microarray technology for identifying Mollicutes species. The microarray included individual species-specific oligonucleotide probes for characterizing human Mollicutes species and other species known to be common cell line contaminants. Evaluation of the microarray was conducted using multiple, previously characterized, Mollicutes species. The microarray analysis of the samples used demonstrated a highly specific assay, which is capable of rapid and accurate discrimination among Mollicutes species.

  19. The RNA-binding protein Gemin5 binds directly to the ribosome and regulates global translation

    PubMed Central

    Francisco-Velilla, Rosario; Fernandez-Chamorro, Javier; Ramajo, Jorge; Martinez-Salas, Encarnación

    2016-01-01

    RNA-binding proteins (RBPs) play crucial roles in all organisms. The protein Gemin5 harbors two functional domains. The N-terminal domain binds to snRNAs targeting them for snRNPs assembly, while the C-terminal domain binds to IRES elements through a non-canonical RNA-binding site. Here we report a comprehensive view of the Gemin5 interactome; most partners copurified with the N-terminal domain via RNA bridges. Notably, Gemin5 sediments with the subcellular ribosome fraction, and His-Gemin5 binds to ribosome particles via its N-terminal domain. The interaction with the ribosome was lost in F381A and Y474A Gemin5 mutants, but not in W14A and Y15A. Moreover, the ribosomal proteins L3 and L4 bind directly with Gemin5, and conversely, Gemin5 mutants impairing the binding to the ribosome are defective in the interaction with L3 and L4. The overall polysome profile was affected by Gemin5 depletion or overexpression, concomitant to an increase or a decrease, respectively, of global protein synthesis. Gemin5, and G5-Nter as well, were detected on the polysome fractions. These results reveal the ribosome-binding capacity of the N-ter moiety, enabling Gemin5 to control global protein synthesis. Our study uncovers a crosstalk between this protein and the ribosome, and provides support for the view that Gemin5 may control translation elongation. PMID:27507887

  20. Identification of the gene encoding the 5S ribosomal RNA maturase in Bacillus subtilis: mature 5S rRNA is dispensable for ribosome function.

    PubMed Central

    Condon, C; Brechemier-Baey, D; Beltchev, B; Grunberg-Manago, M; Putzer, H

    2001-01-01

    Over 25 years ago, Pace and coworkers described an activity called RNase M5 in Bacillus subtilis cell extracts responsible for 5S ribosomal RNA maturation (Sogin & Pace, Nature, 1974, 252:598-600). Here we show that RNase M5 is encoded by a gene of previously unknown function that is highly conserved among the low G + C gram-positive bacteria. We propose that the gene be named rnmV. The rnmV gene is nonessential. B. subtilis strains lacking RNase M5 do not make mature 5S rRNA, indicating that this process is not necessary for ribosome function. 5S rRNA precursors can, however, be found in both free and translating ribosomes. In contrast to RNase E, which cleaves the Escherichia coli 5S precursor in a single-stranded region, which is then trimmed to yield mature 5S RNA, RNase M5 cleaves the B. subtilis equivalent in a double-stranded region to yield mature 5S rRNA in one step. For the most part, eubacteria contain one or the other system for 5S rRNA production, with an imperfect division along gram-negative and gram-positive lines. A potential correlation between the presence of RNase E or RNase M5 and the single- or double-stranded nature of the predicted cleavage sites is explored. PMID:11233981

  1. Escherichia coli and Enterococcus spp. in rainwater tank samples: comparison of culture-based methods and 23S rRNA gene quantitative PCR assays.

    PubMed

    Ahmed, W; Richardson, K; Sidhu, J P S; Toze, S

    2012-10-16

    In this study, culture-based methods and quantitative PCR (qPCR) assays were compared with each other for the measurement of Escherichia coli and Enterococcus spp. in water samples collected from rainwater tanks in Southeast Queensland, Australia. Among the 50 rainwater tank samples tested, 26 (52%) and 46 (92%) samples yielded E. coli numbers as measured by EPA Method 1603 and E. coli 23S rRNA gene qPCR assay, respectively. Similarly, 49 (98%) and 47 (94%) samples yielded Enterococcus spp. numbers as measured by EPA Method 1600 and Enterococcus spp. 23S rRNA gene qPCR assay, respectively. The mean E. coli (2.49 ± 0.85) log(10) and Enterococcus spp. (2.72 ± 0.32) log(10) numbers as measured by qPCR assays were significantly (P < 0001) different than E. coli (0.91 ± 0.80) log(10) and Enterococcus spp. (1.86 ± 0.60) log(10) numbers as measured by culture-based method. Weak but significant correlations were observed between both EPA Method 1603 and the E. coli qPCR assay (r = 0.47, P = 0.0009), and EPA Method 1600 and the Enterococcus spp. qPCR assay (r = 0.42, P = 0.002). Good qualitative agreement was found between the culture-based method and the Enterococcus spp. qPCR assay in terms of detecting fecal pollution in water samples from the studied rainwater tanks. More research studies, however, are needed to shed some light on the discrepancies associated with the culture-based methods and qPCR assays for measuring fecal indicator bacteria.

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

    PubMed Central

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

    2009-01-01

    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. PACS: 87.10.Pq; 87.15.bk; 87.15.kj; 87.16.dj; 87.16.dr PMID:19029596

  3. Domain organization and crystal structure of the catalytic domain of E.coli RluF, a pseudouridine synthase that acts on 23S rRNA

    SciTech Connect

    Sunita,S.; Zhenxing, H.; Swaathi, J.; Cygler, M.; Matte, A.; Sivaraman, J.

    2006-01-01

    Pseudouridine synthases catalyze the isomerization of uridine to pseudouridine ({psi}) in rRNA and tRNA. The pseudouridine synthase RluF from Escherichia coli (E.C. 4.2.1.70) modifies U2604 in 23S rRNA, and belongs to a large family of pseudouridine synthases present in all kingdoms of life. Here we report the domain architecture and crystal structure of the catalytic domain of E. coli RluF at 2.6 Angstroms resolution. Limited proteolysis, mass spectrometry and N-terminal sequencing indicate that RluF has a distinct domain architecture, with the catalytic domain flanked at the N and C termini by additional domains connected to it by flexible linkers. The structure of the catalytic domain of RluF is similar to those of RsuA and TruB. RluF is a member of the RsuA sequence family of {psi}-synthases, along with RluB and RluE. Structural comparison of RluF with its closest structural homologues, RsuA and TruB, suggests possible functional roles for the N-terminal and C-terminal domains of RluF.

  4. Genetic modulation of RNA metabolism in Drosophilia. I. Increased rate of ribosomal RNA synthesis.

    PubMed

    Clark, S H; Strausbaugh, L D; Kiefer, B I

    1977-08-01

    It has been suggested that a particular Y chromosome which is rDNA-deficient (YbbSuVar-5) may be associated with an increased utilization of rDNA template in adult testes (Shermoen and Kiefer 1975). To extend the observations on this chromosome, experiments were designed to determine if the chromosome has an effect on rRNA synthesis in bobbed adults and on classic bobbed phenotypes (shortened and thinner scutellar bristles and delayed development). Specific activity measurements were made on rRNA extracted from adult males of the genotypes car bb/YbbSuVar-5, which are rDNA-deficient to the same extent, and from Samarkand+ isogenic (Sam+ iso), which is a wild-type stock. The resulting data demonstrated that the presence of the YbbSuVar-5 chromosome increases the rate of ribosomal RNA synthesis in adult flies. In addition, it was found that the presence of this particular Y chromosome restores wild-type bristle phenotype and development time. Appropriate genetic crosses indicate that the observed effects (increased rRNA synthesis, restoration of wild-type phenotype) are a function of this particular Y chromosome, and are not due to autosomal factors. The results of these experiments suggest that the rate of rRNA accumulation is under genetic control.

  5. Use of 16S Ribosomal RNA Sequences to Infer Relationships among Archaebacteria.

    DTIC Science & Technology

    1987-04-16

    FIELD GROUP SUB-GROUP Archaebacteria; Eubacteria ; Eukaryotes; 16S Ribosomal RNA; 08 I Phylogeny; rRNA; RNA Sequencing; Molecular Clock; Urkingdoms; r...16S rRNA data were used to infer the relat onships among the archaebacteria, and of the archaebacteria to the eubacteria and eukaryotes. ur programs for...been published (1, 2, 16, 18). The analyses render untenable the suggestions of Lake and colleagues (Lake et al., 1985) that the eubacteria derive from

  6. The complex of tmRNA-SmpB and EF-G on translocating ribosomes.

    PubMed

    Ramrath, David J F; Yamamoto, Hiroshi; Rother, Kristian; Wittek, Daniela; Pech, Markus; Mielke, Thorsten; Loerke, Justus; Scheerer, Patrick; Ivanov, Pavel; Teraoka, Yoshika; Shpanchenko, Olga; Nierhaus, Knud H; Spahn, Christian M T

    2012-05-06

    Bacterial ribosomes stalled at the 3' end of malfunctioning messenger RNAs can be rescued by transfer-messenger RNA (tmRNA)-mediated trans-translation. The SmpB protein forms a complex with the tmRNA, and the transfer-RNA-like domain (TLD) of the tmRNA then enters the A site of the ribosome. Subsequently, the TLD-SmpB module is translocated to the P site, a process that is facilitated by the elongation factor EF-G, and translation is switched to the mRNA-like domain (MLD) of the tmRNA. Accurate loading of the MLD into the mRNA path is an unusual initiation mechanism. Despite various snapshots of different ribosome-tmRNA complexes at low to intermediate resolution, it is unclear how the large, highly structured tmRNA is translocated and how the MLD is loaded. Here we present a cryo-electron microscopy reconstruction of a fusidic-acid-stalled ribosomal 70S-tmRNA-SmpB-EF-G complex (carrying both of the large ligands, that is, EF-G and tmRNA) at 8.3 Å resolution. This post-translocational intermediate (TI(POST)) presents the TLD-SmpB module in an intrasubunit ap/P hybrid site and a tRNA(fMet) in an intrasubunit pe/E hybrid site. Conformational changes in the ribosome and tmRNA occur in the intersubunit space and on the solvent side. The key underlying event is a unique extra-large swivel movement of the 30S head, which is crucial for both tmRNA-SmpB translocation and MLD loading, thereby coupling translocation to MLD loading. This mechanism exemplifies the versatile, dynamic nature of the ribosome, and it shows that the conformational modes of the ribosome that normally drive canonical translation can also be used in a modified form to facilitate more complex tasks in specialized non-canonical pathways.

  7. Reduced expression of the mouse ribosomal protein Rpl17 alters the diversity of mature ribosomes by enhancing production of shortened 5.8S rRNA

    PubMed Central

    Wang, Minshi; Parshin, Andrey V.; Shcherbik, Natalia; Pestov, Dimitri G.

    2015-01-01

    Processing of rRNA during ribosome assembly can proceed through alternative pathways but it is unclear whether this could affect the structure of the ribosome. Here, we demonstrate that shortage of a ribosomal protein can change pre-rRNA processing in a way that over time alters ribosome diversity in the cell. Reducing the amount of Rpl17 in mouse cells led to stalled 60S subunit maturation, causing degradation of most of the synthesized precursors. A fraction of pre-60S subunits, however, were able to complete maturation, but with a 5′-truncated 5.8S rRNA, which we named 5.8SC. The 5′ exoribonuclease Xrn2 is involved in the generation of both 5.8SC and the canonical long form of 5.8S rRNA. Ribosomes containing 5.8SC rRNA are present in various mouse and human cells and engage in translation. These findings uncover a previously undescribed form of mammalian 5.8S rRNA and demonstrate that perturbations in ribosome assembly can be a source of heterogeneity in mature ribosomes. PMID:25995445

  8. Reduced expression of the mouse ribosomal protein Rpl17 alters the diversity of mature ribosomes by enhancing production of shortened 5.8S rRNA.

    PubMed

    Wang, Minshi; Parshin, Andrey V; Shcherbik, Natalia; Pestov, Dimitri G

    2015-07-01

    Processing of rRNA during ribosome assembly can proceed through alternative pathways but it is unclear whether this could affect the structure of the ribosome. Here, we demonstrate that shortage of a ribosomal protein can change pre-rRNA processing in a way that over time alters ribosome diversity in the cell. Reducing the amount of Rpl17 in mouse cells led to stalled 60S subunit maturation, causing degradation of most of the synthesized precursors. A fraction of pre-60S subunits, however, were able to complete maturation, but with a 5'-truncated 5.8S rRNA, which we named 5.8SC. The 5' exoribonuclease Xrn2 is involved in the generation of both 5.8S(C) and the canonical long form of 5.8S rRNA. Ribosomes containing 5.8S(C) rRNA are present in various mouse and human cells and engage in translation. These findings uncover a previously undescribed form of mammalian 5.8S rRNA and demonstrate that perturbations in ribosome assembly can be a source of heterogeneity in mature ribosomes.

  9. Ribosome-associated Asc1/RACK1 is required for endonucleolytic cleavage induced by stalled ribosome at the 3' end of nonstop mRNA.

    PubMed

    Ikeuchi, Ken; Inada, Toshifumi

    2016-06-17

    Dom34-Hbs1 stimulates degradation of aberrant mRNAs lacking termination codons by dissociating ribosomes stalled at the 3' ends, and plays crucial roles in Nonstop Decay (NSD) and No-Go Decay (NGD). In the dom34Δ mutant, nonstop mRNA is degraded by sequential endonucleolytic cleavages induced by a stalled ribosome at the 3' end. Here, we report that ribosome-associated Asc1/RACK1 is required for the endonucleolytic cleavage of nonstop mRNA by stalled ribosome at the 3' end of mRNA in dom34Δ mutant cells. Asc1/RACK1 facilitates degradation of truncated GFP-Rz mRNA in the absence of Dom34 and exosome-dependent decay. Asc1/RACK1 is required for the sequential endonucleolytic cleavages by the stalled ribosome in the dom34Δ mutant, depending on its ribosome-binding activity. The levels of peptidyl-tRNA derived from nonstop mRNA were elevated in dom34Δasc1Δ mutant cells, and overproduction of nonstop mRNA inhibited growth of mutant cells. E3 ubiquitin ligase Ltn1 degrades the arrest products from truncated GFP-Rz mRNA in dom34Δ and dom34Δasc1Δ mutant cells, and Asc1/RACK1 represses the levels of substrates for Ltn1-dependent degradation. These indicate that ribosome-associated Asc1/RACK1 facilitates endonucleolytic cleavage of nonstop mRNA by stalled ribosomes and represses the levels of aberrant products even in the absence of Dom34. We propose that Asc1/RACK1 acts as a fail-safe in quality control for nonstop mRNA.

  10. Ribosome-associated Asc1/RACK1 is required for endonucleolytic cleavage induced by stalled ribosome at the 3′ end of nonstop mRNA

    PubMed Central

    Ikeuchi, Ken; Inada, Toshifumi

    2016-01-01

    Dom34-Hbs1 stimulates degradation of aberrant mRNAs lacking termination codons by dissociating ribosomes stalled at the 3′ ends, and plays crucial roles in Nonstop Decay (NSD) and No-Go Decay (NGD). In the dom34Δ mutant, nonstop mRNA is degraded by sequential endonucleolytic cleavages induced by a stalled ribosome at the 3′ end. Here, we report that ribosome-associated Asc1/RACK1 is required for the endonucleolytic cleavage of nonstop mRNA by stalled ribosome at the 3′ end of mRNA in dom34Δ mutant cells. Asc1/RACK1 facilitates degradation of truncated GFP-Rz mRNA in the absence of Dom34 and exosome-dependent decay. Asc1/RACK1 is required for the sequential endonucleolytic cleavages by the stalled ribosome in the dom34Δ mutant, depending on its ribosome-binding activity. The levels of peptidyl-tRNA derived from nonstop mRNA were elevated in dom34Δasc1Δ mutant cells, and overproduction of nonstop mRNA inhibited growth of mutant cells. E3 ubiquitin ligase Ltn1 degrades the arrest products from truncated GFP-Rz mRNA in dom34Δ and dom34Δasc1Δ mutant cells, and Asc1/RACK1 represses the levels of substrates for Ltn1-dependent degradation. These indicate that ribosome-associated Asc1/RACK1 facilitates endonucleolytic cleavage of nonstop mRNA by stalled ribosomes and represses the levels of aberrant products even in the absence of Dom34. We propose that Asc1/RACK1 acts as a fail-safe in quality control for nonstop mRNA. PMID:27312062

  11. Group II intron–ribosome association protects intron RNA from degradation

    PubMed Central

    Contreras, Lydia M.; Huang, Tao; Piazza, Carol Lyn; Smith, Dorie; Qu, Guosheng; Gelderman, Grant; Potratz, Jeffrey P.; Russell, Rick; Belfort, Marlene

    2013-01-01

    The influence of the cellular environment on the structures and properties of catalytic RNAs is not well understood, despite great interest in ribozyme function. Here we report on ribosome association of group II introns, which are ribozymes that are important because of their putative ancestry to spliceosomal introns and retrotransposons, their retromobility via an RNA intermediate, and their application as gene delivery agents. We show that group II intron RNA, in complex with the intron-encoded protein from the native Lactoccocus lactis host, associates strongly with ribosomes in vivo. Ribosomes have little effect on intron ribozyme activities; rather, the association with host ribosomes protects the intron RNA against degradation by RNase E, an enzyme previously shown to be a silencer of retromobility in Escherichia coli. The ribosome interacts strongly with the intron, exerting protective effects in vivo and in vitro, as demonstrated by genetic and biochemical experiments. These results are consistent with the ribosome influencing the integrity of catalytic RNAs in bacteria in the face of degradative nucleases that regulate intron mobility. PMID:24046482

  12. Group II intron-ribosome association protects intron RNA from degradation.

    PubMed

    Contreras, Lydia M; Huang, Tao; Piazza, Carol Lyn; Smith, Dorie; Qu, Guosheng; Gelderman, Grant; Potratz, Jeffrey P; Russell, Rick; Belfort, Marlene

    2013-11-01

    The influence of the cellular environment on the structures and properties of catalytic RNAs is not well understood, despite great interest in ribozyme function. Here we report on ribosome association of group II introns, which are ribozymes that are important because of their putative ancestry to spliceosomal introns and retrotransposons, their retromobility via an RNA intermediate, and their application as gene delivery agents. We show that group II intron RNA, in complex with the intron-encoded protein from the native Lactoccocus lactis host, associates strongly with ribosomes in vivo. Ribosomes have little effect on intron ribozyme activities; rather, the association with host ribosomes protects the intron RNA against degradation by RNase E, an enzyme previously shown to be a silencer of retromobility in Escherichia coli. The ribosome interacts strongly with the intron, exerting protective effects in vivo and in vitro, as demonstrated by genetic and biochemical experiments. These results are consistent with the ribosome influencing the integrity of catalytic RNAs in bacteria in the face of degradative nucleases that regulate intron mobility.

  13. Identification of virulence factors in 16S-23S rRNA intergenic spacer genotyped Staphylococcus aureus isolated from water buffaloes and small ruminants.

    PubMed

    Cremonesi, P; Zottola, T; Locatelli, C; Pollera, C; Castiglioni, B; Scaccabarozzi, L; Moroni, P

    2013-01-01

    Staphylococcus aureus is an important human and animal pathogen, and is regarded as an important cause of intramammary infection (IMI) in ruminants. Staphylococcus aureus genetic variability and virulence factors have been well studied in veterinary medicine, especially in cows as support for control and management of IMI. The aim of the present study was to genotype 71 Staph. aureus isolates from the bulk tank and foremilk of water buffaloes (n=40) and from udder tissue (n=7) and foremilk (n=24) from small ruminants. The method used was previously applied to bovine Staph. aureus and is based on the amplification of the 16S-23S rRNA intergenic spacer region. The technique applied was able to identify different Staph. aureus genotypes isolated from dairy species other than the bovine species, and cluster the genotypes according to species and herds. Virulence gene distribution was consistent with genotype differentiation. The isolates were also characterized through determination of the presence of 19 virulence-associated genes by specific PCR. Enterotoxins A, C, D, G, I, J, and L were associated with Staph. aureus isolates from buffaloes, whereas enterotoxins C and L were linked to small ruminants. Genes coding for methicillin resistance, Panton-Valentine leukocidin, exfoliative toxins A and B, and enterotoxins B, E, and H were undetected. These findings indicate that RNA template-specific PCR is a valid technique for typing Staph. aureus from buffaloes and small ruminants and is a useful tool for understanding udder infection epidemiology.

  14. Flipping of the ribosomal A-site adenines provides a basis for tRNA selection

    PubMed Central

    Zeng, Xiancheng; Chugh, Jeetender; Casiano-Negroni, Anette; Al-Hashimi, Hashim M.; Brooks, Charles L.

    2014-01-01

    Ribosomes control the missense error rate of ~10−4 during translation though quantitative contributions of individual mechanistic steps of the conformational changes yet to be fully determined. Biochemical and biophysical studies led to a qualitative tRNA selection model in which ribosomal A-site residues A1492 and A1493 (A1492/3) flip out in response to cognate tRNA binding, promoting the subsequent reactions, but not in the case of near cognate or non-cognate tRNA. However, this model was recently questioned by X-ray structures revealing conformations of extrahelical A1492/3 and domain closure of the decoding center in both cognate and near-cognate tRNA bound ribosome complexes, suggesting that the non-specific flipping of A1492/3 has no active role in tRNA selection. We explore this question by carrying out molecular dynamics (MD) simulations, aided with fluorescence and NMR experiments, to probe the free energy cost of extrahelical flipping of 1492/3 and the strain energy associated with domain conformational change. Our rigorous calculations demonstrate that the A1492/3 flipping is indeed a specific response to the binding of cognate tRNA, contributing 3 kcal/mol to the specificity of tRNA selection. Furthermore, the different A-minor interactions in cognate and near-cognate complexes propagate into the conformational strain and contribute another 4 kcal/mol in domain closure. The recent structure of ribosome with features of extrahelical A1492/3 and closed domain in near-cognate complex is reconciled by possible tautomerization of the wobble base pair in mRNA-tRNA. These results quantitatively rationalize other independent experimental observations and explain the ribosomal discrimination mechanism of selecting cognate versus near-cognate tRNA. PMID:24813122

  15. Initiation factor 2, tRNA, and 50S subunits cooperatively stabilize mRNAs on the ribosome during initiation

    PubMed Central

    Masuda, Tomoaki; Petrov, Alexey N.; Iizuka, Ryo; Funatsu, Takashi; Puglisi, Joseph D.; Uemura, Sotaro

    2012-01-01

    Initiation factor 2 (IF2) is a key factor in initiation of bacterial protein synthesis. It recruits initiator tRNA to the small ribosomal subunit and facilitates joining of the large ribosomal subunit. Using reconstituted translation system of Escherichia coli and optical tweezers, we directly measure the rupture force between single ribosomal complexes and mRNAs for initiation complexes in the presence and the absence of IF2. We demonstrate that IF2 together with codon recognition by initiator tRNA increases the force required to dislocate mRNA from the ribosome complexes; mRNA stabilization by IF2 required the presence of a joined 50S subunit, and was independent of bound guanine nucleotide. IF2 thus helps lock the 70S ribosome over the start codon during initiation, thus maintaining reading frame. Our results show how mRNA is progressively stabilized on the ribosome through distinct steps of initiation. PMID:22411833

  16. Piecemeal Buildup of the Genetic Code, Ribosomes, and Genomes from Primordial tRNA Building Blocks

    PubMed Central

    Caetano-Anollés, Derek; Caetano-Anollés, Gustavo

    2016-01-01

    The origin of biomolecular machinery likely centered around an ancient and central molecule capable of interacting with emergent macromolecular complexity. tRNA is the oldest and most central nucleic acid molecule of the cell. Its co-evolutionary interactions with aminoacyl-tRNA synthetase protein enzymes define the specificities of the genetic code and those with the ribosome their accurate biosynthetic interpretation. Phylogenetic approaches that focus on molecular structure allow reconstruction of evolutionary timelines that describe the history of RNA and protein structural domains. Here we review phylogenomic analyses that reconstruct the early history of the synthetase enzymes and the ribosome, their interactions with RNA, and the inception of amino acid charging and codon specificities in tRNA that are responsible for the genetic code. We also trace the age of domains and tRNA onto ancient tRNA homologies that were recently identified in rRNA. Our findings reveal a timeline of recruitment of tRNA building blocks for the formation of a functional ribosome, which holds both the biocatalytic functions of protein biosynthesis and the ability to store genetic memory in primordial RNA genomic templates. PMID:27918435

  17. Research Techniques Made Simple: Bacterial 16S Ribosomal RNA Gene Sequencing in Cutaneous Research.

    PubMed

    Jo, Jay-Hyun; Kennedy, Elizabeth A; Kong, Heidi H

    2016-03-01

    Skin serves as a protective barrier and also harbors numerous microorganisms collectively comprising the skin microbiome. As a result of recent advances in sequencing (next-generation sequencing), our understanding of microbial communities on skin has advanced substantially. In particular, the 16S ribosomal RNA gene sequencing technique has played an important role in efforts to identify the global communities of bacteria in healthy individuals and patients with various disorders in multiple topographical regions over the skin surface. Here, we describe basic principles, study design, and a workflow of 16S ribosomal RNA gene sequencing methodology, primarily for investigators who are not familiar with this approach. This article will also discuss some applications and challenges of 16S ribosomal RNA sequencing as well as directions for future development.

  18. Archaeal aminoacyl-tRNA synthetases interact with the ribosome to recycle tRNAs.

    PubMed

    Godinic-Mikulcic, Vlatka; Jaric, Jelena; Greber, Basil J; Franke, Vedran; Hodnik, Vesna; Anderluh, Gregor; Ban, Nenad; Weygand-Durasevic, Ivana

    2014-04-01

    Aminoacyl-tRNA synthetases (aaRS) are essential enzymes catalyzing the formation of aminoacyl-tRNAs, the immediate precursors for encoded peptides in ribosomal protein synthesis. Previous studies have suggested a link between tRNA aminoacylation and high-molecular-weight cellular complexes such as the cytoskeleton or ribosomes. However, the structural basis of these interactions and potential mechanistic implications are not well understood. To biochemically characterize these interactions we have used a system of two interacting archaeal aaRSs: an atypical methanogenic-type seryl-tRNA synthetase and an archaeal ArgRS. More specifically, we have shown by thermophoresis and surface plasmon resonance that these two aaRSs bind to the large ribosomal subunit with micromolar affinities. We have identified the L7/L12 stalk and the proteins located near the stalk base as the main sites for aaRS binding. Finally, we have performed a bioinformatics analysis of synonymous codons in the Methanothermobacter thermautotrophicus genome that supports a mechanism in which the deacylated tRNAs may be recharged by aaRSs bound to the ribosome and reused at the next occurrence of a codon encoding the same amino acid. These results suggest a mechanism of tRNA recycling in which aaRSs associate with the L7/L12 stalk region to recapture the tRNAs released from the preceding ribosome in polysomes.

  19. Determining RNA quality for NextGen sequencing: some exceptions to the gold standard rule of 23S to 16S rRNA ratio

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Using next-generation-sequencing technology to assess entire transcriptomes requires high quality starting RNA. Currently, RNA quality is routinely judged using automated microfluidic gel electrophoresis platforms and associated algorithms. Here we report that such automated methods generate false-n...

  20. RBF1, a Plant Homolog of the Bacterial Ribosome-Binding Factor RbfA, Acts in Processing of the Chloroplast 16S Ribosomal RNA1[W

    PubMed Central

    Fristedt, Rikard; Scharff, Lars B.; Clarke, Cornelia A.; Wang, Qin; Lin, Chentao; Merchant, Sabeeha S.; Bock, Ralph

    2014-01-01

    Plastids (chloroplasts) possess 70S ribosomes that are very similar in structure and function to the ribosomes of their bacterial ancestors. While most components of the bacterial ribosome (ribosomal RNAs [rRNAs] and ribosomal proteins) are well conserved in the plastid ribosome, little is known about the factors mediating the biogenesis of plastid ribosomes. Here, we have investigated a putative homolog of the bacterial RbfA (for ribosome-binding factor A) protein that was identified as a cold-shock protein and an auxiliary factor acting in the 5′ maturation of the 16S rRNA. The unicellular green alga Chlamydomonas reinhardtii and the vascular plant Arabidopsis (Arabidopsis thaliana) both encode a single RbfA-like protein in their nuclear genomes. By generating specific antibodies against this protein, we show that the plant RbfA-like protein functions exclusively in the plastid, where it is associated with thylakoid membranes. Analysis of mutants for the corresponding gene (termed RBF1) reveals that the gene function is essential for photoautotrophic growth. Weak mutant alleles display reduced levels of plastid ribosomes, a specific depletion in 30S ribosomal subunits, and reduced activity of plastid protein biosynthesis. Our data suggest that, while the function in ribosome maturation and 16S rRNA 5′ end processing is conserved, the RBF1 protein has assumed an additional role in 3′ end processing. Together with the apparent absence of a homologous protein from plant mitochondria, our findings illustrate that the assembly process of the 70S ribosome is not strictly conserved and has undergone some modifications during organelle evolution. PMID:24214533

  1. The influence of base identity and base pairing on the function of the alpha-sarcin loop of 23S rRNA.

    PubMed Central

    O'Connor, M; Dahlberg, A E

    1996-01-01

    The alpha-sarcin loop of large subunit rRNAs is one of the sites of interaction of elongation factors with the ribosome, and the target of the cytotoxins alpha-sarcin and ricin. Using a genetic selection for increased frameshifting in a reporter gene, we have isolated a C --> U mutation at position 2666 in the alpha-sarcin loop. In the NMR-derived structure of the loop, bases equivalent to 2666 and 2654 are paired via a non-canonical base pairing interaction. Each of the three base substitutions at C2666 and A2654 was constructed by site-directed mutagenesis of a plasmid borne copy of the rrnB operon of Escherichia coli. Only the C2666 --> U and A2654 --> G mutations that resulted in the formation of canonical A-U and C-G base pairs respectively, increased the levels of stop codon readthrough and frameshifting. The effects of different base pair combinations at positions 2666 and 2654 on ribosome function were then tested by constructing and analyzing all possible base combinations at these sites. All A --> G base substitution mutations at position 2654 and C --> U substitutions at position 2666 increased the levels of translational errors. However, these effects were greatest when G2654 and U2666 had the potential to engage in standard Watson-Crick base pairing interactions. These data indicate that base identity as well as base pairing interactions are important for the function of this essential component of the large subunit rRNA. PMID:8758999

  2. Reformation of functional liver polyribosomes from ribosome monomers in the absence of RNA synthesis.

    PubMed

    Stewart, G A; Farber, E

    1967-07-07

    The administration to rats of the ethyl analog of methionine, ethionine, results in the rapid decrease in the hepatic concentration of adenosine triphosphate followed by an extensive disaggregation of polysomes to ribosome monomers and a concomitant inhibition of protein synthesis. These effects are readily reversed by the injection of methionine or precursors of adenine nucleotides such as adenine. The reformation of liver polyribosomes in such animals following the administration of adenine plus methionine was found to occur under conditions in which new RNA synthesis was markedly inhibited. Free messenger RNA without attached ribosomes must be capable of remaining functionally active in the liver cytoplasm for many hours.

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

    PubMed

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

    2015-12-01

    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.

  4. 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

  5. Intrageneric structure of the genus Gluconobacter analyzed by the 16S rRNA gene and 16S-23S rRNA gene internal transcribed spacer sequences.

    PubMed

    Takahashi, Mai; Yukphan, Pattaraporn; Yamada, Yuzo; Suzuki, Ken-ichiro; Sakane, Takeshi; Nakagawa, Yasuyoshi

    2006-06-01

    Forty-nine strains belonging to the genus Gluconobacter were re-examined with respect to their species identification based on the sequences of the 16S rDNA and 16S-23S rDNA internal transcribed spacer regions (ITS). A phylogenetic tree constructed from the 16S rDNA sequences indicated the presence of five clusters corresponding, respectively, to the major five species of the genus Gluconobacter, namely G. albidus, G. cerinus, G. frateurii, G. oxydans (type species), and G. thailandicus. The type strain of G. asaii, NBRC 3276T (T=type strain) was included in the G. cerinus cluster, which is consistent with the report that G. asaii is a junior subjective synonym of G. cerinus. Existence of the G. albidus, G. cerinus, G. frateurii, G. oxydans, and G. thailandicus clusters was also recognized by the ITS sequence analysis. Both sequence analyses revealed that the G. cerinus and G. frateurii clusters were heterogeneous. The G. cerinus cluster comprised three strains of G. cerinus and one strain of G. frateurii, while the G. frateurii cluster included ten strains of G. frateurii, three of G. cerinus, and eleven of G. oxydans. These results suggest that phenotypic differences among Gluconobacter species are ambiguous and the species definition must be re-evaluated. The 16S rDNA and ITS sequences determined in this study are valuable for the identification and phylogenetic analysis of Gluconobacter species.

  6. Direct detection of Brucella spp. in raw milk by PCR and reverse hybridization with 16S-23S rRNA spacer probes.

    PubMed Central

    Rijpens, N P; Jannes, G; Van Asbroeck, M; Rossau, R; Herman, L M

    1996-01-01

    The 16S-23S rRNA spacer regions of Brucella abortus, B. melitensis, and B. suis were cloned and subcloned after PCR amplification. Sequence analysis of the inserts revealed a spacer of about 800 bp with very high ( > 99%) homology among the three species examined. Two genus-specific primer pairs, BRU-P5-BRU-P8 and BRU-P6-BRU-P7, that could be used in a nested PCR format and three genus-specific DNA probes, BRU-ICG2, BRU-ICG3, and BRU-ICG4, were deduced from this spacer. The specificity and sensitivity of both primer sets and probes were examined by testing them against a collection of 18 Brucella strains and 56 strains from other relevant taxa by using PCR and the Line Probe Assay (LiPA), respectively. A method for direct detection of Brucella spp. in 1 ml of raw milk was developed on the basis of enzymatic treatment of the milk components and subsequent PCR and LiPA hybridization. After a single PCR, sensitivities of 2.8 x 10(5) and 2.8 x 10(4) CFU/ml were obtained for detection by agarose gel electrophoresis and LiPA, respectively. Nested PCR yielded a sensitivity of 2.8 x 10(2) CFU/ml for both methods. PMID:8633866

  7. Simulating movement of tRNA through the ribosome during hybrid-state formation

    NASA Astrophysics Data System (ADS)

    Whitford, Paul C.; Sanbonmatsu, Karissa Y.

    2013-09-01

    Biomolecular simulations provide a means for exploring the relationship between flexibility, energetics, structure, and function. With the availability of atomic models from X-ray crystallography and cryoelectron microscopy (cryo-EM), and rapid increases in computing capacity, it is now possible to apply molecular dynamics (MD) simulations to large biomolecular machines, and systematically partition the factors that contribute to function. A large biomolecular complex for which atomic models are available is the ribosome. In the cell, the ribosome reads messenger RNA (mRNA) in order to synthesize proteins. During this essential process, the ribosome undergoes a wide range of conformational rearrangements. One of the most poorly understood transitions is translocation: the process by which transfer RNA (tRNA) molecules move between binding sites inside of the ribosome. The first step of translocation is the adoption of a "hybrid" configuration by the tRNAs, which is accompanied by large-scale rotations in the ribosomal subunits. To illuminate the relationship between these rearrangements, we apply MD simulations using a multi-basin structure-based (SMOG) model, together with targeted molecular dynamics protocols. From 120 simulated transitions, we demonstrate the viability of a particular route during P/E hybrid-state formation, where there is asynchronous movement along rotation and tRNA coordinates. These simulations not only suggest an ordering of events, but they highlight atomic interactions that may influence the kinetics of hybrid-state formation. From these simulations, we also identify steric features (H74 and surrounding residues) encountered during the hybrid transition, and observe that flexibility of the single-stranded 3'-CCA tail is essential for it to reach the endpoint. Together, these simulations provide a set of structural and energetic signatures that suggest strategies for modulating the physical-chemical properties of protein synthesis by the

  8. PTRF/Cavin-1 promotes efficient ribosomal RNA transcription in response to metabolic challenges

    PubMed Central

    Liu, Libin; Pilch, Paul F

    2016-01-01

    Ribosomal RNA transcription mediated by RNA polymerase I represents the rate-limiting step in ribosome biogenesis. In eukaryotic cells, nutrients and growth factors regulate ribosomal RNA transcription through various key factors coupled to cell growth. We show here in mature adipocytes, ribosomal transcription can be acutely regulated in response to metabolic challenges. This acute response is mediated by PTRF (polymerase I transcription and release factor, also known as cavin-1), which has previously been shown to play a critical role in caveolae formation. The caveolae–independent rDNA transcriptional role of PTRF not only explains the lipodystrophy phenotype observed in PTRF deficient mice and humans, but also highlights its crucial physiological role in maintaining adipocyte allostasis. Multiple post-translational modifications of PTRF provide mechanistic bases for its regulation. The role of PTRF in ribosomal transcriptional efficiency is likely relevant to many additional physiological situations of cell growth and organismal metabolism. DOI: http://dx.doi.org/10.7554/eLife.17508.001 PMID:27528195

  9. Computational and Experimental Characterization of Ribosomal DNA and RNA G-Quadruplexes

    NASA Astrophysics Data System (ADS)

    Cho, Samuel

    DNA G-quadruplexes in human telomeres and gene promoters are being extensively studied for their role in controlling the growth of cancer cells. Recent studies strongly suggest that guanine (G)-rich genes encoding pre-ribosomal RNA (pre-rRNA) are a potential anticancer target through the inhibition of RNA polymerase I (Pol I) in ribosome biogenesis. However, the structures of ribosomal G-quadruplexes at atomic resolution are unknown, and very little biophysical characterization has been performed on them to date. Here, we have modeled two putative rDNA G-quadruplex structures, NUC 19P and NUC 23P, which we observe via circular dichroism (CD) spectroscopy to adopt a predominantly parallel topology, and their counterpart rRNA. To validate and refine the putative ribosomal G-quadruplex structures, we performed all-atom molecular dynamics (MD) simulations using the CHARMM36 force field in the presence and absence of stabilizing K + or Na + ions. We optimized the CHARMM36 force field K + parameters to be more consistent with quantum mechanical calculations (and the polarizable Drude model force field) so that the K + ion is predominantly in the G-quadruplex channel. Our MD simulations show that the rDNA G-quadruplex have more well-defined, predominantly parallel-topology structures than rRNA and NUC 19P is more structured than NUC 23P, which features extended loops. Our study demonstrates that they are both potential targets for the design of novel chemotherapeutics.

  10. Protein synthesis factors (RF1, RF2, RF3, RRF, and tmRNA) and peptidyl-tRNA hydrolase rescue stalled ribosomes at sense codons.

    PubMed

    Vivanco-Domínguez, Serafín; Bueno-Martínez, José; León-Avila, Gloria; Iwakura, Nobuhiro; Kaji, Akira; Kaji, Hideko; Guarneros, Gabriel

    2012-04-13

    During translation, ribosomes stall on mRNA when the aminoacyl-tRNA to be read is not readily available. The stalled ribosomes are deleterious to the cell and should be rescued to maintain its viability. To investigate the contribution of some of the cellular translation factors on ribosome rescuing, we provoked stalling at AGA codons in mutants that affected the factors and then analyzed the accumulation of oligopeptidyl (peptides of up to 6 amino acid residues, oligopep-)-tRNA or polypeptidyl (peptides of more than 300 amino acids in length, polypep-)-tRNA associated with ribosomes. Stalling was achieved by starvation for aminoacyl-tRNA(Arg4) upon induced expression of engineered lacZ (β-galactosidase) reporter gene harboring contiguous AGA codons close to the initiation codon or at internal codon positions together with minigene ATGAGATAA accompanied by reduced peptidyl-tRNA hydrolase (Pth). Our results showed accumulations of peptidyl-tRNA associated with ribosomes in mutants for release factors (RF1, RF2, and RF3), ribosome recycling factor (RRF), Pth, and transfer-messenger RNA (tmRNA), implying that each of these factors cooperate in rescuing stalled ribosomes. The role of these factors in ribosome releasing from the stalled complex may vary depending on the length of the peptide in the peptidyl-tRNA. RF3 and RRF rescue stalled ribosomes by "drop-off" of peptidyl-tRNA, while RF1, RF2 (in the absence of termination codon), or Pth may rescue by hydrolyzing the associated peptidyl-tRNA. This is followed by the disassembly of the ribosomal complex of tRNA and mRNA by RRF and elongation factor G.

  11. High-Resolution Analysis of Coronavirus Gene Expression by RNA Sequencing and Ribosome Profiling

    PubMed Central

    Jones, Joshua D.; Chung, Betty Y.-W.; Siddell, Stuart G.; Brierley, Ian

    2016-01-01

    Members of the family Coronaviridae have the largest genomes of all RNA viruses, typically in the region of 30 kilobases. Several coronaviruses, such as Severe acute respiratory syndrome-related coronavirus (SARS-CoV) and Middle East respiratory syndrome-related coronavirus (MERS-CoV), are of medical importance, with high mortality rates and, in the case of SARS-CoV, significant pandemic potential. Other coronaviruses, such as Porcine epidemic diarrhea virus and Avian coronavirus, are important livestock pathogens. Ribosome profiling is a technique which exploits the capacity of the translating ribosome to protect around 30 nucleotides of mRNA from ribonuclease digestion. Ribosome-protected mRNA fragments are purified, subjected to deep sequencing and mapped back to the transcriptome to give a global “snap-shot” of translation. Parallel RNA sequencing allows normalization by transcript abundance. Here we apply ribosome profiling to cells infected with Murine coronavirus, mouse hepatitis virus, strain A59 (MHV-A59), a model coronavirus in the same genus as SARS-CoV and MERS-CoV. The data obtained allowed us to study the kinetics of virus transcription and translation with exquisite precision. We studied the timecourse of positive and negative-sense genomic and subgenomic viral RNA production and the relative translation efficiencies of the different virus ORFs. Virus mRNAs were not found to be translated more efficiently than host mRNAs; rather, virus translation dominates host translation at later time points due to high levels of virus transcripts. Triplet phasing of the profiling data allowed precise determination of translated reading frames and revealed several translated short open reading frames upstream of, or embedded within, known virus protein-coding regions. Ribosome pause sites were identified in the virus replicase polyprotein pp1a ORF and investigated experimentally. Contrary to expectations, ribosomes were not found to pause at the ribosomal

  12. Is wheat mitochondrial 5S ribosomal RNA prokaryotic in nature?

    PubMed Central

    Gray, M W; Spencer, D F

    1981-01-01

    Küntzel et al. (1981) (Nucleic Acids Res. 9, 1451-1461) recently concluded that the sequence of wheat mitochondrial 5S rRNA is significantly more related to prokaryotic than to eukaryotic 5S rRNA sequences, and displays an especially high affinity to that of the thermophilic Gram-negative bacterium, Thermus aquaticus. However, the sequence on which this conclusion was based, although attributed to us, differs in several places from the one determined by us. We show here that the correct sequence (Spencer, D.F., Bonen, L. and Gray, M.W. (1981) Biochemistry, in press) does not support the conclusions of Küntzel et al. about potential secondary structure in wheat mitochondrial 5S rRNA and its phylogenetic significance. We further show that when the wheat mitochondrial 5S rRNA sequence is matched against published alignments for E. coli, T. aquaticus, and wheat cytosol 5S rRNAs, the mitochondrial sequence shows no greater homology to the T. aquaticus sequence than to the E. coli sequence, and only slightly more homology to these two sequences than to wheat cytosol 5S rRNA. This analysis confirms our original view (Biochemistry, in press) that wheat mitochondrial 5S rRNA is neither obviously prokaryotic nor eukaryotic in nature, but shows characteristics of both classes of 5S rRNA, as well as some unique features. PMID:7024917

  13. The Ribosome Shape Directs mRNA Translocation through Entrance and Exit Dynamics

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The protein-synthesizing ribosome undergoes large motions to effect the translocation of tRNAs (transfer ribonucleic acids) and mRNA (messenger ribonucleic acid); here the domain motions of this system are explored with a coarse-grained elastic network model using normal mode analysis. Crystal struc...

  14. Role of messenger RNA-ribosome complex in complementary DNA display.

    PubMed

    Naimuddin, Mohammed; Ohtsuka, Isao; Kitamura, Koichiro; Kudou, Motonori; Kimura, Shinnosuke

    2013-07-15

    In vitro display technologies such as ribosome display and messenger RNA (mRNA)/complementary DNA (cDNA) display are powerful methods that can generate library diversities on the order of 10(10-14). However, in mRNA and cDNA display methods, the end use diversity is two orders of magnitude lower than initial diversity and is dependent on the downstream processes that act as limiting factors. We found that in our previous cDNA display protocol, the purification of protein fusions by the use of streptavidin matrices from cell-free translation mixtures had poor efficiency (∼10-15%) that seriously affected the diversity of the purified library. Here, we have investigated and optimized the protocols that provided remarkable purification efficiencies. The stalled ribosome in the mRNA-ribosome complex was found to impede this purification efficiency. Among the various conditions tested, destabilization of ribosomes by appropriate concentration of metal chelating agents in combination with an optimal temperature of 30°C were found to be crucial and effective for nearly complete isolation of protein fusions from the cell-free translation system. Thus, this protocol provided 8- to 10-fold increased efficiency of purification over the previous method and results in retaining the diversity of the library by approximately an order of magnitude-important for directed evolution. We also discuss the possible effects in the fabrication of protein chips.

  15. Yeast ribosomal protein L10 helps coordinate tRNA movement through the large subunit

    PubMed Central

    Petrov, Alexey N.; Meskauskas, Arturas; Roshwalb, Sara C.; Dinman, Jonathan D.

    2008-01-01

    Yeast ribosomal protein L10 (E. coli L16) is located at the center of a topological nexus that connects many functional regions of the large subunit. This essential protein has previously been implicated in processes as diverse as ribosome biogenesis, translational fidelity and mRNA stability. Here, the inability to maintain the yeast Killer virus was used as a proxy for large subunit defects to identify a series of L10 mutants. These mapped to roughly four discrete regions of the protein. A detailed analysis of mutants located in the N-terminal ‘hook’ of L10, which inserts into the bulge of 25S rRNA helix 89, revealed strong effects on rRNA structure corresponding to the entire path taken by the tRNA 3′ end as it moves through the large subunit during the elongation cycle. The mutant-induced structural changes are wide-ranging, affecting ribosome biogenesis, elongation factor binding, drug resistance/hypersensitivity, translational fidelity and virus maintenance. The importance of L10 as a potential transducer of information through the ribosome, and of a possible role of its N-terminal domain in switching between the pre- and post-translocational states are discussed. PMID:18824477

  16. Identification and sequence of the initiation site for rat 45S ribosomal RNA synthesis.

    PubMed Central

    Harrington, C A; Chikaraishi, D M

    1983-01-01

    The transcription initiation site for rat 45S precursor ribosomal RNA synthesis was determined by nuclease protection mapping with two single-strand endonucleases. S1 and mung bean, and one single-strand exonuclease, ExoVII. These experiments were performed with end-labeled ribosomal DNA from double-stranded pBR322 recombinants and from single-stranded M13 recombinants. Results from experiments using both kinds of DNA and all three enzymes showed that the 5' end of 45S RNA mapped to a unique site 125 bases upstream from the Hind III site in the ribosomal DNA gene. The DNA surrounding this site (designated +1) was sequenced from -281 to +641. The entire sequence of this region shows extensive homology to the comparable region of mouse. This includes three stretches of T residues in the non-coding strand between +300 and +630. Two sets of direct repeats adjacent to these T-rich regions are observed. Comparison of the mouse and human ribosomal DNA transcription initiation sites with the rat sequence reported in this paper demonstrates a conserved sequence at +2 to +16, CTGACACGCTGTCCT. This suggests that this region may be important for the initiation of transcription on mammalian ribosomal DNAs. Images PMID:6304628

  17. Fluctuations in protein synthesis from a single RNA template: Stochastic kinetics of ribosomes

    NASA Astrophysics Data System (ADS)

    Garai, Ashok; Chowdhury, Debashish; Ramakrishnan, T. V.

    2009-01-01

    Proteins are polymerized by cyclic machines called ribosomes, which use their messenger RNA (mRNA) track also as the corresponding template, and the process is called translation. We explore, in depth and detail, the stochastic nature of the translation. We compute various distributions associated with the translation process; one of them—namely, the dwell time distribution—has been measured in recent single-ribosome experiments. The form of the distribution, which fits best with our simulation data, is consistent with that extracted from the experimental data. For our computations, we use a model that captures both the mechanochemistry of each individual ribosome and their steric interactions. We also demonstrate the effects of the sequence inhomogeneities of real genes on the fluctuations and noise in translation. Finally, inspired by recent advances in the experimental techniques of manipulating single ribosomes, we make theoretical predictions on the force-velocity relation for individual ribosomes. In principle, all our predictions can be tested by carrying out in vitro experiments.

  18. Identification and structural analysis of a ribosomal RNA gene promoter from Thiobacillus ferrooxidans.

    PubMed

    Takamiya, M; Salazar, O; Vargas, D; Jedlicki, E; Orellana, O

    1990-10-15

    The 5'-terminus of a rRNA operon (rrnT2) from Thiobacillus ferrooxidans was characterized. The rRNA promoters from this microorganism were identified by means of a functional assay in Escherichia coli. DNA sequencing of the promoter region, upstream the 16 S rRNA gene, showed the presence of a consensus sequence for bacterial ribosomal promoters. Other features such as a 'discriminator' sequence, antiterminator elements and an upstream hexanucleotide common to several rRNA operons were also found. Two other putative transcription promoters were also identified.

  19. Cloning and determination of the transcription termination site of ribosomal RNA gene of the mouse.

    PubMed Central

    Kominami, R; Mishima, Y; Urano, Y; Sakai, M; Muramatsu, M

    1982-01-01

    A Eco RI 6.6 kb DNA fragment containing the 3'-end of 28S ribosomal RNA gene of the mouse was detected by Southern blot hybridization, and cloned in a lambda-phage vector. The site of transcription termination and the processed 3'-end of 28S RNA were determined on the cloned fragment and the surrounding nucleotide sequence determined. The 3'-terminal nucleotides of mouse 28S RNA are similar to those of yeast, Drosophila and Xenopus although the homology was lost drastically beyond the 3'-end of 28S RNA. 45S precursor RNA terminated at 30 nucleotides downstream from the 3'-end of 28S RNA gene. A structure of a dyad symmetry with a loop was found immediately prior to the termination site of 45S RNA. The rDNA termination site thus shares some common features with termination sites recognized by other RNA polymerases. Images PMID:6281727

  20. Can we estimate bacterial growth rates from ribosomal RNA content?

    SciTech Connect

    Kemp, P.F.

    1995-12-31

    Several studies have demonstrated a strong relationship between the quantity of RNA in bacterial cells and their growth rate under laboratory conditions. It may be possible to use this relationship to provide information on the activity of natural bacterial communities, and in particular on growth rate. However, if this approach is to provide reliably interpretable information, the relationship between RNA content and growth rate must be well-understood. In particular, a requisite of such applications is that the relationship must be universal among bacteria, or alternately that the relationship can be determined and measured for specific bacterial taxa. The RNA-growth rate relationship has not been used to evaluate bacterial growth in field studies, although RNA content has been measured in single cells and in bulk extracts of field samples taken from coastal environments. These measurements have been treated as probable indicators of bacterial activity, but have not yet been interpreted as estimators of growth rate. The primary obstacle to such interpretations is a lack of information on biological and environmental factors that affect the RNA-growth rate relationship. In this paper, the available data on the RNA-growth rate relationship in bacteria will be reviewed, including hypotheses regarding the regulation of RNA synthesis and degradation as a function of growth rate and environmental factors; i.e. the basic mechanisms for maintaining RNA content in proportion to growth rate. An assessment of the published laboratory and field data, the current status of this research area, and some of the remaining questions will be presented.

  1. Characterization of recombinant bacteriophages containing mosquito ribosomal RNA genes

    SciTech Connect

    Park, Y.J.

    1988-01-01

    A family of nine recombinant bacteriophages containing rRNA genes from cultured cells of the mosquito, Aedes albopictus, has been isolated by screening two different genomic DNA libraries - Charon 30 and EMBL 3 using {sup 32}P-labeled 18S and 28S rRNA as probes. These nine recombinant bacteriophages were characterized by restriction mapping, Southern blotting, and S1 nuclease analysis. The 18S rRNA coding region contains an evolutionarily conserved EcoRI site near the 3{prime}-end, and measures 1800 bp. The 28S rRNA genes were divided into {alpha} and {beta} coding regions measuring 1750 bp and 2000 bp, respectively. The gap between these two regions measures about 340 bp. No insertion sequences were found in the rRNA coding regions. The entire rDNA repeat unit had a minimum length of 15.6 kb, including a nontranscribed spacer region. The non-transcribed spacer region of cloned A. albopictus rDNA contained a common series of seven PvuI sites within a 1250 bp region upstream of the 18S rRNA coding region, and a proportion of this region also showed heterogeneity both in the length and in the restriction sites.

  2. The CRM domain: an RNA binding module derived from an ancient ribosome-associated protein.

    PubMed

    Barkan, Alice; Klipcan, Larik; Ostersetzer, Oren; Kawamura, Tetsuya; Asakura, Yukari; Watkins, Kenneth P

    2007-01-01

    The CRS1-YhbY domain (also called the CRM domain) is represented as a stand-alone protein in Archaea and Bacteria, and in a family of single- and multidomain proteins in plants. The function of this domain is unknown, but structural data and the presence of the domain in several proteins known to interact with RNA have led to the proposal that it binds RNA. Here we describe a phylogenetic analysis of the domain, its incorporation into diverse proteins in plants, and biochemical properties of a prokaryotic and eukaryotic representative of the domain family. We show that a bacterial member of the family, Escherichia coli YhbY, is associated with pre-50S ribosomal subunits, suggesting that YhbY functions in ribosome assembly. GFP fused to a single-domain CRM protein from maize localizes to the nucleolus, suggesting that an analogous activity may have been retained in plants. We show further that an isolated maize CRM domain has RNA binding activity in vitro, and that a small motif shared with KH RNA binding domains, a conserved "GxxG" loop, contributes to its RNA binding activity. These and other results suggest that the CRM domain evolved in the context of ribosome function prior to the divergence of Archaea and Bacteria, that this function has been maintained in extant prokaryotes, and that the domain was recruited to serve as an RNA binding module during the evolution of plant genomes.

  3. Relationships between 16S-23S rRNA gene internal transcribed spacer DNA and genomic DNA similarities in the taxonomy of phototrophic bacteria

    NASA Astrophysics Data System (ADS)

    Okamura, K.; Hisada, T.; Takata, K.; Hiraishi, A.

    2013-04-01

    Rapid and accurate identification of microbial species is essential task in microbiology and biotechnology. In prokaryotic systematics, genomic DNA-DNA hybridization is the ultimate tool to determine genetic relationships among bacterial strains at the species level. However, a practical problem in this assay is that the experimental procedure is laborious and time-consuming. In recent years, information on the 16S-23S rRNA gene internal transcribed spacer (ITS) region has been used to classify bacterial strains at the species and intraspecies levels. It is unclear how much information on the ITS region can reflect the genome that contain it. In this study, therefore, we evaluate the quantitative relationship between ITS DNA and entire genomic DNA similarities. For this, we determined ITS sequences of several species of anoxygenic phototrophic bacteria belonging to the order Rhizobiales, and compared with DNA-DNA relatedness among these species. There was a high correlation between the two genetic markers. Based on the regression analysis of this relationship, 70% DNA-DNA relatedness corresponded to 92% ITS sequence similarity. This suggests the usefulness of the ITS sequence similarity as a criterion for determining the genospecies of the phototrophic bacteria. To avoid the effects of polymorphism bias of ITS on similarities, PCR products from all loci of ITS were used directly as genetic probes for comparison. The results of ITS DNA-DNA hybridization coincided well with those of genomic DNA-DNA relatedness. These collective data indicate that the whole ITS DNA-DNA similarity can be used as an alternative to genomic DNA-DNA similarity.

  4. Transcription of a yeast ribosomal RNA minigene in Saccharomyces cerevisiae.

    PubMed Central

    Quincey, R V; Arnold, R E

    1984-01-01

    A transcription system using intact yeast has been developed for investigating which sequences are implicated in the initiation of transcription of yeast rRNA genes. The system employs an rRNA minigene that consists of the initiation and termination sites for rRNA biosynthesis separated by approx. 700 base pairs of vector DNA in the Escherichia coli-yeast shuttle vector, pJDB207. Two recombinants containing this minigene were constructed; one retained all of the nontranscribed spacer DNA upstream from the initiation site, the other retained 208 base pairs of this DNA. Transcripts of this structurally unique minigene in RNA from yeast transformed with these recombinants were readily detected by nuclease S1 mapping. These transcripts were initiated at the site used by the host rRNA genes, were approx. 3-fold more abundant in the recombinant retaining all of the nontranscribed spacer and were less abundant when the yeast was not growing. Images Fig. 4. Fig. 5. Fig. 6. PMID:6097222

  5. Secondary structure features of ribosomal RNA species within intact ribosomal subunits and efficiency of RNA-protein interactions in thermoacidophilic (Caldariella acidophila, Bacillus acidocaldarius) and mesophilic (Escherichia coli) bacteria.

    PubMed

    Cammarano, P; Mazzei, F; Londei, P; Teichner, A; de Rosa, M; Gambacorta, A

    1983-08-02

    Ribosomal subunits of Caldariella acidophila (max.growth temp., 90 degrees C) have been compared to subunits of Bacillus acidocaldarius (max. growth temp., 70 degrees C) and Escherichia coli (max. growth temp., 47 degrees C) with respect to (a) bihelical content of rRNA; (b) G . C content of bihelical domains and (c) tightness of rRNA-protein interactions. The principal results are as follows. Subunits of C. acidophilia ribosomes (Tm = 90-93 degrees C) exhibit considerable thermal tolerance over their B. acidocaldarius (Tm = 77 degrees C) and E. coli counterparts (Tm = 72 degrees C). Based on the "melting' hyperchromicities of the intact ribosomal subunits a 51-55% fraction of the nucleotides appears to participate in hydrogen-bonded base pairing regardless of ribosome source, whereas a larger fraction, 67-70%, appears to be involved in hydrogen bonding in the naked rRNA species. The G . C content of bihelical domains of both free and ribosome-bound rRNA increases with increasing thermophily; based on hyperchromicity dispersion spectra of intact subunits and free rRNA, the bihelical parts of C. acidophila rRNA are estimated to contain 63-64% G . C, compared to 58.5% G . C for B. acidocaldarius and 55% G . C for E. coli. The increment of ribosome Tm values with increasing thermophily is greater than the increase in Tm for the free rRNA, indicating that within ribosomes bihelical domains of the thermophile rRNA species are stabilized more efficiently than their mesophile counterparts by proteins or/ and other component(s). The efficiency of the rRNA-protein interactions in the mesophile and thermophile ribosomes has been probed by comparing the releases, with LiCl-urea, of the rRNA species from the corresponding ribosomal subunits stuck to a Celite column through their protein moiety; it has been established that the release of C. acidophila rRNA from the Celite-bound ribosomes occurs at salt-urea concentrations about 4-fold higher than those required to release rRNA

  6. Validation of two ribosomal RNA removal methods for microbial metatranscriptomics

    SciTech Connect

    He, Shaomei; Wurtzel, Omri; Singh, Kanwar; Froula, Jeff L; Yilmaz, Suzan; Tringe, Susannah G; Wang, Zhong; Chen, Feng; Lindquist, Erika A; Sorek, Rotem; Hugenholtz, Philip

    2010-10-01

    The predominance of rRNAs in the transcriptome is a major technical challenge in sequence-based analysis of cDNAs from microbial isolates and communities. Several approaches have been applied to deplete rRNAs from (meta)transcriptomes, but no systematic investigation of potential biases introduced by any of these approaches has been reported. Here we validated the effectiveness and fidelity of the two most commonly used approaches, subtractive hybridization and exonuclease digestion, as well as combinations of these treatments, on two synthetic five-microorganism metatranscriptomes using massively parallel sequencing. We found that the effectiveness of rRNA removal was a function of community composition and RNA integrity for these treatments. Subtractive hybridization alone introduced the least bias in relative transcript abundance, whereas exonuclease and in particular combined treatments greatly compromised mRNA abundance fidelity. Illumina sequencing itself also can compromise quantitative data analysis by introducing a G+C bias between runs.

  7. RNA editing in six mitochondrial ribosomal protein genes of Didymium iridis.

    PubMed

    Hendrickson, Peter G; Silliker, Margaret E

    2010-06-01

    Similarity searches with Didymium iridis mitochondrial genomic DNA identified six possible ribosomal protein-coding regions, however, each region contained stop codons that would need to be removed by RNA editing to produce functional transcripts. RT-PCR was used to amplify these regions from total RNA for cloning and sequencing. Six functional transcripts were verified for the following ribosomal protein genes: rpS12, rpS7, rpL2, rpS19, rpS3, and rpL16. The editing events observed, such as single C and U nucleotide insertions and a dinucleotide insertion, were consistent with previously observed editing patterns seen in D. iridis. Additionally, a new form of insertional editing, a single A insertion, was observed in a conserved region of the rpL16 gene. While the majority of codons created by editing specify hydrophobic amino acids, a greater proportion of the codons created in these hydrophilic ribosomal proteins called for positively charged amino acids in comparison to the previously characterized hydrophobic respiratory protein genes. This first report of edited soluble mitochondrial ribosomal proteins in myxomycetes expands upon the RNA editing patterns previously seen; there was: a greater proportion of created codons specifying positively charged amino acids, a shift in the codon position edited, and the insertion of single A nucleotides.

  8. TIF-IA: An oncogenic target of pre-ribosomal RNA synthesis.

    PubMed

    Jin, Rui; Zhou, Wei

    2016-12-01

    Cancer cells devote the majority of their energy consumption to ribosome biogenesis, and pre-ribosomal RNA transcription accounts for 30-50% of all transcriptional activity. This aberrantly elevated biological activity is an attractive target for cancer therapeutic intervention if approaches can be developed to circumvent the development of side effects in normal cells. TIF-IA is a transcription factor that connects RNA polymerase I with the UBF/SL-1 complex to initiate the transcription of pre-ribosomal RNA. Its function is conserved in eukaryotes from yeast to mammals, and its activity is promoted by the phosphorylation of various oncogenic kinases in cancer cells. The depletion of TIF-IA induces cell death in lung cancer cells and mouse embryonic fibroblasts but not in several other normal tissue types evaluated in knock-out studies. Furthermore, the nuclear accumulation of TIF-IA under UTP down-regulated conditions requires the activity of LKB1 kinase, and LKB1-inactivated cancer cells are susceptible to cell death under such stress conditions. Therefore, TIF-IA may be a unique target to suppress ribosome biogenesis without significantly impacting the survival of normal tissues.

  9. Structure of the Ribosomal RNA Decoding Site Containing a Selenium-Modified Responsive Fluorescent Ribonucleoside Probe.

    PubMed

    Nuthanakanti, Ashok; Boerneke, Mark A; Hermann, Thomas; Srivatsan, Seergazhi G

    2017-03-01

    Comprehensive understanding of the structure-function relationship of RNA both in real time and at atomic level will have a profound impact in advancing our understanding of RNA functions in biology. Here, we describe the first example of a multifunctional nucleoside probe, containing a conformation-sensitive fluorophore and an anomalous X-ray diffraction label (5-selenophene uracil), which enables the correlation of RNA conformation and recognition under equilibrium and in 3D. The probe incorporated into the bacterial ribosomal RNA decoding site, fluorescently reports antibiotic binding and provides diffraction information in determining the structure without distorting native RNA fold. Further, by comparing solution binding data and crystal structure, we gained insight on how the probe senses ligand-induced conformational change in RNA. Taken together, our nucleoside probe represents a new class of biophysical tool that would complement available tools for functional RNA investigations.

  10. Functional conformations of the L11–ribosomal RNA complex revealed by correlative analysis of cryo-EM and molecular dynamics simulations

    PubMed Central

    Li, Wen; Sengupta, Jayati; Rath, Bimal K.; Frank, Joachim

    2006-01-01

    The interaction between the GTPase-associated center (GAC) and the aminoacyl-tRNA·EF-Tu·GTP ternary complex is of crucial importance in the dynamic process of decoding and tRNA accommodation. The GAC includes protein L11 and helices 43–44 of 23S rRNA (referred to as L11–rRNA complex). In this study, a method of fitting based on a systematic comparison between cryo-electron microscopy (cryo-EM) density maps and structures obtained by molecular dynamics simulations has been developed. This method has led to the finding of atomic models of the GAC that fit the EM maps with much improved cross-correlation coefficients compared with the fitting of the X-ray structure. Two types of conformations of the L11–rRNA complex, produced by the simulations, match the cryo-EM maps representing the states either bound or unbound to the aa-tRNA·EF-Tu·GTP ternary complex. In the bound state, the N-terminal domain of L11 is extended from its position in the crystal structure, and the base of nucleotide A1067 in the 23S ribosomal RNA is flipped out. This position of the base allows the RNA to reach the elbow region of the aminoacyl-tRNA when the latter is bound in the A/T site. In the unbound state, the N-terminal domain of L11 is rotated only slightly, and A1067 of the RNA is flipped back into the less-solvent-exposed position, as in the crystal structure. By matching our experimental cryo-EM maps with much improved cross-correlation coefficients compared to the crystal structure, these two conformations prove to be strong candidates of the two functional states. PMID:16682558

  11. A tRNA methyltransferase paralog is important for ribosome stability and cell division in Trypanosoma brucei

    PubMed Central

    Fleming, Ian M. C.; Paris, Zdeněk; Gaston, Kirk W.; Balakrishnan, R.; Fredrick, Kurt; Rubio, Mary Anne T.; Alfonzo, Juan D.

    2016-01-01

    Most eukaryotic ribosomes contain 26/28S, 5S, and 5.8S large subunit ribosomal RNAs (LSU rRNAs) in addition to the 18S rRNA of the small subunit (SSU rRNA). However, in kinetoplastids, a group of organisms that include medically important members of the genus Trypanosoma and Leishmania, the 26/28S large subunit ribosomal RNA is uniquely composed of 6 rRNA fragments. In addition, recent studies have shown the presence of expansion segments in the large ribosomal subunit (60S) of Trypanosoma brucei. Given these differences in structure, processing and assembly, T. brucei ribosomes may require biogenesis factors not found in other organisms. Here, we show that one of two putative 3-methylcytidine methyltransferases, TbMTase37 (a homolog of human methyltransferase-like 6, METTL6), is important for ribosome stability in T. brucei. TbMTase37 localizes to the nucleolus and depletion of the protein results in accumulation of ribosomal particles lacking srRNA 4 and reduced levels of polysome associated ribosomes. We also find that TbMTase37 plays a role in cytokinesis, as loss of the protein leads to multi-flagellated and multi-nucleated cells. PMID:26888608

  12. The Circadian Clock Modulates Global Daily Cycles of mRNA Ribosome Loading[OPEN

    PubMed Central

    Missra, Anamika; Ernest, Ben; Jia, Qidong; Ke, Kenneth

    2015-01-01

    Circadian control of gene expression is well characterized at the transcriptional level, but little is known about diel or circadian control of translation. Genome-wide translation state profiling of mRNAs in Arabidopsis thaliana seedlings grown in long day was performed to estimate ribosome loading per mRNA. The experiments revealed extensive translational regulation of key biological processes. Notably, translation of mRNAs for ribosomal proteins and mitochondrial respiration peaked at night. Central clock mRNAs are among those subject to fluctuations in ribosome loading. There was no consistent phase relationship between peak translation states and peak transcript levels. The overlay of distinct transcriptional and translational cycles can be expected to alter the waveform of the protein synthesis rate. Plants that constitutively overexpress the clock gene CCA1 showed phase shifts in peak translation, with a 6-h delay from midnight to dawn or from noon to evening being particularly common. Moreover, cycles of ribosome loading that were detected under continuous light in the wild type collapsed in the CCA1 overexpressor. Finally, at the transcript level, the CCA1-ox strain adopted a global pattern of transcript abundance that was broadly correlated with the light-dark environment. Altogether, these data demonstrate that gene-specific diel cycles of ribosome loading are controlled in part by the circadian clock. PMID:26392078

  13. The Circadian Clock Modulates Global Daily Cycles of mRNA Ribosome Loading.

    PubMed

    Missra, Anamika; Ernest, Ben; Lohoff, Tim; Jia, Qidong; Satterlee, James; Ke, Kenneth; von Arnim, Albrecht G

    2015-09-01

    Circadian control of gene expression is well characterized at the transcriptional level, but little is known about diel or circadian control of translation. Genome-wide translation state profiling of mRNAs in Arabidopsis thaliana seedlings grown in long day was performed to estimate ribosome loading per mRNA. The experiments revealed extensive translational regulation of key biological processes. Notably, translation of mRNAs for ribosomal proteins and mitochondrial respiration peaked at night. Central clock mRNAs are among those subject to fluctuations in ribosome loading. There was no consistent phase relationship between peak translation states and peak transcript levels. The overlay of distinct transcriptional and translational cycles can be expected to alter the waveform of the protein synthesis rate. Plants that constitutively overexpress the clock gene CCA1 showed phase shifts in peak translation, with a 6-h delay from midnight to dawn or from noon to evening being particularly common. Moreover, cycles of ribosome loading that were detected under continuous light in the wild type collapsed in the CCA1 overexpressor. Finally, at the transcript level, the CCA1-ox strain adopted a global pattern of transcript abundance that was broadly correlated with the light-dark environment. Altogether, these data demonstrate that gene-specific diel cycles of ribosome loading are controlled in part by the circadian clock.

  14. An intron within the 16S ribosomal RNA gene of the archaeon Pyrobaculum aerophilum

    NASA Technical Reports Server (NTRS)

    Burggraf, S.; Larsen, N.; Woese, C. R.; Stetter, K. O.

    1993-01-01

    The 16S rRNA genes of Pyrobaculum aerophilum and Pyrobaculum islandicum were amplified by the polymerase chain reaction, and the resulting products were sequenced directly. The two organisms are closely related by this measure (over 98% similar). However, they differ in that the (lone) 16S rRNA gene of Pyrobaculum aerophilum contains a 713-bp intron not seen in the corresponding gene of Pyrobaculum islandicum. To our knowledge, this is the only intron so far reported in the small subunit rRNA gene of a prokaryote. Upon excision the intron is circularized. A secondary structure model of the intron-containing rRNA suggests a splicing mechanism of the same type as that invoked for the tRNA introns of the Archaea and Eucarya and 23S rRNAs of the Archaea. The intron contains an open reading frame whose protein translation shows no certain homology with any known protein sequence.

  15. Structure and Function of the Ribosomal Frameshifting Pseudoknot RNA from Beet Western Yellow Virus

    SciTech Connect

    Egli, M.; Sarkhel, S.; Minasov, G.; Rich, A.

    2010-03-05

    Many viruses reprogram ribosomes to produce two different proteins from two different reading frames. So-called -1 frameshifting often involves pairwise alignment of two adjacent tRNAs at a 'slippery' sequence in the ribosomal A and P sites such that an overlapping codon is shifted upstream by one base relative to the zero frame. In the majority of cases, an RNA pseudoknot located downstream stimulates this type of frameshift. Crystal structures of the frameshifting RNA pseudoknot from Beet Western Yellow Virus (BWYV) have provided a detailed picture of the tertiary interactions stabilizing this folding motif, including a minor-groove triplex and quadruple-base interactions. The structure determined at atomic resolution revealed the locations of several magnesium ions and provided insights into the role of structured water stabilizing the RNA. Systematic in vitro and in vivo mutational analyses based on the structural results revealed specific tertiary interactions and regions in the pseudoknot that drastically change frameshifting efficiency. Here, we summarize recent advances in our understanding of pseudoknot-mediated ribosomal frameshifting on the basis of the insights gained from structural and functional studies of the RNA pseudoknot from BWYV.

  16. RiboDiff: detecting changes of mRNA translation efficiency from ribosome footprints

    PubMed Central

    Zhong, Yi; Karaletsos, Theofanis; Drewe, Philipp; Sreedharan, Vipin T.; Kuo, David; Singh, Kamini; Wendel, Hans-Guido; Rätsch, Gunnar

    2017-01-01

    Motivation: Deep sequencing based ribosome footprint profiling can provide novel insights into the regulatory mechanisms of protein translation. However, the observed ribosome profile is fundamentally confounded by transcriptional activity. In order to decipher principles of translation regulation, tools that can reliably detect changes in translation efficiency in case–control studies are needed. Results: We present a statistical framework and an analysis tool, RiboDiff, to detect genes with changes in translation efficiency across experimental treatments. RiboDiff uses generalized linear models to estimate the over-dispersion of RNA-Seq and ribosome profiling measurements separately, and performs a statistical test for differential translation efficiency using both mRNA abundance and ribosome occupancy. Availability and Implementation: RiboDiff webpage http://bioweb.me/ribodiff. Source code including scripts for preprocessing the FASTQ data are available at http://github.com/ratschlab/ribodiff. Contacts: zhongy@cbio.mskcc.org or raetsch@inf.ethz.ch Supplementary information: Supplementary data are available at Bioinformatics online. PMID:27634950

  17. A proton wire to couple aminoacyl-tRNA accommodation and peptide-bond formation on the ribosome.

    PubMed

    Polikanov, Yury S; Steitz, Thomas A; Innis, C Axel

    2014-09-01

    During peptide-bond formation on the ribosome, the α-amine of an aminoacyl-tRNA attacks the ester carbonyl carbon of a peptidyl-tRNA to yield a peptide lengthened by one amino acid. Although the ribosome's contribution to catalysis is predominantly entropic, the lack of high-resolution structural data for the complete active site in complex with full-length ligands has made it difficult to assess how the ribosome might influence the pathway of the reaction. Here, we present crystal structures of preattack and postcatalysis complexes of the Thermus thermophilus 70S ribosome at ~2.6-Å resolution. These structures reveal a network of hydrogen bonds along which proton transfer could take place to ensure the concerted, rate-limiting formation of a tetrahedral intermediate. We propose that, unlike earlier models, the ribosome and the A-site tRNA facilitate the deprotonation of the nucleophile through the activation of a water molecule.

  18. Multicolor fluorescence detection of ribosomal RNA in microchannels

    NASA Astrophysics Data System (ADS)

    Balberg, Michal; Hristova, Krassimira; Mau, Margit; Frigon, Dominic; Zeringue, Henry C.; Brady, David J.; Beebe, David J.; Raskin, Lutgarde

    2000-03-01

    A micro fluidic device capable of detecting the abundance of bacteria in an environmental solution is described. The micro channels are made of poly(dimethylsioxane) (PDMS) elastomer integrated with fused silica capillaries coated with Aluminum. The detection of specific bacteria is based on molecular probes (beacons) that emit a fluorescent signal only when hybridized to the target. This method allows hybridization in solution, without immobilization, and avoids washing of the unbound probes. By marking 16S rDNA oligonucleotide probes (different genetic sequences) with different color dyes, and detecting the spectral intensity of light in the micro- channel, different micro-organisms can be detected in one sample. Miniaturization of the analytic device allows the use of small quantities of RNA molecules, as target molecules, and improves the detection limits. Future devices should incorporate a parallel array of micro-channels, and enable fast and parallel processing of the molecular signals.

  19. Nuclear Export of Pre-Ribosomal Subunits Requires Dbp5, but Not as an RNA-Helicase as for mRNA Export.

    PubMed

    Neumann, Bettina; Wu, Haijia; Hackmann, Alexandra; Krebber, Heike

    2016-01-01

    The DEAD-box RNA-helicase Dbp5/Rat8 is known for its function in nuclear mRNA export, where it displaces the export receptor Mex67 from the mRNA at the cytoplasmic side of the nuclear pore complex (NPC). Here we show that Dbp5 is also required for the nuclear export of both pre-ribosomal subunits. Yeast temperature-sensitive dbp5 mutants accumulate both ribosomal particles in their nuclei. Furthermore, Dbp5 genetically and physically interacts with known ribosomal transport factors such as Nmd3. Similar to mRNA export we show that also for ribosomal transport Dbp5 is required at the cytoplasmic side of the NPC. However, unlike its role in mRNA export, Dbp5 does not seem to undergo its ATPase cycle for this function, as ATPase-deficient dbp5 mutants that selectively inhibit mRNA export do not affect ribosomal transport. Furthermore, mutants of GLE1, the ATPase stimulating factor of Dbp5, show no major ribosomal export defects. Consequently, while Dbp5 uses its ATPase cycle to displace the export receptor Mex67 from the translocated mRNAs, Mex67 remains bound to ribosomal subunits upon transit to the cytoplasm, where it is detectable on translating ribosomes. Therefore, we propose a model, in which Dbp5 supports ribosomal transport by capturing ribosomal subunits upon their cytoplasmic appearance at the NPC, possibly by binding export factors such as Mex67. Thus, our findings reveal that although different ribonucleoparticles, mRNAs and pre-ribosomal subunits, use shared export factors, they utilize different transport mechanisms.

  20. Kinetoplastid Specific RNA-Protein Interactions in Trypanosoma cruzi Ribosome Biogenesis.

    PubMed

    Umaer, Khan; Williams, Noreen

    2015-01-01

    RNA binding proteins (RBP) play essential roles in the highly conserved and coordinated process of ribosome biogenesis. Our laboratory has previously characterized two essential and abundant RBPs, P34 and P37, in Trypanosoma brucei which are required for several critical steps in ribosome biogenesis. The genes for these proteins have only been identified in kinetoplastid organisms but not in the host genome. We have identified a homolog of the TbP34 and TbP37 in a T. cruzi strain (termed TcP37/NRBD). Although the N-terminal APK-rich domain and RNA recognition motifs are conserved, the C-terminal region which contains putative nuclear and nucleolar localization signals in TbP34 and TbP37 is almost entirely missing from TcP37/NRBD. We have shown that TcP37/NRBD is expressed in T. cruzi epimastigotes at the level of mature mRNA and protein. Despite the loss of the C-terminal domain, TcP37/NRBD is present in the nucleus, including the nucleolus, and the cytoplasm. TcP37/NRBD interacts directly with Tc 5S rRNA, but does not associate with polyadenylated RNA. TcP37/NRBD also associates in vivo and in vitro with large ribosomal protein TcL5 and, unlike the case of T. brucei, this association is strongly enhanced by the presence of 5S rRNA, suggesting that the loss of the C-terminal domain of TcP37/NRBD may alter the interactions within the complex. These results indicate that the unique preribosomal complex comprised of L5, 5S rRNA, and the trypanosome-specific TcP37/NRBD or TbP34 and TbP37 is functionally conserved in trypanosomes despite the differences in the C-termini of the trypanosome-specific protein components.

  1. Quantitative studies of mRNA recruitment to the eukaryotic ribosome

    PubMed Central

    Fraser, Christopher S.

    2015-01-01

    The process of peptide bond synthesis by ribosomes is conserved between species, but the initiation step differs greatly between the three kingdoms of life. This is illustrated by the evolution of roughly an order of magnitude more initiation factor mass found in humans compared with bacteria. Eukaryotic initiation of translation is comprised of a number of sub-steps: (i) recruitment of an mRNA and initiator methionyl-tRNA to the 40S ribosomal subunit; (ii) migration of the 40S subunit along the 5′ UTR to locate the initiation codon; and (iii) recruitment of the 60S subunit to form the 80S initiation complex. Although the mechanism and regulation of initiation has been studied for decades, many aspects of the pathway remain unclear. In this review, I will focus discussion on what is known about the mechanism of mRNA selection and its recruitment to the 40S subunit. I will summarize how the 43S preinitiation complex (PIC) is formed and stabilized by interactions between its components. I will discuss what is known about the mechanism of mRNA selection by the eukaryotic initiation factor 4F (eIF4F) complex and how the selected mRNA is recruited to the 43S PIC. The regulation of this process by secondary structure located in the 5′ UTR of an mRNA will also be discussed. Finally, I present a possible kinetic model with which to explain the process of mRNA selection and recruitment to the eukaryotic ribosome. PMID:25742741

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

    NASA Technical Reports Server (NTRS)

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

    1999-01-01

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

  3. Characterization of nitrogen-fixing Paenibacillus species by polymerase chain reaction-restriction fragment length polymorphism analysis of part of genes encoding 16S rRNA and 23S rRNA and by multilocus enzyme electrophoresis.

    PubMed

    Coelho, Marcia Reed Rodrigues; von der Weid, Irene; Zahner, Viviane; Seldin, Lucy

    2003-05-28

    Forty-two strains representing the eight recognized nitrogen-fixing Paenibacillus species and 12 non-identified strains were examined by restriction fragment length polymorphism (RFLP) analysis of part of 16S and 23S rRNA genes amplified by polymerase chain reaction (PCR). Eleven different 16S rDNA genotypes were obtained from the combined data of RFLP analysis with four endonucleases and they were in agreement with the established taxonomic classification. Only one group of unclassified strains (Group I) was assigned in a separate genotype, suggesting they belong to a new species. Using the 23S PCR-RFLP method only six genotypes were detected, showing that this method is less discriminative than the 16S PCR-RFLP. Using the multilocus enzyme electrophoresis (MLEE) assay, the 48 strains tested could be classified into 35 zymovars. The seven enzymatic loci tested were polymorphic and the different profiles obtained among strains allowed the grouping of strains into 10 clusters. The PCR-RFLP methods together with the MLEE assay provide a rapid tool for the characterization and the establishment of the taxonomic position of isolates belonging to this nitrogen-fixing group, which shows a great potentiality in promoting plant growth.

  4. Antibacterial discovery in actinomycetes strains with mutations in RNA polymerase or ribosomal protein S12.

    PubMed

    Hosaka, Takeshi; Ohnishi-Kameyama, Mayumi; Muramatsu, Hideyuki; Murakami, Kana; Tsurumi, Yasuhisa; Kodani, Shinya; Yoshida, Mitsuru; Fujie, Akihiko; Ochi, Kozo

    2009-05-01

    We show that selection of drug-resistant bacterial mutants allows the discovery of antibacterial compounds. Mutant strains of a soil-isolated Streptomyces species that does not produce antibacterials synthesize a previously unknown class of antibacterial, which we name piperidamycin. Overall, 6% of non-Streptomyces actinomycetes species and 43% of Streptomyces species that do not produce antibacterials are activated to produce them. The antibacterial-producing mutants all carried mutations in RNA polymerase and/or the ribosomal protein S12.

  5. The mitochondrial genome of the entomopathogenic fungus Beauveria bassiana: analysis of the ribosomal RNA region.

    PubMed

    Pfeifer, T A; Hegedus, D D; Khachatourians, G G

    1993-01-01

    The 28.5-kbp mitochondrial (mt) genome from the entomopathogenic fungus Beauveria bassiana was studied using restriction enzyme analysis, gene probe hybridization, and DNA sequence comparisons. A detailed restriction enzyme map allowed cloning of the entire genome into a number of segments. Hybridization of heterologous gene probes to the mtDNA resulted in the identification of the large ribosomal RNA (lrRNA) and small ribosomal RNA (srRNA) genes. Gene probes derived from several yeasts and fungi failed to identify any additional genes. However, partial DNA sequence analysis revealed the lrRNA and srRNA genes as well as four protein-encoding genes: the NADH dehydrogenase subunit 1 (NAD1), NADH dehydrogenase subunit 6 (NAD6), cytochrome oxidase subunit 3 (CO3), and ATPase subunit 6 (ATP6) genes. The ATPase subunit 9 (ATP9) gene was not identified by hybridization to mtDNA, but could be detected by hybridization to total cellular DNA. The portions of the genes sequenced were homologous to the equivalent genes from yeast and other filamentous fungi, most notably Aspergillus nidulans. No introns were identified in these regions. The organization of the sequenced region of the B. bassiana mt genome more closely resembled that of A. nidulans than that of Podospora anserina or Neurospora crassa.

  6. Sequence of the 16S ribosomal RNA from Halobacterium volcanii, an archaebacterium

    NASA Technical Reports Server (NTRS)

    Gupta, R.; Lanter, J. M.; Woese, C. R.

    1983-01-01

    The sequence of the 16S ribosomal RNA (rRNA) from the archaebacterium Halobacterium volcanii has been determined by DNA sequencing methods. The archaebacterial rRNA is similar to its eubacterial counterpart in secondary structure. Although it is closer in sequence to the eubacterial 16S rRNA than to the eukaryotic 16S-like rRNA, the H. volcanii sequence also shows certain points of specific similarity to its eukaryotic counterpart. Since the H. volcanii sequence is closer to both the eubacterial and the eukaryotic sequences than these two are to one another, it follows that the archaebacterial sequence resembles their common ancestral sequence more closely than does either of the other two versions.

  7. 25S ribosomal RNA homologies of basidiomycetous yeasts: taxonomic and phylogenetic implications

    NASA Technical Reports Server (NTRS)

    Baharaeen, S.; Vishniac, H. S.

    1984-01-01

    Genera, families, and possibly orders of basidiomycetous yeasts can be defined by 25S rRNA homology and correlated phenotypic characters. The teleomorphic genera Filobasidium, Leucosporidium, and Rhodosporidium have greater than 96 relative binding percent (rb%) intrageneric 25S rRNA homology and significant intergeneric separation from each other and from Filobasidiella. The anamorphic genus Cryptococcus can be defined by morphology (monopolar budding), colony color, and greater than 75 rb% intrageneric homology; Vanrija is heterogeneous. Agaricostilbum (Phragmobasidiomycetes, Auriculariales), Hansenula (Ascomycotera, Endomycota), Tremella (Phragmobasidiomycetes, Tremellales), and Ustilago (Ustomycota, Ustilaginales) appear equally unrelated to the Cryptococcus, Filobasidiella, and Rhodosporidium spp. used as probes. The Filobasidiaceae and Sporidiaceae, Filobasidiales and Sporidiales, form coherent homology groups which appear to have undergone convergent 25S rRNA evolution, since their relatedness is much greater than that indicated by 5S rRNA homology. Ribosomal RNA homologies do not appear to measure evolutionary distance.

  8. Substitution rate calibration of small subunit ribosomal RNA identifies chlorarachniophyte endosymbionts as remnants of green algae.

    PubMed Central

    Van de Peer, Y; Rensing, S A; Maier, U G; De Wachter, R

    1996-01-01

    Chlorarachniophytes are amoeboid algae with chlorophyll a and b containing plastids that are surrounded by four membranes instead of two as in plants and green algae. These extra membranes form important support for the hypothesis that chlorarachniophytes have acquired their plastids by the ingestion of another eukaryotic plastid-containing alga. Chlorarachniophytes also contain a small nucleus-like structure called the nucleomorph situated between the two inner and the two outer membranes surrounding the plastid. This nucleomorph is a remnant of the endosymbiont's nucleus and encodes, among other molecules, small subunit ribosomal RNA. Previous phylogenetic analyses on the basis of this molecule provided unexpected and contradictory evidence for the origin of the chlorarachniophyte endosymbiont. We developed a new method for measuring the substitution rates of the individual nucleotides of small subunit ribosomal RNA. From the resulting substitution rate distribution, we derived an equation that gives a more realistic relationship between sequence dissimilarity and evolutionary distance than equations previously available. Phylogenetic trees constructed on the basis of evolutionary distances computed by this new method clearly situate the chlorarachniophyte nucleomorphs among the green algae. Moreover, this relationship is confirmed by transversion analysis of the Chlorarachnion plastid small subunit ribosomal RNA. PMID:8755544

  9. Detection of bacterial 16S ribosomal RNA genes for forensic identification of vaginal fluid.

    PubMed

    Akutsu, Tomoko; Motani, Hisako; Watanabe, Ken; Iwase, Hirotaro; Sakurada, Koichi

    2012-05-01

    To preliminarily evaluate the applicability of bacterial DNA as a marker for the forensic identification of vaginal fluid, we developed and performed PCR-based detection of 16S ribosomal RNA genes of Lactobacillus spp. dominating the vagina and of bacterial vaginosis-related bacteria from DNA extracted from body fluids and stains. As a result, 16S ribosomal RNA genes of Lactobacillus crispatus, Lactobacillus jensenii and Atopobium vaginae were specifically detected in vaginal fluid and female urine samples. Bacterial genes detected in female urine might have originated from contaminated vaginal fluid. In addition, those of Lactobacillus iners, Lactobacillus gasseri and Gardnerella vaginalis were also detected in non-vaginal body fluids such as semen. Because bacterial genes were successfully amplified in DNA samples extracted by using the general procedure for animal tissues without any optional treatments, DNA samples prepared for the identification of vaginal fluid can also be used for personal identification. In conclusion, 16S ribosomal RNA genes of L. crispatus, L. jensenii and A. vaginae could be effective markers for forensic identification of vaginal fluid.

  10. Role of a ribosomal RNA phosphate oxygen during the EF-G-triggered GTP hydrolysis.

    PubMed

    Koch, Miriam; Flür, Sara; Kreutz, Christoph; Ennifar, Eric; Micura, Ronald; Polacek, Norbert

    2015-05-19

    Elongation factor-catalyzed GTP hydrolysis is a key reaction during the ribosomal elongation cycle. Recent crystal structures of G proteins, such as elongation factor G (EF-G) bound to the ribosome, as well as many biochemical studies, provide evidence that the direct interaction of translational GTPases (trGTPases) with the sarcin-ricin loop (SRL) of ribosomal RNA (rRNA) is pivotal for hydrolysis. However, the precise mechanism remains elusive and is intensively debated. Based on the close proximity of the phosphate oxygen of A2662 of the SRL to the supposedly catalytic histidine of EF-G (His87), we probed this interaction by an atomic mutagenesis approach. We individually replaced either of the two nonbridging phosphate oxygens at A2662 with a methyl group by the introduction of a methylphosphonate instead of the natural phosphate in fully functional, reconstituted bacterial ribosomes. Our major finding was that only one of the two resulting diastereomers, the SP methylphosphonate, was compatible with efficient GTPase activation on EF-G. The same trend was observed for a second trGTPase, namely EF4 (LepA). In addition, we provide evidence that the negative charge of the A2662 phosphate group must be retained for uncompromised activity in GTP hydrolysis. In summary, our data strongly corroborate that the nonbridging proSP phosphate oxygen at the A2662 of the SRL is critically involved in the activation of GTP hydrolysis. A mechanistic scenario is supported in which positioning of the catalytically active, protonated His87 through electrostatic interactions with the A2662 phosphate group and H-bond networks are key features of ribosome-triggered activation of trGTPases.

  11. An RNA Element That Facilitates Programmed Ribosomal Readthrough in Turnip Crinkle Virus Adopts Multiple Conformations

    PubMed Central

    Kuhlmann, Micki M.; Chattopadhyay, Maitreyi; Stupina, Vera A.; Gao, Feng

    2016-01-01

    ABSTRACT Ribosome recoding is used by RNA viruses for translational readthrough or frameshifting past termination codons for the synthesis of extension products. Recoding sites, along with downstream recoding stimulatory elements (RSEs), have long been studied in reporter constructs, because these fragments alone mediate customary levels of recoding and are thus assumed to contain complete instructions for establishment of the proper ratio of termination to recoding. RSEs from the Tombusviridae and Luteoviridae are thought to be exceptions, since they contain a long-distance RNA-RNA connection with the 3′ end. This interaction has been suggested to substitute for pseudoknots, thought to be missing in tombusvirid RSEs. We provide evidence that the phylogenetically conserved RSE of the carmovirus Turnip crinkle virus (TCV) adopts an alternative, smaller structure that extends an upstream conserved hairpin and that this alternative structure is the predominant form of the RSE within nascent viral RNA in plant cells and when RNA is synthesized in vitro. The TCV RSE also contains an internal pseudoknot along with the long-distance interaction, and the pseudoknot is not compatible with the phylogenetically conserved structure. Conserved residues just past the recoding site are important for recoding, and these residues are also conserved in the RSEs of gammaretroviruses. Our data demonstrate the dynamic nature of the TCV RSE and suggest that studies using reporter constructs may not be effectively recapitulating RSE-mediated recoding within viral genomes. IMPORTANCE Ribosome recoding is used by RNA viruses to enable ribosomes to extend translation past termination codons for the synthesis of longer products. Recoding sites and a downstream recoding stimulatory element (RSE) mediate expected levels of recoding when excised and placed in reporter constructs and thus are assumed to contain complete instructions for the establishment of the proper ratio of termination to

  12. Identification of Novel RNA-Protein Contact in Complex of Ribosomal Protein S7 and 3'-Terminal Fragment of 16S rRNA in E. coli.

    PubMed

    Golovin, A V; Khayrullina, G A; Kraal, B; Kopylov, Capital A Cyrillic М

    2012-10-01

    For prokaryotes in vitro, 16S rRNA and 20 ribosomal proteins are capable of hierarchical self- assembly yielding a 30S ribosomal subunit. The self-assembly is initiated by interactions between 16S rRNA and three key ribosomal proteins: S4, S8, and S7. These proteins also have a regulatory function in the translation of their polycistronic operons recognizing a specific region of mRNA. Therefore, studying the RNA-protein interactions within binary complexes is obligatory for understanding ribosome biogenesis. The non-conventional RNA-protein contact within the binary complex of recombinant ribosomal protein S7 and its 16S rRNA binding site (236 nucleotides) was identified. UV-induced RNA-protein cross-links revealed that S7 cross-links to nucleotide U1321 of 16S rRNA. The careful consideration of the published RNA- protein cross-links for protein S7 within the 30S subunit and their correlation with the X-ray data for the 30S subunit have been performed. The RNA - protein cross-link within the binary complex identified in this study is not the same as the previously found cross-links for a subunit both in a solution, and in acrystal. The structure of the binary RNA-protein complex formed at the initial steps of self-assembly of the small subunit appears to be rearranged during the formation of the final structure of the subunit.

  13. The pre-existing population of 5S rRNA effects p53 stabilization during ribosome biogenesis inhibition.

    PubMed

    Onofrillo, Carmine; Galbiati, Alice; Montanaro, Lorenzo; Derenzini, Massimo

    2017-01-17

    Pre-ribosomal complex RPL5/RPL11/5S rRNA (5S RNP) is considered the central MDM2 inhibitory complex that control p53 stabilization during ribosome biogenesis inhibition. Despite its role is well defined, the dynamic of 5S RNP assembly still requires further characterization. In the present work, we report that MDM2 inhibition is dependent by a pre-existing population of 5S rRNA.

  14. Ribosome mediated specificity in Hox mRNA translation and vertebrate tissue patterning

    PubMed Central

    Kondrashov, Nadya; Shimizu, Kunihiko; Hsieh, Andrew C.; Ishijima, Junko; Shiroishi, Toshihiko; Barna, Maria

    2015-01-01

    Historically, the ribosome has been viewed as a complex ribozyme with constitutive rather than regulatory capacity in mRNA translation. Here we identify mutations of the Ribosomal Protein L38 (Rpl38) gene in mice exhibiting surprising tissue specific patterning defects, including pronounced homeotic transformations of the axial skeleton. In Rpl38 mutant embryos, global protein synthesis is unchanged however the translation of a select subset of Homeobox mRNAs is perturbed. Our data reveal that RPL38 facilitates 80S complex formation on these mRNAs as a regulatory component of the ribosome to confer transcript-specific translational control. We further show that Rpl38 expression is markedly enriched in regions of the embryo where loss-of-function phenotypes occur. Unexpectedly, a ribosomal protein (RP) expression screen reveals dynamic regulation of individual RPs within the vertebrate embryo. Collectively, these findings suggest that RP activity may be highly regulated to impart a new layer of specificity in the control of gene expression and mammalian development. PMID:21529712

  15. Does HIV-1 mRNA 5'-untranslated region bear an internal ribosome entry site?

    PubMed

    Smirnova, Victoria V; Terenin, Ilya M; Khutornenko, Anastasia A; Andreev, Dmitri E; Dmitriev, Sergey E; Shatsky, Ivan N

    2016-02-01

    Unspliced human immunodeficiency virus-1 (HIV-1) mRNA is capped and therefore can be translated via conventional scanning mechanism. In addition, its 5' untranslated region (5'UTR) is thought to function as an internal ribosome entry site (IRES) during G2/M-phase of cell cycle or when cap-dependent translation is inhibited. Recently, customary methods of internal initiation demonstrating have been challenged, and consequently existence of certain IRESs of cellular origin has been put under question. Since a precise knowledge of translation initiation mechanism used by HIV may be important for cure development, presence of the IRES in HIV-1 mRNA demands a careful reexamination using contemporary stringent criteria. The key point of our strategy is to compare translation efficiency of bicistronic mRNA bearing HIV-1 unspliced mRNA 5' UTR in the intercistronic position to that of the corresponding capped monocistronic mRNA. This approach allows determination of internal initiation contribution into the overall level of particular mRNA translation. We found that both in cell-free systems and in cultured cells monocistronic mRNA with HIV-1 unspliced mRNA 5'UTR is translated significantly better than bicistronic one. Importantly, it is also true for G2/M-phase stalled cells or for cells under conditions of inhibited cap-dependent translation. Thus, in our hands contribution of internal ribosome entry into the overall level of translation driven by HIV-1 unspliced mRNA 5'UTR is negligible, and 5'-dependent scanning is a primary mechanism of its translation initiation.

  16. 18S Ribosomal RNA Evaluation as Preanalytical Quality Control for Animal DNA

    PubMed Central

    Meli, Marina L.; Novacco, Marilisa; Borel, Nicole

    2016-01-01

    The 18S ribosomal RNA (rRNA) gene is present in all eukaryotic cells. In this study, we evaluated the use of this gene to verify the presence of PCR-amplifiable host (animal) DNA as an indicator of sufficient sample quality for quantitative real-time PCR (qPCR) analysis. We compared (i) samples from various animal species, tissues, and sample types, including swabs; (ii) multiple DNA extraction methods; and (iii) both fresh and formalin-fixed paraffin-embedded (FFPE) samples. Results showed that 18S ribosomal RNA gene amplification was possible from all tissue samples evaluated, including avian, reptile, and FFPE samples and most swab samples. A single swine rectal swab, which showed sufficient DNA quantity and the demonstrated lack of PCR inhibitors, nonetheless was negative by 18S qPCR. Such a sample specifically illustrates the improvement of determination of sample integrity afforded by inclusion of 18S rRNA gene qPCR analysis in addition to spectrophotometric analysis and the use of internal controls for PCR inhibition. Other possible applications for the described 18S rRNA qPCR are preselection of optimal tissue specimens for studies or preliminary screening of archived samples prior to acceptance for biobanking projects. PMID:27672657

  17. Efficient hammerhead ribozyme and antisense RNA targeting in a slow ribosome Escherichia coli mutant.

    PubMed

    Chen, H; Ferbeyre, G; Cedergren, R

    1997-05-01

    We have evaluated inhibition of the plasmid-born chloramphenicol acetyl transferase gene (CAT) by the hammerhead ribozyme and antisense RNA in Escherichia coli where the translation and transcription rates have been modified. Whereas neither antisense nor the hammerhead had an inhibitory effect on CAT activity in wild-type E. coli, both reduced the level of the messenger RNA and the activity of the CAT gene by almost 60% in a slow ribosome mutant. Streptomycin, which increases the speed of translation in this mutant strain, restored full CAT activity. The level of CAT activity expressed from a T7 RNA polymerase promoter was not affected by the presence of either antisense RNA or the hammerhead ribozyme. When the target gene was expressed from a chromosomal locus in wild-type E. coli, both antisense RNA and the hammerhead ribozyme showed some inhibitory activity, but the level of inhibition was significantly increased in the slow ribosome strain. This bacterial system offers a unique entry to the study of cellular factors which mediate the activity of ribozymes in vivo.

  18. Universal and domain-specific sequences in 23S–28S ribosomal RNA identified by computational phylogenetics

    PubMed Central

    Doris, Stephen M.; Smith, Deborah R.; Beamesderfer, Julia N.; Raphael, Benjamin J.; Nathanson, Judith A.; Gerbi, Susan A.

    2015-01-01

    Comparative analysis of ribosomal RNA (rRNA) sequences has elucidated phylogenetic relationships. However, this powerful approach has not been fully exploited to address ribosome function. Here we identify stretches of evolutionarily conserved sequences, which correspond with regions of high functional importance. For this, we developed a structurally aligned database, FLORA (full-length organismal rRNA alignment) to identify highly conserved nucleotide elements (CNEs) in 23S–28S rRNA from each phylogenetic domain (Eukarya, Bacteria, and Archaea). Universal CNEs (uCNEs) are conserved in sequence and structural position in all three domains. Those in regions known to be essential for translation validate our approach. Importantly, some uCNEs reside in areas of unknown function, thus identifying novel sequences of likely great importance. In contrast to uCNEs, domain-specific CNEs (dsCNEs) are conserved in just one phylogenetic domain. This is the first report of conserved sequence elements in rRNA that are domain-specific; they are largely a eukaryotic phenomenon. The locations of the eukaryotic dsCNEs within the structure of the ribosome suggest they may function in nascent polypeptide transit through the ribosome tunnel and in tRNA exit from the ribosome. Our findings provide insights and a resource for ribosome function studies. PMID:26283689

  19. Development of an endpoint genotyping assay to detect the Mycoplasma pneumoniae 23S rRNA gene and distinguish the existence of macrolide resistance-associated mutations at position 2063.

    PubMed

    Suzuki, Yu; Seto, Junji; Shimotai, Yoshitaka; Ikeda, Tatsuya; Yahagi, Kazue; Mizuta, Katsumi; Matsuzaki, Yoko; Hongo, Seiji

    2016-12-01

    The prevalence of macrolide-resistant Mycoplasma pneumoniae harboring a mutation in the 23S rRNA gene is increasing, and rapid detection assays are needed for clinical management. We developed an endpoint genotyping assay to detect the M. pneumoniae 23S rRNA gene and determine the existence of macrolide resistance-associated mutations at position 2063 (A2063G, A2063T and A2063C mutations). This A2063B genotyping assay detected more than 50 copies/reaction of the M. pneumoniae gene in every nucleotide mutation at position 2063. Of 42 clinical specimens, 3 were positive without mutation, 6 were positive with the A2063G mutation, and 33 were negative. The results were confirmed using nested PCR with the sequencing of the M. pneumoniae 23S rRNA gene, and a high sensitivity (90%), specificity (100%), and coincidence ratio (kappa coefficient=0.93) were obtained. Therefore, the A2063B genotyping assay is useful for the rapid discrimination of macrolide resistance mutations at position 2063.

  20. The SILVA ribosomal RNA gene database project: improved data processing and web-based tools.

    PubMed

    Quast, Christian; Pruesse, Elmar; Yilmaz, Pelin; Gerken, Jan; Schweer, Timmy; Yarza, Pablo; Peplies, Jörg; Glöckner, Frank Oliver

    2013-01-01

    SILVA (from Latin silva, forest, http://www.arb-silva.de) is a comprehensive web resource for up to date, quality-controlled databases of aligned ribosomal RNA (rRNA) gene sequences from the Bacteria, Archaea and Eukaryota domains and supplementary online services. The referred database release 111 (July 2012) contains 3 194 778 small subunit and 288 717 large subunit rRNA gene sequences. Since the initial description of the project, substantial new features have been introduced, including advanced quality control procedures, an improved rRNA gene aligner, online tools for probe and primer evaluation and optimized browsing, searching and downloading on the website. Furthermore, the extensively curated SILVA taxonomy and the new non-redundant SILVA datasets provide an ideal reference for high-throughput classification of data from next-generation sequencing approaches.

  1. Evaluating bacterial activity from cell-specific ribosomal RNA content measured with oligonucleotide probes

    SciTech Connect

    Kemp, P.F.; Lee, S.; LaRoche, J.

    1992-10-01

    We describe a procedure for measuring the cell-specific quantity of ribosomal RNA (rRNA) and DNA in order to evaluate the frequency distribution of activity among cells. The procedure is inherently quantitative, does not require sample incubation and potentially can be taxon-specific. Fluorescently-labelled oligonucleotide probes are hybridized to the complementary 16S rRNA sequences in preserved, intact cells. The resulting cell fluorescence is proportional to cellular rRNA content and can be measured with a microscope-mounted photometer system, by image analysis, or by flow cytometry. Similarly, DNA content is measured as fluorescence of cells stained with the DNA specific fluorochrome DAPI. These are either prepared as separate samples for purposes of enumeration and DNA measurements, or are dual-labelled cells which are also hybridized with oligonucleotide probes.

  2. Evaluating bacterial activity from cell-specific ribosomal RNA content measured with oligonucleotide probes

    SciTech Connect

    Kemp, P.F.; Lee, S.; LaRoche, J.

    1992-01-01

    We describe a procedure for measuring the cell-specific quantity of ribosomal RNA (rRNA) and DNA in order to evaluate the frequency distribution of activity among cells. The procedure is inherently quantitative, does not require sample incubation and potentially can be taxon-specific. Fluorescently-labelled oligonucleotide probes are hybridized to the complementary 16S rRNA sequences in preserved, intact cells. The resulting cell fluorescence is proportional to cellular rRNA content and can be measured with a microscope-mounted photometer system, by image analysis, or by flow cytometry. Similarly, DNA content is measured as fluorescence of cells stained with the DNA specific fluorochrome DAPI. These are either prepared as separate samples for purposes of enumeration and DNA measurements, or are dual-labelled cells which are also hybridized with oligonucleotide probes.

  3. The SILVA ribosomal RNA gene database project: improved data processing and web-based tools

    PubMed Central

    Quast, Christian; Pruesse, Elmar; Yilmaz, Pelin; Gerken, Jan; Schweer, Timmy; Yarza, Pablo; Peplies, Jörg; Glöckner, Frank Oliver

    2013-01-01

    SILVA (from Latin silva, forest, http://www.arb-silva.de) is a comprehensive web resource for up to date, quality-controlled databases of aligned ribosomal RNA (rRNA) gene sequences from the Bacteria, Archaea and Eukaryota domains and supplementary online services. The referred database release 111 (July 2012) contains 3 194 778 small subunit and 288 717 large subunit rRNA gene sequences. Since the initial description of the project, substantial new features have been introduced, including advanced quality control procedures, an improved rRNA gene aligner, online tools for probe and primer evaluation and optimized browsing, searching and downloading on the website. Furthermore, the extensively curated SILVA taxonomy and the new non-redundant SILVA datasets provide an ideal reference for high-throughput classification of data from next-generation sequencing approaches. PMID:23193283

  4. Sequence characterization of 5S ribosomal RNA from eight gram positive procaryotes

    NASA Technical Reports Server (NTRS)

    Woese, C. R.; Luehrsen, K. R.; Pribula, C. D.; Fox, G. E.

    1976-01-01

    Complete nucleotide sequences are presented for 5S rRNA from Bacillus subtilis, B. firmus, B. pasteurii, B. brevis, Lactobacillus brevis, and Streptococcus faecalis, and 5S rRNA oligonucleotide catalogs and partial sequence data are given for B. cereus and Sporosarcina ureae. These data demonstrate a striking consistency of 5S rRNA primary and secondary structure within a given bacterial grouping. An exception is B. brevis, in which the 5S rRNA sequence varies significantly from that of other bacilli in the tuned helix and the procaryotic loop. The localization of these variations suggests that B. brevis occupies an ecological niche that selects such changes. It is noted that this organism produces antibiotics which affect ribosome function.

  5. Analysis of two domains with novel RNA-processing activities throws light on the complex evolution of ribosomal RNA biogenesis

    PubMed Central

    Burroughs, A. Maxwell; Aravind, L.

    2014-01-01

    Ribosomal biogenesis has been extensively investigated, especially to identify the elusive nucleases and cofactors involved in the complex rRNA processing events in eukaryotes. Large-scale screens in yeast identified two biochemically uncharacterized proteins, TSR3 and TSR4, as being key players required for rRNA maturation. Using multiple computational approaches we identify the conserved domains comprising these proteins and establish sequence and structural features providing novel insights regarding their roles. TSR3 is unified with the DTW domain into a novel superfamily of predicted enzymatic domains, with the balance of the available evidence pointing toward an RNase role with the archaeo-eukaryotic TSR3 proteins processing rRNA and the bacterial versions potentially processing tRNA. TSR4, its other eukaryotic homologs PDCD2/rp-8, PDCD2L, Zfrp8, and trus, the predominantly bacterial DUF1963 proteins, and other uncharacterized proteins are unified into a new domain superfamily, which arose from an ancient duplication event of a strand-swapped, dimer-forming all-beta unit. We identify conserved features mediating protein-protein interactions (PPIs) and propose a potential chaperone-like function. While contextual evidence supports a conserved role in ribosome biogenesis for the eukaryotic TSR4-related proteins, there is no evidence for such a role for the bacterial versions. Whereas TSR3-related proteins can be traced to the last universal common ancestor (LUCA) with a well-supported archaeo-eukaryotic branch, TSR4-related proteins of eukaryotes are derived from within the bacterial radiation of this superfamily, with archaea entirely lacking them. This provides evidence for “systems admixture,” which followed the early endosymbiotic event, playing a key role in the emergence of the uniquely eukaryotic ribosome biogenesis process. PMID:25566315

  6. Nucleotide sequence of the rrnG ribosomal RNA promoter region of Escherichia coli.

    PubMed Central

    Shen, W F; Squires, C; Squires, C L

    1982-01-01

    The primary structure of the promoter region for a ribosomal RNA transcription unit (rrnG) of Escherichia coli K12 has been determined. The sequence was obtained from 1 1.5 kbp EcoRI fragment derived from the hybrid plasmid pLC23-30. This fragment contains 455 bp preceding P1 of the rrnG promoter region and 674 bp of the rrnG 16S RNA gene. The sequence before the rrnG promoter region contains an open reading frame (ORF-BG) followed by a possible hairpin structure that resembles other known transcription terminators. The sequence of the rrnG promoter region is similar but not identical to that of rrnA and rrnB. Several minor differences between the sequences of the 16S RNA genes of rrnG and rrnB were also noted. In addition, sequences were found that could generate special structures involving the promoter regions of rrn loci. Such structures are described and their possible involvement in the regulation of ribosomal RNA synthesis is discussed. PMID:6285294

  7. Are stop codons recognized by base triplets in the large ribosomal RNA subunit?

    PubMed

    Liang, Han; Landweber, Laura F; Fresco, Jacques R

    2005-10-01

    The precise mechanism of stop codon recognition in translation termination is still unclear. A previously published study by Ivanov and colleagues proposed a new model for stop codon recognition in which 3-nucleotide Ter-anticodons within the loops of hairpin helices 69 (domain IV) and 89 (domain V) in large ribosomal subunit (LSU) rRNA recognize stop codons to terminate protein translation in eubacteria and certain organelles. We evaluated this model by extensive bioinformatic analysis of stop codons and their putative corresponding Ter-anticodons across a much wider range of species, and found many cases for which it cannot explain the stop codon usage without requiring the involvement of one or more of the eight possible noncomplementary base pairs. Involvement of such base pairs may not be structurally or thermodynamically damaging to the model. However, if, according to the model, Ter-anticodon interaction with stop codons occurs within the ribosomal A-site, the structural stringency which that site imposes on sense codon.tRNA anticodon interaction should also extend to stop codon.Ter-anticodon interactions. Moreover, with Ter-tRNA in place of an aminoacyl-tRNA, for each of the various Ter-anticodons there is a sense codon that can interact with it preferentially by complementary and wobble base-pairing. Both these considerations considerably weaken the arguments put forth previously.

  8. Global shape mimicry of tRNA within a viral internal ribosome entry site mediates translational reading frame selection

    PubMed Central

    Au, Hilda H.; Cornilescu, Gabriel; Mouzakis, Kathryn D.; Ren, Qian; Burke, Jordan E.; Lee, Seonghoon; Butcher, Samuel E.; Jan, Eric

    2015-01-01

    The dicistrovirus intergenic region internal ribosome entry site (IRES) adopts a triple-pseudoknotted RNA structure and occupies the core ribosomal E, P, and A sites to directly recruit the ribosome and initiate translation at a non-AUG codon. A subset of dicistrovirus IRESs directs translation in the 0 and +1 frames to produce the viral structural proteins and a +1 overlapping open reading frame called ORFx, respectively. Here we show that specific mutations of two unpaired adenosines located at the core of the three-helical junction of the honey bee dicistrovirus Israeli acute paralysis virus (IAPV) IRES PKI domain can uncouple 0 and +1 frame translation, suggesting that the structure adopts distinct conformations that contribute to 0 or +1 frame translation. Using a reconstituted translation system, we show that ribosomes assembled on mutant IRESs that direct exclusive 0 or +1 frame translation lack reading frame fidelity. Finally, a nuclear magnetic resonance/small-angle X-ray scattering hybrid approach reveals that the PKI domain of the IAPV IRES adopts an RNA structure that resembles a complete tRNA. The tRNA shape-mimicry enables the viral IRES to gain access to the ribosome tRNA-binding sites and form intermolecular contacts with the ribosome that are necessary for initiating IRES translation in a specific reading frame. PMID:26554019

  9. Structures of human SRP72 complexes provide insights into SRP RNA remodeling and ribosome interaction

    PubMed Central

    Becker, Matthias M. M.; Lapouge, Karine; Segnitz, Bernd; Wild, Klemens; Sinning, Irmgard

    2017-01-01

    Co-translational protein targeting and membrane protein insertion is a fundamental process and depends on the signal recognition particle (SRP). In mammals, SRP is composed of the SRP RNA crucial for SRP assembly and function and six proteins. The two largest proteins SRP68 and SRP72 form a heterodimer and bind to a regulatory site of the SRP RNA. Despite their essential roles in the SRP pathway, structural information has been available only for the SRP68 RNA-binding domain (RBD). Here we present the crystal structures of the SRP68 protein-binding domain (PBD) in complex with SRP72-PBD and of the SRP72-RBD bound to the SRP S domain (SRP RNA, SRP19 and SRP68) detailing all interactions of SRP72 within SRP. The SRP72-PBD is a tetratricopeptide repeat, which binds an extended linear motif of SRP68 with high affinity. The SRP72-RBD is a flexible peptide crawling along the 5e- and 5f-loops of SRP RNA. A conserved tryptophan inserts into the 5e-loop forming a novel type of RNA kink-turn stabilized by a potassium ion, which we define as K+-turn. In addition, SRP72-RBD remodels the 5f-loop involved in ribosome binding and visualizes SRP RNA plasticity. Docking of the S domain structure into cryo-electron microscopy density maps reveals multiple contact sites between SRP68/72 and the ribosome, and explains the role of SRP72 in the SRP pathway. PMID:27899666

  10. Ccr4-Not Regulates RNA Polymerase I Transcription and Couples Nutrient Signaling to the Control of Ribosomal RNA Biogenesis

    PubMed Central

    Laribee, R. Nicholas; Hosni-Ahmed, Amira; Workman, Jason J.; Chen, Hongfeng

    2015-01-01

    Ribosomal RNA synthesis is controlled by nutrient signaling through the mechanistic target of rapamycin complex 1 (mTORC1) pathway. mTORC1 regulates ribosomal RNA expression by affecting RNA Polymerase I (Pol I)-dependent transcription of the ribosomal DNA (rDNA) but the mechanisms involved remain obscure. This study provides evidence that the Ccr4-Not complex, which regulates RNA Polymerase II (Pol II) transcription, also functions downstream of mTORC1 to control Pol I activity. Ccr4-Not localizes to the rDNA and physically associates with the Pol I holoenzyme while Ccr4-Not disruption perturbs rDNA binding of multiple Pol I transcriptional regulators including core factor, the high mobility group protein Hmo1, and the SSU processome. Under nutrient rich conditions, Ccr4-Not suppresses Pol I initiation by regulating interactions with the essential transcription factor Rrn3. Additionally, Ccr4-Not disruption prevents reduced Pol I transcription when mTORC1 is inhibited suggesting Ccr4-Not bridges mTORC1 signaling with Pol I regulation. Analysis of the non-essential Pol I subunits demonstrated that the A34.5 subunit promotes, while the A12.2 and A14 subunits repress, Ccr4-Not interactions with Pol I. Furthermore, ccr4Δ is synthetically sick when paired with rpa12Δ and the double mutant has enhanced sensitivity to transcription elongation inhibition suggesting that Ccr4-Not functions to promote Pol I elongation. Intriguingly, while low concentrations of mTORC1 inhibitors completely inhibit growth of ccr4Δ, a ccr4Δ rpa12Δ rescues this growth defect suggesting that the sensitivity of Ccr4-Not mutants to mTORC1 inhibition is at least partially due to Pol I deregulation. Collectively, these data demonstrate a novel role for Ccr4-Not in Pol I transcriptional regulation that is required for bridging mTORC1 signaling to ribosomal RNA synthesis. PMID:25815716

  11. Occlusion of the Ribosome Binding Site Connects the Translational Initiation Frequency, mRNA Stability and Premature Transcription Termination

    PubMed Central

    Eriksen, Mette; Sneppen, Kim; Pedersen, Steen; Mitarai, Namiko

    2017-01-01

    Protein production is controlled by ribosome binding to the messenger RNA (mRNA), quantified in part by the binding affinity between the ribosome and the ribosome binding sequence on the mRNA. Using the E. coli lac operon as model, Ringquist et al. (1992) found a more than 1,000-fold difference in protein yield when varying the Shine-Dalgarno sequence and its distance to the translation start site. Their proposed model accounted for this large variation by only a variation in the binding affinity and the subsequent initiation rate. Here we demonstrate that the decrease in protein yield with weaker ribosome binding sites in addition is caused by a decreased mRNA stability, and by an increased rate of premature transcription termination. Using different ribosome binding site sequences of the E. coli lacZ gene, we found that an approximately 100-fold span in protein expression could be subdivided into three mechanisms that each affected expression 3- to 6-fold. Our experiments is consistent with a two-step ribosome initiation model, in which occlusion of the initial part of the mRNA by a ribosome simultaneously protects the mRNA from both premature transcription termination and degradation: The premature termination we suggest is coupled to the absence of occlusion that allows binding of transcription termination factor, possibly Rho. The mRNA stability is explained by occlusion that prevents binding of the degrading enzymes. In our proposed scenario, a mRNA with lower translation initiation rate would at the same time be “hit” by an increased premature termination and a shorter life-time. Our model further suggests that the transcription from most if not all natural promoters is substantially influenced by premature termination. PMID:28382022

  12. A rapid and simple pipeline for synthesis of mRNA-ribosome-V(H)H complexes used in single-domain antibody ribosome display.

    PubMed

    Bencurova, Elena; Pulzova, Lucia; Flachbartova, Zuzana; Bhide, Mangesh

    2015-06-01

    The single-domain antibody (VHH) is a promising building block for a number of antibody-based applications. Ribosome display can successfully be used in the production of VHH. However, the construction of the expression cassette, confirmation of the translation and proper folding of the nascent chain, and the purification of the ribosome complexes, remain cumbersome tasks. Additionally, selection of the most suitable expression system can be challenging. We have designed primers that will amplify virtually all Camelidae VHH. With the help of a double-overlap extension (OE) polymerase chain reaction (PCR) we have fused VHH with the F1 fragment (T7 promoter and species-independent translation sequence) and the F2 fragment (mCherry, Myc-tag, tether, SecM arrest sequence and 3' stem loop) to generate a full-length DNA cassette. OE-PCR generated fragments were incubated directly with cell-free lysates (Leishmania torentolae, rabbit reticulocyte or E. coli) for the synthesis of mRNA-VHH-mCherry-ribosome complexes in vitro. Alternatively, the cassette was ligated in pQE-30 vector and transformed into E. coli to produce ribosome complexes in vivo. The results showed that the same expression cassette could be used to synthesize ribosome complexes with different expression systems. mCherry reporter served to confirm the synthesis and proper folding of the nascent chain, Myc-tag was useful in the rapid purification of ribosome complexes, and combination of the SecM sequence and 3' stem loop made the cassette universal, both for cells-free and E. coli in vivo. This rapid and universal pipeline can effectively be used in antibody ribosome display and VHH production.

  13. Human Ribosomal RNA-Derived Resident MicroRNAs as the Transmitter of Information upon the Cytoplasmic Cancer Stress

    PubMed Central

    2016-01-01

    Dysfunction of ribosome biogenesis induces divergent ribosome-related diseases including ribosomopathy and occasionally results in carcinogenesis. Although many defects in ribosome-related genes have been investigated, little is known about contribution of ribosomal RNA (rRNA) in ribosome-related disorders. Meanwhile, microRNA (miRNA), an important regulator of gene expression, is derived from both coding and noncoding region of the genome and is implicated in various diseases. Therefore, we performed in silico analyses using M-fold, TargetScan, GeneCoDia3, and so forth to investigate RNA relationships between rRNA and miRNA against cellular stresses. We have previously shown that miRNA synergism is significantly correlated with disease and the miRNA package is implicated in memory for diseases; therefore, quantum Dynamic Nexus Score (DNS) was also calculated using MESer program. As a result, seventeen RNA sequences identical with known miRNAs were detected in the human rRNA and termed as rRNA-hosted miRNA analogs (rmiRNAs). Eleven of them were predicted to form stem-loop structures as pre-miRNAs, and especially one stem-loop was completely identical with hsa-pre-miR-3678 located in the non-rDNA region. Thus, these rmiRNAs showed significantly high DNS values, participation in regulation of cancer-related pathways, and interaction with nucleolar RNAs, suggesting that rmiRNAs may be stress-responsible resident miRNAs which transmit stress-tuning information in multiple levels. PMID:27517048

  14. RNA dimerization plays a role in ribosomal frameshifting of the SARS coronavirus.

    PubMed

    Ishimaru, Daniella; Plant, Ewan P; Sims, Amy C; Yount, Boyd L; Roth, Braden M; Eldho, Nadukkudy V; Pérez-Alvarado, Gabriela C; Armbruster, David W; Baric, Ralph S; Dinman, Jonathan D; Taylor, Deborah R; Hennig, Mirko

    2013-02-01

    Messenger RNA encoded signals that are involved in programmed -1 ribosomal frameshifting (-1 PRF) are typically two-stemmed hairpin (H)-type pseudoknots (pks). We previously described an unusual three-stemmed pseudoknot from the severe acute respiratory syndrome (SARS) coronavirus (CoV) that stimulated -1 PRF. The conserved existence of a third stem-loop suggested an important hitherto unknown function. Here we present new information describing structure and function of the third stem of the SARS pseudoknot. We uncovered RNA dimerization through a palindromic sequence embedded in the SARS-CoV Stem 3. Further in vitro analysis revealed that SARS-CoV RNA dimers assemble through 'kissing' loop-loop interactions. We also show that loop-loop kissing complex formation becomes more efficient at physiological temperature and in the presence of magnesium. When the palindromic sequence was mutated, in vitro RNA dimerization was abolished, and frameshifting was reduced from 15 to 5.7%. Furthermore, the inability to dimerize caused by the silent codon change in Stem 3 of SARS-CoV changed the viral growth kinetics and affected the levels of genomic and subgenomic RNA in infected cells. These results suggest that the homodimeric RNA complex formed by the SARS pseudoknot occurs in the cellular environment and that loop-loop kissing interactions involving Stem 3 modulate -1 PRF and play a role in subgenomic and full-length RNA synthesis.

  15. Epigenetic repression of ribosomal RNA transcription by ROCK-dependent aberrant cytoskeletal organization

    PubMed Central

    Wu, Tse-Hsiang; Kuo, Yuan-Yeh; Lee, Hsiao-Hui; Kuo, Jean-Cheng; Ou, Meng-Hsin; Chang, Zee-Fen

    2016-01-01

    It is known that ribosomal RNA (rRNA) synthesis is regulated by cellular energy and proliferation status. In this study, we investigated rRNA gene transcription in response to cytoskeletal stress. Our data revealed that the cell shape constrained by isotropic but not elongated micropatterns in HeLa cells led to a significant reduction in rRNA transcription dependent on ROCK. Expression of a dominant-active form of ROCK also repressed rRNA transcription. Isotropic constraint and ROCK over-activation led to different types of aberrant F-actin organization, but their suppression effects on rRNA transcription were similarly reversed by inhibition of histone deacetylase (HDAC) or overexpression of a dominant negative form of Nesprin, which shields the signal transmitted from actin filament to the nuclear interior. We further showed that the binding of HDAC1 to the active fraction of rDNA genes is increased by ROCK over-activation, thus reducing H3K9/14 acetylation and suppressing transcription. Our results demonstrate an epigenetic control of active rDNA genes that represses rRNA transcription in response to the cytoskeletal stress. PMID:27350000

  16. How are tRNAs and mRNA arranged in the ribosome? An attempt to correlate the stereochemistry of the tRNA-mRNA interaction with constraints imposed by the ribosomal topography.

    PubMed Central

    Lim, V; Venclovas, C; Spirin, A; Brimacombe, R; Mitchell, P; Müller, F

    1992-01-01

    Two tRNA molecules at the ribosomal A- and P-sites, with a relatively small angle between the planes of the L-shaped molecules, can be arranged in two mutually exclusive orientations. In one (the 'R'-configuration), the T-loop of the A-site tRNA faces the D-loop of the P-site tRNA, whereas in the other (the 'S'-configuration) the D-loop of the A-site tRNA faces the T-loop of the P-site tRNA. A number of stereochemical arguments, based on the crystal structure of 'free' tRNA, favour the R-configuration. In the ribosome, the CCA-ends of the tRNA molecules are 'fixed' at the base of the central protuberance (the peptidyl transferase centre) of the 50S subunit, and the anticodon loops lie in the neck region (the decoding site) of the 30S subunit. The translocation step is essentially a rotational movement of the tRNA from the A- to the P-site, and there is convincing evidence that the A-site must be located nearest to the L7/L12 protuberance of the 50S subunit. The mRNA in the two codon-anticodon duplexes lies on the 'inside' of the 'elbows' of the tRNA molecules (in both the S-type and R-type configurations), and runs up between the two molecules from the A- to the P-site in the 3' to 5'-direction. These considerations have the consequence that in the S-configuration the mRNA in the codon-anticodon duplexes is directed towards the 50S subunit, whereas in the R-configuration it is directed towards the 30S subunit. The results of site-directed cross-linking experiments, in particular cross-links to mRNA at positions within or very close to the codons interacting with A- or P-site tRNA, favour the latter situation. This conclusion is in direct contradiction to other current models for the arrangement of mRNA and tRNA on the ribosome. Images PMID:1614849

  17. MMB-GUI: a fast morphing method demonstrates a possible ribosomal tRNA translocation trajectory.

    PubMed

    Tek, Alex; Korostelev, Andrei A; Flores, Samuel Coulbourn

    2016-01-08

    Easy-to-use macromolecular viewers, such as UCSF Chimera, are a standard tool in structural biology. They allow rendering and performing geometric operations on large complexes, such as viruses and ribosomes. Dynamical simulation codes enable modeling of conformational changes, but may require considerable time and many CPUs. There is an unmet demand from structural and molecular biologists for software in the middle ground, which would allow visualization combined with quick and interactive modeling of conformational changes, even of large complexes. This motivates MMB-GUI. MMB uses an internal-coordinate, multiscale approach, yielding as much as a 2000-fold speedup over conventional simulation methods. We use Chimera as an interactive graphical interface to control MMB. We show how this can be used for morphing of macromolecules that can be heterogeneous in biopolymer type, sequence, and chain count, accurately recapitulating structural intermediates. We use MMB-GUI to create a possible trajectory of EF-G mediated gate-passing translocation in the ribosome, with all-atom structures. This shows that the GUI makes modeling of large macromolecules accessible to a wide audience. The morph highlights similarities in tRNA conformational changes as tRNA translocates from A to P and from P to E sites and suggests that tRNA flexibility is critical for translocation completion.

  18. Crystal Structure of a Luteoviral RNA Pseudoknot and Model for a Minimal Ribosomal Frameshifting Motif

    SciTech Connect

    Pallan, Pradeep S.; Marshall, William S.; Harp, Joel; Jewett III, Frederic C.; Wawrzak, Zdzislaw; Brown II, Bernard A.; Rich, Alexander; Egli, Martin

    2010-03-08

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

  19. MMB-GUI: a fast morphing method demonstrates a possible ribosomal tRNA translocation trajectory

    PubMed Central

    Tek, Alex; Korostelev, Andrei A.; Flores, Samuel Coulbourn

    2016-01-01

    Easy-to-use macromolecular viewers, such as UCSF Chimera, are a standard tool in structural biology. They allow rendering and performing geometric operations on large complexes, such as viruses and ribosomes. Dynamical simulation codes enable modeling of conformational changes, but may require considerable time and many CPUs. There is an unmet demand from structural and molecular biologists for software in the middle ground, which would allow visualization combined with quick and interactive modeling of conformational changes, even of large complexes. This motivates MMB-GUI. MMB uses an internal-coordinate, multiscale approach, yielding as much as a 2000-fold speedup over conventional simulation methods. We use Chimera as an interactive graphical interface to control MMB. We show how this can be used for morphing of macromolecules that can be heterogeneous in biopolymer type, sequence, and chain count, accurately recapitulating structural intermediates. We use MMB-GUI to create a possible trajectory of EF-G mediated gate-passing translocation in the ribosome, with all-atom structures. This shows that the GUI makes modeling of large macromolecules accessible to a wide audience. The morph highlights similarities in tRNA conformational changes as tRNA translocates from A to P and from P to E sites and suggests that tRNA flexibility is critical for translocation completion. PMID:26673695

  20. RNA chaperones stimulate formation and yield of the U3 snoRNA-pre-rRNA duplexes needed for eukaryotic ribosome biogenesis

    PubMed Central

    Gérczei, Tímea; Shah, Binal N.; Manzo, Anthony J.; Walter, Nils G.; Correll, Carl C.

    2010-01-01

    To satisfy the high demand for ribosome synthesis in rapidly growing eukaryotic cells, short duplexes between the U3 small nucleolar RNA (snoRNA) and the precursor ribosomal RNA (pre-rRNA) must form quickly and with high yield. These interactions, designated the U3-ETS and U3-18S duplexes, are essential to initiate the processing of small subunit rRNA. Previously, we showed in vitro that duplexes corresponding to those in Saccharomyces cerevisiae are only observed after addition of one of two proteins: Imp3p or Imp4p. Here, we used fluorescence-based and other in vitro assays to determine whether these proteins possess RNA chaperone activities and to assess whether these activities are sufficient to satisfy the duplex yield and rate requirements expected in vivo. Assembly of both proteins with the U3 snoRNA into a chaperone complex destabilizes a U3-stem structure, apparently to expose its 18S base-pairing site. As a result, the chaperone complex accelerates formation of the U3-18S duplex from an undetectable rate to one comparable to the intrinsic rate observed for hybridizing short duplexes. The chaperone complex also stabilizes the U3-ETS duplex by 2.7 kcal/mol. These chaperone activities provide high U3-ETS duplex yield and rapid U3-18S duplex formation over a broad concentration range to help ensure that the U3-pre-rRNA interactions limit neither ribosome biogenesis nor rapid cell growth. The thermodynamic and kinetic framework used is general and thus suitable to investigate the mechanism of action of other RNA chaperones. PMID:19482034

  1. Mechanical insights into ribosomal progression overcoming RNA G-quadruplex from periodical translation suppression in cells

    NASA Astrophysics Data System (ADS)

    Endoh, Tamaki; Sugimoto, Naoki

    2016-03-01

    G-quadruplexes formed on DNA and RNA can be roadblocks to movement of polymerases and ribosome on template nucleotides. Although folding and unfolding processes of the G-quadruplexes are deliberately studied in vitro, how the mechanical and physical properties of the G-quadruplexes affect intracellular biological systems is still unclear. In this study, mRNAs with G-quadruplex forming sequences located either in the 5‧ untranslated region (UTR) or in the open reading frame (ORF) were constructed to evaluate positional effects of the G-quadruplex on translation suppression in cells. Periodic fluctuation of translation suppression was observed at every three nucleotides within the ORF but not within the 5‧ UTR. The results suggested that difference in motion of ribosome at the 5‧ UTR and the ORF determined the ability of the G-quadruplex structure to act as a roadblock to translation in cells and provided mechanical insights into ribosomal progression to overcome the roadblock.

  2. Abiotrophia defectiva bleb-associated endophthalmitis confirmed with 16s ribosomal RNA sequencing.

    PubMed

    Hugo Lee, Ming-Han; Lawlor, Mitchell; Lee, Anne J

    2015-01-01

    One recognized complication of trabeculectomy with visually devastating potential is blebitis. We present a case of a 74-year-old woman with a culture and polymerase chain reaction-positive Abiotrophia defectiva bleb-associated endophthalmitis. Abiotrophia defectiva is a rare but possible cause of endophthalmitis secondary to blebitis and should be considered in culture-negative cases. Prompt identification, hence directed eradication, of the causative organism in such visually threatening cases may be facilitated by requesting polymerase chain reaction and 16S ribosomal RNA sequencing.

  3. Chemical probing of adenine residues within the secondary structure of rabbit /sup 18/S ribosomal RNA

    SciTech Connect

    Rairkar, A.; Rubino, H.M.; Lockard, R.E.

    1988-01-26

    The location of unpaired adenine residues within the secondary structure of rabbit /sup 18/S ribosomal RNA was determined by chemical probing. Naked /sup 18/S rRNA was first prepared by digestion of purified 40S subunits with matrix-bound proteinase K in sodium dodecyl sulfate, thereby omitting the use of nucleic acid denaturants. Adenines within naked /sup 18/S rRNA were chemically probed by using either diethyl pyrocarbonate or dimethyl sulfate, which specifically react with unpaired nucleotides. Adenine modification sites were identified by polyacrylamide sequencing gel electrophoresis either upon aniline-induced strand scission of /sup 32/P-end-labeled intact and fragmented rRNA or by primer extension using sequence-specific DNA oligomers with reverse transcriptase. The data indicate good agreement between the general pattern of adenine reactivity and the location of unpaired regions in /sup 18/S rRNA determined by comparative sequence analysis. The overall reactivity of adenine residues toward single-strand-specific chemical probes was, also, similar for both rabbit and Escherichia coli small rRNA. The number of strongly reactive adenines appearing within phylogenetically determined helical segments, however, was greater in rabbit /sup 18/S rRNA than for E. coli /sup 16/S rRNA. Some of these adenines were found clustered in specific helices. Such differences suggest a greater irregularity of many of the helical elements within mammalian /sup 18/S rRNA, as compared with prokaryotic /sup 16/S rRNA. These helical irregularities could be important for protein association and also may represent biologically relevant flexible regions of the molecule.

  4. Mapping of the RNA recognition site of Escherichia coli ribosomal protein S7.

    PubMed Central

    Robert, F; Gagnon, M; Sans, D; Michnick, S; Brakier-Gingras, L

    2000-01-01

    Bacterial ribosomal protein S7 initiates the folding of the 3' major domain of 16S ribosomal RNA by binding to its lower half. The X-ray structure of protein S7 from thermophilic bacteria was recently solved and found to be a modular structure, consisting of an alpha-helical domain with a beta-ribbon extension. To gain further insights into its interaction with rRNA, we cloned the S7 gene from Escherichia coli K12 into a pET expression vector and introduced 4 deletions and 12 amino acid substitutions in the protein sequence. The binding of each mutant to the lower half of the 3' major domain of 16S rRNA was assessed by filtration on nitrocellulose membranes. Deletion of the N-terminal 17 residues or deletion of the B hairpins (residues 72-89) severely decreased S7 affinity for the rRNA. Truncation of the C-terminal portion (residues 138-178), which includes part of the terminal alpha-helix, significantly affected S7 binding, whereas a shorter truncation (residues 148-178) only marginally influenced its binding. Severe effects were also observed with several strategic point mutations located throughout the protein, including Q8A and F17G in the N-terminal region, and K35Q, G54S, K113Q, and M115G in loops connecting the alpha-helices. Our results are consistent with the occurrence of several sites of contact between S7 and the 16S rRNA, in line with its role in the folding of the 3' major domain. PMID:11105763

  5. Molecular and cytological characterization of ribosomal RNA genes in Chenopodium quinoa and Chenopodium berlandieri.

    PubMed

    Maughan, P J; Kolano, B A; Maluszynska, J; Coles, N D; Bonifacio, A; Rojas, J; Coleman, C E; Stevens, M R; Fairbanks, D J; Parkinson, S E; Jellen, E N

    2006-07-01

    The nucleolus organizer region (NOR) and 5S ribosomal RNA (rRNA) genes are valuable as chromosome landmarks and in evolutionary studies. The NOR intergenic spacers (IGS) and 5S rRNA nontranscribed spacers (NTS) were PCR-amplified and sequenced from 5 cultivars of the Andean grain crop quinoa (Chenopodium quinoa Willd., 2n = 4x = 36) and a related wild ancestor (C. berlandieri Moq. subsp. zschackei (Murr) A. Zobel, 2n = 4x = 36). Length heterogeneity observed in the IGS resulted from copy number difference in subrepeat elements, small re arrangements, and species-specific indels, though the general sequence composition of the 2 species was highly similar. Fifteen of the 41 sequence polymorphisms identified among the C. quinoa lines were synapomorphic and clearly differentiated the highland and lowland ecotypes. Analysis of the NTS sequences revealed 2 basic NTS sequence classes that likely originated from the 2 allopolyploid subgenomes of C. quinoa. Fluorescence in situ hybridization (FISH) analysis showed that C. quinoa possesses an interstitial and a terminal pair of 5S rRNA loci and only 1 pair of NOR, suggesting a reduction in the number of rRNA loci during the evolution of this species. C. berlandieri exhibited variation in both NOR and 5S rRNA loci without changes in ploidy.

  6. Ribosomal RNA genes of Trypanosoma brucei. Cloning of a rRNA gene containing a mobile element.

    PubMed Central

    Hasan, G; Turner, M J; Cordingley, J S

    1982-01-01

    An ordered restriction map of the ribosomal RNA genes of Trypanosoma brucei brucei is presented. Bgl II fragments of T.b.brucei genomic DNA were cloned into pAT 153, and the clones containing rDNA identified. Restriction maps were established and the sense strands identified. One clone was shown by heteroduplex mapping to contain a 1.1 kb inserted sequence which was demonstrated to be widely distributed throughout the genomes of members of the subgenus Trypanozoon. However, in two other subgenera of Trypanosoma, Nannomonas and Schizotrypanum, the sequence is far less abundant. Analysis of the genomic DNA from two serodemes of T.b.brucei showed that the sequence was present in the rRNA of only one of them, implying that the sequence is a mobile element and that its appearance in rDNA is a comparitively recent occurrence. Images PMID:6294613

  7. Human Argonaute 2 Is Tethered to Ribosomal RNA through MicroRNA Interactions.

    PubMed

    Atwood, Blake L; Woolnough, Jessica L; Lefevre, Gaelle M; Saint Just Ribeiro, Mariana; Felsenfeld, Gary; Giles, Keith E

    2016-08-19

    The primary role of the RNAi machinery is to promote mRNA degradation within the cytoplasm in a microRNA-dependent manner. However, both Dicer and the Argonaute protein family have expanded roles in gene regulation within the nucleus. To further our understanding of this role, we have identified chromatin binding sites for AGO2 throughout the 45S region of the human rRNA gene. The location of these sites was mirrored by the positions of AGO2 cross-linking sites identified via PAR-CLIP-seq. AGO2 binding to the rRNA within the nucleus was confirmed by RNA immunoprecipitation and quantitative-PCR. To explore a possible mechanism by which AGO2 could be recruited to the rRNA, we identified 1174 regions within the 45S rRNA transcript that have the ability to form a perfect duplex with position 2-6 (seed sequence) of each microRNA expressed in HEK293T cells. Of these potential AGO2 binding sites, 479 occurred within experimentally verified AGO2-rRNA cross-linking sites. The ability of AGO2 to cross-link to rRNA was almost completely lost in a DICER knock-out cell line. The transfection of miR-92a-2-3p into the noDICE cell line facilitated AGO2 cross-linking at a region of the rRNA that has a perfect seed match at positions 3-8, including a single G-U base pair. Knockdown of AGO2 within HEK293T cells causes a slight, but statistically significant increase in the overall rRNA synthesis rate but did not impact the ratio of processing intermediates or the recruitment of the Pol I transcription factor UBTF.

  8. Crystallographic Studies of the Ribosomal A-Site Molecular Switches by Using Model RNA Oligomers.

    PubMed

    Kondo, Jiro

    2016-01-01

    An RNA molecular switch in the aminoacyl-tRNA decoding site (A site) of the ribosome plays a key role in the decoding process of the protein biosynthesis. The switch discriminates a single cognate-tRNA from near-cognate tRNAs by changing its conformation from "off" to "on" states and recognizing the first two base pairs of codon-anticodon mini-helix to check whether these base pairs are of the canonical Watson-Crick type or not. Aminoglycoside antibiotics specifically target the "on" state of the bacterial A-site molecular switch and disturb the fidelity of the decoding process, resulting to cell death. If it occurs in human who was given aminoglycosides, it can lead to undesirable side effects. In order to understand the molecular bases of the decoding and the antibacterial and toxic side effects of aminoglycosides, it is necessary to determine the three-dimensional structures of the A-site molecular switches both in the presence and absence of aminoglycosides. This chapter focuses on methods in crystallographic studies of the A-site switches by using model RNA oligomers. The methods can be utilized in crystallographic studies of any DNA/RNA oligomers.

  9. Akt activation enhances ribosomal RNA synthesis through casein kinase II and TIF-IA.

    PubMed

    Nguyen, Le Xuan Truong; Mitchell, Beverly S

    2013-12-17

    Transcription initiation factor I (TIF-IA) plays an essential role in regulating ribosomal RNA (rRNA) synthesis by tethering RNA polymerase I (Pol I) to the rDNA promoter. We have found that activated Akt enhances rRNA synthesis through the phosphorylation of casein kinase IIα (CK2α) on a threonine residue near its N terminus. CK2 in turn phosphorylates TIF-IA, thereby increasing rDNA transcription. Activated Akt also stabilizes TIF-IA, induces its translocation to the nucleolus, and enhances its interaction with Pol I. Treatment with AZD8055, an inhibitor of both Akt and mammalian target of rapamycin phosphorylation, but not with rapamycin, disrupts Akt-mediated TIF-IA stability, translocation, and activity. These data support a model in which activated Akt enhances rRNA synthesis both by preventing TIF-IA degradation and phosphorylating CK2α, which in turn phosphorylates TIF-IA. This model provides an explanation for the ability of activated Akt to promote cell proliferation and, potentially, transformation.

  10. Investigating the ion dependence of the first unfolding step of GTPase-Associating Center ribosomal RNA.

    PubMed

    Hayatshahi, Hamed S; Bergonzo, Christina; Cheatham Iii, Thomas E

    2017-04-13

    The interactions in the tertiary structure of a ribosomal RNA fragment in the GTPase Associating Center (GAC) have been experimentally studied, but the roles of the bound and diffuse cations in its folding pathway have not yet been fully elucidated. Melting experiments have shown that the temperature of the first of the two distinguishable transitions in the unfolding pathway of the GAC RNA can be regulated by altering the magnesium concentration, yet the physical interpretation of such ion-dependent effects on folding have not been clearly understood in spite of the availability of crystal structures that depict many GAC RNA-ion interactions. Here, we use umbrella sampling and molecular dynamics (MD) simulations to provide a physical description for the first transition in this unfolding pathway, with a focus on the role of a chelated magnesium ion. Our results indicate that the presence of cations mediating the local interaction of two loops stabilizes the folded state relative to the unfolded or partially folded states. Also, our findings suggest that a bridging magnesium ion between the two loops improves the stabilizing effect. This is consistent with the multistep unfolding pathway proposed for the GAC RNA and highlights the importance of ions in the first unfolding step. The results suggest how MD simulations can provide insight into RNA unfolding pathways as a complementary approach to experiments.

  11. Molecular Identification of Ptychodera flava (Hemichordata: Enteropneusta): Reconsideration in Light of Nucleotide Polymorphism in the 18S Ribosomal RNA Gene.

    PubMed

    Urata, Makoto

    2015-06-01

    Seven nuclear and mitochondrial DNA markers were examined in 12 specimens of Ptychodera flava, a model acorn worm used in molecular biology, collected in Japan from three local populations with different modes of living. A comparison of intraspecific results did not show genetically isolated populations despite the species' enclave habitats and asexual reproduction. Moreover, both the nuclear 18S ribosomal RNA gene and mitochondrial 16S ribosomal RNA gene sequences were identical to those from Moorea in French Polynesia, nearly 10,000 kilometers away from Japan. I also provide the first definitive information regarding polymorphisms in 18S ribosomal RNA gene, the external transcribed spacer (ETS), internal transcribed spacers (ITS), and mitochondrial cytochrome c oxidase subunit 1 (mtCO1) sequence in hemichordates using newly designed primer sets, and I show both high larval vagility and certain criteria for the molecular identification of this species.

  12. Antibiotics that bind to the A site of the large ribosomal subunit can induce mRNA translocation.

    PubMed

    Ermolenko, Dmitri N; Cornish, Peter V; Ha, Taekjip; Noller, Harry F

    2013-02-01

    In the absence of elongation factor EF-G, ribosomes undergo spontaneous, thermally driven fluctuation between the pre-translocation (classical) and intermediate (hybrid) states of translocation. These fluctuations do not result in productive mRNA translocation. Extending previous findings that the antibiotic sparsomycin induces translocation, we identify additional peptidyl transferase inhibitors that trigger productive mRNA translocation. We find that antibiotics that bind the peptidyl transferase A site induce mRNA translocation, whereas those that do not occupy the A site fail to induce translocation. Using single-molecule FRET, we show that translocation-inducing antibiotics do not accelerate intersubunit rotation, but act solely by converting the intrinsic, thermally driven dynamics of the ribosome into translocation. Our results support the idea that the ribosome is a Brownian ratchet machine, whose intrinsic dynamics can be rectified into unidirectional translocation by ligand binding.

  13. The ribosome triggers the stringent response by RelA via a highly distorted tRNA

    PubMed Central

    Agirrezabala, Xabier; Fernández, Israel S; Kelley, Ann C; Cartón, David Gil; Ramakrishnan, Venki; Valle, Mikel

    2013-01-01

    The bacterial stringent response links nutrient starvation with the transcriptional control of genes. This process is initiated by the stringent factor RelA, which senses the presence of deacylated tRNA in the ribosome as a symptom of amino-acid starvation to synthesize the alarmone (p)ppGpp. Here we report a cryo-EM study of RelA bound to ribosomes bearing cognate, deacylated tRNA in the A-site. The data show that RelA on the ribosome stabilizes an unusual distorted form of the tRNA, with the acceptor arm making contact with RelA and far from its normal location in the peptidyl transferase centre. PMID:23877429

  14. The bacterial toxin RelE induces specific mRNA cleavage in the A site of the eukaryote ribosome

    PubMed Central

    Andreev, Dmitri; Hauryliuk, Vasili; Terenin, Ilya; Dmitriev, Sergey; Ehrenberg, Måns; Shatsky, Ivan

    2008-01-01

    RelE/RelB is a well-characterized toxin–anti-toxin pair involved in nutritional stress responses in Bacteria and Archae. RelE lacks any eukaryote homolog, but we demonstrate here that it efficiently and specifically cleaves mRNA in the A site of the eukaryote ribosome. The cleavage mechanism is similar to that in bacteria, showing the feasibility of A-site cleavage of mRNA for regulatory purposes also in eukaryotes. RelE cleavage in the A-site codon of a stalled eukaryote ribosome is precise and easily monitored, making “RelE printing” a useful complement to toeprinting to determine the exact mRNA location on the eukaryote ribosome and to probe the occupancy of its A site. PMID:18083838

  15. Ribosomal Protein S12 and Aminoglycoside Antibiotics Modulate A-site mRNA Cleavage and Transfer-Messenger RNA Activity in Escherichia coli*

    PubMed Central

    Holberger, Laura E.; Hayes, Christopher S.

    2009-01-01

    Translational pausing in Escherichia coli can lead to mRNA cleavage within the ribosomal A-site. A-site mRNA cleavage is thought to facilitate transfer-messenger RNA (tmRNA)·SmpB- mediated recycling of stalled ribosome complexes. Here, we demonstrate that the aminoglycosides paromomycin and streptomycin inhibit A-site cleavage of stop codons during inefficient translation termination. Aminoglycosides also induced stop codon read-through, suggesting that these antibiotics alleviate ribosome pausing during termination. Streptomycin did not inhibit A-site cleavage in rpsL mutants, which express streptomycin-resistant variants of ribosomal protein S12. However, rpsL strains exhibited reduced A-site mRNA cleavage compared with rpsL+ cells. Additionally, tmRNA·SmpB-mediated SsrA peptide tagging was significantly reduced in several rpsL strains but could be fully restored in a subset of mutants when treated with streptomycin. The streptomycin-dependent rpsL(P90K) mutant also showed significantly lower levels of A-site cleavage and tmRNA·SmpB activity. Mutations in rpsD (encoding ribosomal protein S4), which suppressed streptomycin dependence, were able to partially restore A-site cleavage to rpsL(P90K) cells but failed to increase tmRNA·SmpB activity. Taken together, these results show that perturbations to A-site structure and function modulate A-site mRNA cleavage and tmRNA·SmpB activity. We propose that tmRNA·SmpB binds to streptomycin-resistant rpsL ribosomes less efficiently, leading to a partial loss of ribosome rescue function in these mutants. PMID:19776006

  16. Pokeweed antiviral protein region Gly209-Lys225 is critical for RNA N-glycosidase activity of the prokaryotic ribosome.

    PubMed

    Nagasawa, Yoshimi; Fujii, Kazuyuki; Yoshikawa, Takafumi; Kobayashi, Yoshinori; Kondo, Toshiya

    2008-05-01

    Pokeweed antiviral protein (PAP) isolated from Phytolacca americana is a ribosome-inactivating protein (RIP) that has RNA N-glycosidase (RNG) activity towards both eukaryotic and prokaryotic ribosomes. In contrast, karasurin-A (KRN), a RIP from Trichosanthes kirilowii var. japonica, is active only on eukaryotic ribosomes. Stepwise selection of chimera proteins between PAP and KRN indicated that the C-terminal region of PAP (residues 209-225) was critical for RNG activity toward prokaryotic ribosomes. When the region of PAP (residues 209-225) was replaced with the corresponding region of KRN the PAP chimera protein, like KRN, was active only on eukaryotic ribosomes. Furthermore, insertion of the region of PAP (residues 209-225) into the KRN chimera protein resulted not only in the detectable RNG activity toward prokaryotic ribosome, but also activity toward the eukaryotic ribosomes as well that was seven-fold higher than for the original KRN. In this study, the possibility of genetic manipulation of the activity and substrate specificity of RIPs is demonstrated.

  17. Evolution of ribosomal RNA gene copy number on the sex chromosomes of Drosophila melanogaster.

    PubMed

    Lyckegaard, E M; Clark, A G

    1991-07-01

    A diverse array of cellular and evolutionary forces--including unequal crossing-over, magnification, compensation, and natural selection--is at play modulating the number of copies of ribosomal RNA (rRNA) genes on the X and Y chromosomes of Drosophila. Accurate estimates of naturally occurring distributions of copy numbers on both the X and Y chromosomes are needed in order to explore the evolutionary end result of these forces. Estimates of relative copy numbers of the ribosomal DNA repeat, as well as of the type I and type II inserts, were obtained for a series of 96 X chromosomes and 144 Y chromosomes by using densitometric measurements of slot blots of genomic DNA from adult D. melanogaster bearing appropriate deficiencies that reveal chromosome-specific copy numbers. Estimates of copy number were put on an absolute scale with slot blots having serial dilutions both of the repeat and of genomic DNA from nonpolytene larval brain and imaginal discs. The distributions of rRNA copy number are decidedly skewed, with a long tail toward higher copy numbers. These distributions were fitted by a population genetic model that posits three different types of exchange events--sister-chromatid exchange, intrachromatid exchange, and interchromosomal crossing-over. In addition, the model incorporates natural selection, because experimental evidence shows that there is a minimum number of functional elements necessary for survival. Adequate fits of the model were found, indicating that either natural selection also eliminates chromosomes with high copy number or that the rate of intrachromatid exchange exceeds the rate of interchromosomal exchange.

  18. Matching the crystallographic structure of ribosomal protein S7 to a three-dimensional model of the 16S ribosomal RNA.

    PubMed Central

    Tanaka, I; Nakagawa, A; Hosaka, H; Wakatsuki, S; Mueller, F; Brimacombe, R

    1998-01-01

    Two recently published but independently derived structures, namely the X-ray crystallographic structure of ribosomal protein S7 and the "binding pocket" for this protein in a three-dimensional model of the 16S rRNA, have been correlated with one another. The known rRNA-protein interactions for S7 include a minimum binding site, a number of footprint sites, and two RNA-protein crosslink sites on the 16S rRNA, all of which form a compact group in the published 16S rRNA model (despite the fact that these interactions were not used as primary modeling constraints in building that model). The amino acids in protein S7 that are involved in the two crosslinks to 16S rRNA have also been determined in previous studies, and here we have used these sites to orient the crystallographic structure of S7 relative to its rRNA binding pocket. Some minor alterations were made to the rRNA model to improve the fit. In the resulting structure, the principal positively charged surface of the protein is in contact with the 16S rRNA, and all of the RNA-protein interaction data are satisfied. The quality of the fit gives added confidence as to the validity of the 16S rRNA model. Protein S7 is furthermore known to be crosslinked both to P site-bound tRNA and to mRNA at positions upstream of the P site codon; the matched S7-16S rRNA structure makes a prediction as to the location of this crosslink site within the protein molecule. PMID:9582096

  19. Cap- and initiator tRNA-dependent initiation of TYMV polyprotein synthesis by ribosomes: evaluation of the Trojan horse model for TYMV RNA translation.

    PubMed

    Matsuda, Daiki; Dreher, Theo W

    2007-01-01

    Turnip yellow mosaic virus (TYMV) RNA directs the translation of two overlapping open reading frames. Competing models have been previously published to explain ribosome access to the downstream polyprotein cistron. The Trojan horse model, based on cell-free experiments, proposes noncanonical cap-independent initiation in which the 3'-terminal tRNA-like structure (TLS) functionally replaces initiator tRNA, and the valine bound to the TLS becomes cis-incorporated into viral protein. The initiation coupling model, based on in vivo expression and ribosome toe-printing studies, proposes a variation of canonical leaky scanning. Here, we have re-examined the wheat germ extract experiments that led to the Trojan horse model, incorporating a variety of controls. We report that (1) translation in vitro from the polyprotein AUG of TYMV RNA is unchanged after removal of the 3' TLS but is stimulated by the presence of a 5'-cap; (2) the presence of free cap analog or edeine (which interferes with initiation at the ribosomal P site and its tRNA(i) (Met) involvement) inhibits translation from the polyprotein AUG; (3) the toe-prints of immediately post-initiation ribosomes on TYMV RNA are similar with and without an intact TLS; and (4) significant deacylation of valyl-TYMV RNA in wheat germ extract can complicate the detection of cis-incorporation. These results favor the initiation coupling model.

  20. Secondary structure of rabbit 18S ribosomal RNA determined from biochemical and phylogenetic data

    SciTech Connect

    Rairkar, A.; Rubino, H.; Lockard, R.E.

    1986-05-01

    To understand the functional role of 18S rRNA in the eukaryotic 40S subunit, its higher order structure must first be determined. Native deproteinized 18S rRNA was isolated from purified rabbit 40S subunits, fractionated on SDS-sucrose density gradients and concentrated using centricon-30 microconcentrators. The structure of native 18S rRNA was probed chemically with both diethylpyrocarbonate (DEPC) and dimethyl sulfate (DMS) which react with unpaired adenosine and guanosine residues, respectively. After /sup 32/P-end-labeling of intact and fragmented RNA, the modified nucleotides were identified by polyacrylamide sequencing gel electrophoresis upon aniline induced strand scission. On the basis of both the biochemical and phylogenetic data, a secondary structure model is proposed which includes the two major G + C rich insertion elements. A comparison of the structure data with previously published phylogenetic models suggests an instability of certain predicted helices. These unstable helices may normally be stabilized by ribosomal proteins and could represent the flexible elements involved in biologically significant conformational switches within 40S subunit.

  1. Simple peptides derived from the ribosomal core potentiate RNA polymerase ribozyme function

    NASA Astrophysics Data System (ADS)

    Tagami, Shunsuke; Attwater, James; Holliger, Philipp

    2017-03-01

    The emergence of functional interactions between nucleic acids and polypeptides was a key transition in the origin of life and remains at the heart of all biology. However, how and why simple non-coded peptides could have become critical for RNA function is unclear. Here, we show that putative ancient peptide segments from the cores of both ribosomal subunits enhance RNA polymerase ribozyme (RPR) function, as do derived homopolymeric peptides comprising lysine or the non-proteinogenic lysine analogues ornithine or, to a lesser extent, diaminobutyric acid, irrespective of chirality or chiral purity. Lysine decapeptides enhance RPR function by promoting holoenzyme assembly through primer-template docking, accelerate RPR evolution, and allow RPR-catalysed RNA synthesis at near physiological (≥1 mM) Mg2+ concentrations, enabling templated RNA synthesis within membranous protocells. Our results outline how compositionally simple, mixed-chirality peptides may have augmented the functional potential of early RNAs and promoted the emergence of the first protocells.

  2. Simple peptides derived from the ribosomal core potentiate RNA polymerase ribozyme function.

    PubMed

    Tagami, Shunsuke; Attwater, James; Holliger, Philipp

    2017-04-01

    The emergence of functional interactions between nucleic acids and polypeptides was a key transition in the origin of life and remains at the heart of all biology. However, how and why simple non-coded peptides could have become critical for RNA function is unclear. Here, we show that putative ancient peptide segments from the cores of both ribosomal subunits enhance RNA polymerase ribozyme (RPR) function, as do derived homopolymeric peptides comprising lysine or the non-proteinogenic lysine analogues ornithine or, to a lesser extent, diaminobutyric acid, irrespective of chirality or chiral purity. Lysine decapeptides enhance RPR function by promoting holoenzyme assembly through primer-template docking, accelerate RPR evolution, and allow RPR-catalysed RNA synthesis at near physiological (≥1 mM) Mg(2+) concentrations, enabling templated RNA synthesis within membranous protocells. Our results outline how compositionally simple, mixed-chirality peptides may have augmented the functional potential of early RNAs and promoted the emergence of the first protocells.

  3. Model of EF4-induced ribosomal state transitions and mRNA translocation

    NASA Astrophysics Data System (ADS)

    Xie, Ping

    2014-08-01

    EF4, a highly conserved protein present in bacteria, mitochondria and chloroplasts, can bind to both the posttranslocation and pretranslocation ribosomal complexes. When binding to the posttranslocation state, it catalyzes backward translocation to a pretranslocation state. When binding to the pretranslocation state, it catalyzes transition to another pretranslocation state that is similar and possibly identical to that resulting from the posttranslocation state bound by EF4, and competes with EF-G to regulate the elongation cycle. However, the molecular mechanism on how EF4 induces state transitions and mRNA translocation remains unclear. Here, we present both the model for state transitions induced by EF4 binding to the posttranslocation state and that by EF4 binding to the pretranslocation state, based on which we study the kinetics of EF4-induced state transitions and mRNA translocation, giving quantitative explanations of the available experimental data. Moreover, we present some predicted results on state transitions and mRNA translocation induced by EF4 binding to the pretranslocation state complexed with the mRNA containing a duplex region.

  4. A functional involvement of ABCE1, eukaryotic ribosome recycling factor, in nonstop mRNA decay in Drosophila melanogaster cells.

    PubMed

    Kashima, Isao; Takahashi, Masaki; Hashimoto, Yoshifumi; Sakota, Eri; Nakamura, Yoshikazu; Inada, Toshifumu

    2014-11-01

    When ribosomes encounter mRNAs lacking stop codons, two quality-control machineries, NSD for nonstop mRNA decay and ribosome quality control (RQC) for co-translational degradation of the nonstop protein by the proteasome, are triggered to eliminate aberrant molecules. In yeast, it is known that Dom34 (a homolog of eRF1) and Ltn1 (an E3 ubiquitin ligase) play crucial roles in NSD and RQC, respectively, by triggering ribosome rescue at the 3' end of nonstop mRNAs and proteasome-dependent polypeptide degradation. Here we confirmed the essential role of Ltn1 in RQC for nonstop products in Drosophila cells, and further uncovered a functional role of ABCE1, a eukaryotic ribosome recycling factor, in NSD in Drosophila cells.

  5. Use of PCR primers and probes based on the 23S rRNA and internal transcription spacer (ITS) gene sequence for the detection and enumerization of Lactobacillus acidophilus and Lactobacillus plantarum in feed supplements.

    PubMed

    Tsai, Cheng-Chih; Lai, Chieh-Hsien; Yu, Bi; Tsen, Hau-Yang

    2010-06-01

    Novel polymerase chain reaction (PCR) primers designed from the 16S-23S internal transcription spacer (ITS) rRNA and 23S rRNA genes, respectively, were used for the specific detection of Lactobacillus acidophilus and Lactobacillus plantarum. Molecular weights of the PCR products were 221 and 599 bp, respectively. Strains of L. acidophilus and L. plantarum obtained from the culture center, dairy products, infant stool and other samples, could be identified with these PCR primers. DNAs from other lactic acid bacteria (LAB) species including strains of Lactobacillus pentosus which was closely related to L. plantarum, and bacteria species other than LAB, would not generate the false positive results. When this PCR primer set was used for the detection of L. acidophilus and L. plantarum in feed supplement or feed starter samples, reliable results were obtained. Furthermore, when these L. acidophilus or L. plantarum specific primers were used as DNA probes for the colony hybridization of L. acidophilus and L. plantarum, the viable cells of these LAB species in culture and feed supplements or starter products could be identified and enumerized. The method described here thus offers a rapid and economic way to inspect and assure the quality of the feed supplements or fermentation starters.

  6. Characterization of the tRNA and ribosome-dependent pppGpp-synthesis by recombinant stringent factor from Escherichia coli.

    PubMed

    Knutsson Jenvert, Rose-Marie; Holmberg Schiavone, Lovisa

    2005-02-01

    Stringent factor is a ribosome-dependent ATP:GTP pyrophosphoryl transferase that synthesizes (p)ppGpp upon nutrient deprivation. It is activated by unacylated tRNA in the ribosomal amino-acyl site (A-site) but it is unclear how activation occurs. A His-tagged stringent factor was isolated by affinity-chromatography and precipitation. This procedure yielded a protein of high purity that displayed (a) a low endogenous pyrophosphoryl transferase activity that was inhibited by the antibiotic tetracycline; (b) a low ribosome-dependent activity that was inhibited by the A-site specific antibiotics thiostrepton, micrococcin, tetracycline and viomycin; (c) a tRNA- and ribosome-dependent activity amounting to 4500 pmol pppGpp per pmol stringent factor per minute. Footprinting analysis showed that stringent factor interacted with ribosomes that contained tRNAs bound in classical states. Maximal activity was seen when the ribosomal A-site was presaturated with unacylated tRNA. Less tRNA was required to reach maximal activity when stringent factor and unacylated tRNA were added simultaneously to ribosomes, suggesting that stringent factor formed a complex with tRNA in solution that had higher affinity for the ribosomal A-site. However, tRNA-saturation curves, performed at two different ribosome/stringent factor ratios and filter-binding assays, did not support this hypothesis.

  7. Regulation of ribosomal RNA expression across the lifespan is fine-tuned by maternal diet before implantation.

    PubMed

    Denisenko, Oleg; Lucas, Emma S; Sun, Congshan; Watkins, Adam J; Mar, Daniel; Bomsztyk, Karol; Fleming, Tom P

    2016-07-01

    Cells and organisms respond to nutrient deprivation by decreasing global rates of transcription, translation and DNA replication. To what extent such changes can be reversed is largely unknown. We examined the effect of maternal dietary restriction on RNA synthesis in the offspring. Low protein diet fed either throughout gestation or for the preimplantation period alone reduced cellular RNA content across fetal somatic tissues during challenge and increased it beyond controls in fetal and adult tissues after challenge release. Changes in transcription of ribosomal RNA, the major component of cellular RNA, were responsible for this phenotype as evidenced by matching alterations in RNA polymerase I density and DNA methylation at ribosomal DNA loci. Cellular levels of the ribosomal transcription factor Rrn3 mirrored the rRNA expression pattern. In cell culture experiments, Rrn3 overexpression reduced rDNA methylation and increased rRNA expression; the converse occurred after inhibition of Rrn3 activity. These observations define novel mechanism where poor nutrition before implantation irreversibly alters basal rates of rRNA transcription thereafter in a process mediated by rDNA methylation and Rrn3 factor.

  8. Ribosomal frameshifting in the CCR5 mRNA is regulated by miRNAs and the NMD pathway

    PubMed Central

    Belew, Ashton Trey; Meskauskas, Arturas; Musalgaonkar, Sharmishtha; Advani, Vivek M.; Sulima, Sergey O.; Kasprzak, Wojciech K.; Shapiro, Bruce A.; Dinman, Jonathan D.

    2015-01-01

    Programmed –1 ribosomal frameshift (–1 PRF) signals redirect translating ribosomes to slip back one base on messenger RNAs. Although well characterized in viruses, how these elements may regulate cellular gene expression is not understood. Here we describe a –1 PRF signal in the human mRNA encoding CCR5, the HIV-1 co-receptor. CCR5 mRNA-mediated –1 PRF is directed by an mRNA pseudoknot, and is stimulated by at least two microRNAs. Mapping the mRNA–miRNA interaction suggests that formation of a triplex RNA structure stimulates –1 PRF. A –1 PRF event on the CCR5 mRNA directs translating ribosomes to a premature termination codon, destabilizing it through the nonsense-mediated mRNA decay pathway. At least one additional mRNA decay pathway is also involved. Functional –1 PRF signals that seem to be regulated by miRNAs are also demonstrated in mRNAs encoding six other cytokine receptors, suggesting a novel mode through which immune responses may be fine-tuned in mammalian cells. PMID:25043019

  9. Genome-wide quantification of 5' phosphorylated mRNA degradation intermediates for analysis of ribosome dynamics

    PubMed Central

    Pelechano, Vicent; Wei, Wu; Steinmetz, Lars M.

    2015-01-01

    Co-translational mRNA degradation is a widespread process in which 5’-3’ exonucleolytic degradation follows the last translating ribosome, producing an in vivo ribosomal footprint of mRNA molecules’ 5’ positions. To study this process, we developed 5PSeq, a method that profiles the genome-wide abundance of mRNA degradation intermediates with 5'-phosphorylated ends and allows the study of ribosome dynamics. The method targets 5’P mRNA ends by ligating an oligonucleotide to the 5’P RNA ends. rRNA molecules are then depleted, and 5’P mRNAs are subject to reverse transcription followed by Illumina high-throughput sequencing. 5PSeq can identify translational pauses at rare codons that are often masked when using alternative methods. This approach can be applied to previously extracted RNA samples, is straightforward, and does not require polyribosome purification or in vitro RNA footprinting. The protocol we describe can be applied to S. cerevisiae and potentially other eukaryotic organisms. 3 days are required to generate 5PSeq libraries. PMID:26820793

  10. Low levels of ribosomal RNA partly account for the very high photosynthetic phosphorus-use efficiency of Proteaceae species

    PubMed Central

    Sulpice, Ronan; Ishihara, Hirofumi; Schlereth, Armin; Cawthray, Gregory R; Encke, Beatrice; Giavalisco, Patrick; Ivakov, Alexander; Arrivault, StÉphanie; Jost, Ricarda; Krohn, Nicole; Kuo, John; Laliberté, Etienne; Pearse, Stuart J; Raven, John A; Scheible, Wolf-rüdiger; Teste, François; Veneklaas, Erik J; Stitt, Mark; Lambers, Hans

    2014-01-01

    Abstract Proteaceae species in south-western Australia occur on phosphorus- (P) impoverished soils. Their leaves contain very low P levels, but have relatively high rates of photosynthesis. We measured ribosomal RNA (rRNA) abundance, soluble protein, activities of several enzymes and glucose 6-phosphate (Glc6P) levels in expanding and mature leaves of six Proteaceae species in their natural habitat. The results were compared with those for Arabidopsis thaliana. Compared with A. thaliana, immature leaves of Proteaceae species contained very low levels of rRNA, especially plastidic rRNA. Proteaceae species showed slow development of the photosynthetic apparatus (‘delayed greening’), with young leaves having very low levels of chlorophyll and Calvin–Benson cycle enzymes. In mature leaves, soluble protein and Calvin–Benson cycle enzyme activities were low, but Glc6P levels were similar to those in A. thaliana. We propose that low ribosome abundance contributes to the high P efficiency of these Proteaceae species in three ways: (1) less P is invested in ribosomes; (2) the rate of growth and, hence, demand for P is low; and (3) the especially low plastidic ribosome abundance in young leaves delays formation of the photosynthetic machinery, spreading investment of P in rRNA. Although Calvin–Benson cycle enzyme activities are low, Glc6P levels are maintained, allowing their effective use. PMID:24895754

  11. Genetic characterization of clinical acanthamoeba isolates from Japan using nuclear and mitochondrial small subunit ribosomal RNA.

    PubMed

    Rahman, Md Moshiur; Yagita, Kenji; Kobayashi, Akira; Oikawa, Yosaburo; Hussein, Amjad I A; Matsumura, Takahiro; Tokoro, Masaharu

    2013-08-01

    Because of an increased number of Acanthamoeba keratitis (AK) along with associated disease burdens, medical professionals have become more aware of this pathogen in recent years. In this study, by analyzing both the nuclear 18S small subunit ribosomal RNA (18S rRNA) and mitochondrial 16S rRNA gene loci, 27 clinical Acanthamoeba strains that caused AK in Japan were classified into 3 genotypes, T3 (3 strains), T4 (23 strains), and T5 (one strain). Most haplotypes were identical to the reference haplotypes reported from all over the world, and thus no specificity of the haplotype distribution in Japan was found. The T4 sub-genotype analysis using the 16S rRNA gene locus also revealed a clear sub-conformation within the T4 cluster, and lead to the recognition of a new sub-genotype T4i, in addition to the previously reported sub-genotypes T4a-T4h. Furthermore, 9 out of 23 strains in the T4 genotype were identified to a specific haplotype (AF479533), which seems to be a causal haplotype of AK. While heterozygous nuclear haplotypes were observed from 2 strains, the mitochondrial haplotypes were homozygous as T4 genotype in the both strains, and suggested a possibility of nuclear hybridization (mating reproduction) between different strains in Acanthamoeba. The nuclear 18S rRNA gene and mitochondrial 16S rRNA gene loci of Acanthamoeba spp. possess different unique characteristics usable for the genotyping analyses, and those specific features could contribute to the establishment of molecular taxonomy for the species complex of Acanthamoeba.

  12. An RNA molecular switch: Intrinsic flexibility of 23S rRNA Helices 40 and 68 5’-UAA/5’-GAN internal loops studied by molecular dynamics methods

    PubMed Central

    Réblová, Kamila; Střelcová, Zora; Kulhánek, Petr; Beššeová, Ivana; Mathews, David H.; Nostrand, Keith Van; Yildirim, Ilyas; Turner, Douglas H.; Šponer, Jiří

    2010-01-01

    Functional RNA molecules such as ribosomal RNAs frequently contain highly conserved internal loops with a 5’-UAA/5’-GAN (UAA/GAN) consensus sequence. The UAA/GAN internal loops adopt distinctive structure inconsistent with secondary structure predictions. The structure has a narrow major groove and forms a trans Hoogsteen/Sugar edge (tHS) A/G base pair followed by an unpaired stacked adenine, a trans Watson-Crick/Hoogsteen (tWH) U/A base pair and finally by a bulged nucleotide (N). The structure is further stabilized by a three-adenine stack and base-phosphate interaction. In the ribosome, the UAA/GAN internal loops are involved in extensive tertiary contacts, mainly as donors of A-minor interactions. Further, this sequence can adopt an alternative 2D/3D pattern stabilized by a four-adenine stack involved in a smaller number of tertiary interactions. The solution structure of an isolated UAA/GAA internal loop shows substantially rearranged base pairing with three consecutive non-Watson-Crick base pairs. Its A/U base pair adopts an incomplete cis Watson-Crick/Sugar edge (cWS) A/U conformation instead of the expected Watson-Crick arrangement. We performed 3.1 µs of explicit solvent molecular dynamics (MD) simulations of the X-ray and NMR UAA/GAN structures, supplemented by MM-PBSA free energy calculations, locally enhanced sampling (LES) runs, targeted MD (TMD) and nudged elastic band (NEB) analysis. We compared parm99 and parmbsc0 force fields and net-neutralizing Na+ vs. excess salt KCl ion environments. Both force fields provide a similar description of the simulated structures, with the parmbsc0 leading to modest narrowing of the major groove. The excess salt simulations also cause a similar effect. While the NMR structure is entirely stable in simulations, the simulated X-ray structure shows considerable widening of the major groove, loss of base-phosphate interaction and other instabilities. The alternative X-ray geometry even undergoes conformational

  13. Ribosome-associated GTPases: the role of RNA for GTPase activation.

    PubMed

    Clementi, Nina; Polacek, Norbert

    2010-01-01

    The GTPase super-family comprises a variety of G proteins found in all three domains of life. Although they are participating in completely different processes like signal transduction, protein biosynthesis and regulation of cell proliferation, they all share a highly conserved G domain and use a common mechanism for GTP hydrolysis. Exact timing in hydrolyzing the bound GTP serves as a molecular switch to initiate diverse cellular reactions. Classical GTPases depend on external proteins to fire GTP hydrolysis (GAPs), and following the GTPase reaction to exchange GDP for GTP (GEFs), converting the GTPase into the active state again. In recent years it became clear that there are many GTPases that do not follow this classical switch mode scheme. Certain ribosome-associated GTPases are not reliant on other GEF proteins to exchange GDP for GTP. Furthermore many of these G proteins are not activated by external GAPs, but by evolutionarily ancient molecules, namely by RNA.

  14. A phylogeny of cockroaches and related insects based on DNA sequence of mitochondrial ribosomal RNA genes.

    PubMed Central

    Kambhampati, S

    1995-01-01

    Cockroaches are among the most ancient winged insects, the earliest fossils dating back to about 400 million years. Several conflicting phylogenies for cockroach families, subfamilies, and genera have been proposed in the past. In addition, the relationship of Cryptocercidae to other cockroach families and the relationship between the cockroach, Cryptocercus punctulatus, and the termite, Mastotermes darwiniensis, have generated debate. In this paper, a phylogeny for cockroaches, mantids, and termites based on DNA sequence of the mitochondrial ribosomal RNA genes is presented. The results indicated that cockroaches are a monophyletic group, whose sister group is Mantoidea. The inferred relationship among cockroach families was in agreement with the presently accepted phylogeny. However, there was only partial congruence at the subfamily and the generic levels. The phylogeny inferred here does not support a close relationship between C. punctulatus and M. darwiniensis. The apparent synapomorphies of these two species are likely a manifestation of convergent evolution because there are similarities in biology and habitat. PMID:7534409

  15. Ribosome bypassing at serine codons as a test of the model of selective transfer RNA charging

    PubMed Central

    Lindsley, Dale; Bonthuis, Paul; Gallant, Jonathan; Tofoleanu, Teodora; Elf, Johan; Ehrenberg, Måns

    2005-01-01

    Recently, a model of the flux of amino acids through transfer RNAs (tRNAs) and into protein has been developed. The model predicts that the charging level of different isoacceptors carrying the same amino acid respond very differently to variation in supply of the amino acid or of the rate of charging. It has also been shown that ribosome bypassing is specifically stimulated at ‘hungry' codons calling for an aminoacyl-tRNA in short supply. We have constructed two reporters of bypassing, which differ only in the identity of the serine codon subjected to starvation. The stimulation of bypassing as a function of starvation differed greatly between the two serine codons, in good agreement with the quantitative predictions of the model. PMID:15678161

  16. Identification of ribosomal RNA methyltransferase gene ermF in Riemerella anatipestifer.

    PubMed

    Luo, Hongyan; Liu, Mafeng; Wang, Lanying; Zhou, Wangshu; Wang, Mingshu; Cheng, Anchun; Jia, Renyong; Chen, Shun; Sun, Kunfeng; Yang, Qiao; Chen, Xiaoyue; Zhu, Dekang

    2015-01-01

    Riemerella anatipestifer is a major bacterial pathogen of waterfowl, globally responsible for avian septicaemic disease. As chemotherapy is the predominant method for the prevention and treatment of R. anatipestifer infection in poultry, the widespread use of antibiotics has favoured the emergence of antibiotic-resistant strains. However, little is known about R. anatipestifer susceptibility to macrolide antibiotics and its resistance mechanism. We report for the first time the identification of a macrolide resistance mechanism in R. anatipestifer that is mediated by the ribosomal RNA methyltransferase ermF. We identified the presence of the ermF gene in 64/206 (31%) R. anatipestifer isolates from different regions in China. An ermF deletion strain was constructed to investigate the function of the ermF gene on the resistance to high levels of macrolides. The ermF mutant strain showed significantly decreased resistance to macrolide and lincosamide, exhibiting 1024-, 1024-, 4- and >2048-fold reduction in the minimum inhibitory concentrations for erythromycin, azithromycin, tylosin and lincomycin, respectively. Furthermore, functional analysis of ermF expression in E. coli XL1-blue showed that the R. anatipestifer ermF gene was functional in E. coli XL1-blue and conferred resistance to high levels of erythromycin (100 µg/ml), supporting the hypothesis that the ermF gene is associated with high-level macrolide resistance. Our work suggests that ribosomal RNA modification mediated by the ermF methyltransferase is the predominant mechanism of resistance to erythromycin in R. anatipestifer isolates.

  17. Combined heat shock protein 90 and ribosomal RNA sequence phylogeny supports multiple replacements of dinoflagellate plastids.

    PubMed

    Shalchian-Tabrizi, Kamran; Minge, Marianne A; Cavalier-Smith, Tom; Nedreklepp, Joachim M; Klaveness, Dag; Jakobsen, Kjetill S

    2006-01-01

    Dinoflagellates harbour diverse plastids obtained from several algal groups, including haptophytes, diatoms, cryptophytes, and prasinophytes. Their major plastid type with the accessory pigment peridinin is found in the vast majority of photosynthetic species. Some species of dinoflagellates have other aberrantly pigmented plastids. We sequenced the nuclear small subunit (SSU) ribosomal RNA (rRNA) gene of the "green" dinoflagellate Gymnodinium chlorophorum and show that it is sister to Lepidodinium viride, indicating that their common ancestor obtained the prasinophyte (or other green alga) plastid in one event. As the placement of dinoflagellate species that acquired green algal or haptophyte plastids is unclear from small and large subunit (LSU) rRNA trees, we tested the usefulness of the heat shock protein (Hsp) 90 gene for dinoflagellate phylogeny by sequencing it from four species with aberrant plastids (G. chlorophorum, Karlodinium micrum, Karenia brevis, and Karenia mikimotoi) plus Alexandrium tamarense, and constructing phylogenetic trees for Hsp90 and rRNAs, separately and together. Analyses of the Hsp90 and concatenated data suggest an ancestral origin of the peridinin-containing plastid, and two independent replacements of the peridinin plastid soon after the early radiation of the dinoflagellates. Thus, the Hsp90 gene seems to be a promising phylogenetic marker for dinoflagellate phylogeny.

  18. End-targeting proteomics of isolated chromatin segments of a mammalian ribosomal RNA gene promoter

    PubMed Central

    Ide, Satoru; Dejardin, Jerome

    2015-01-01

    The unbiased identification of proteins associated with specific loci is crucial for understanding chromatin-based processes. The proteomics of isolated chromatin fragment (PICh) method has previously been developed to purify telomeres and identify associated proteins. This approach is based on the affinity capture of endogenous chromatin segments by hybridization with oligonucleotide containing locked nucleic acids. However, PICh is only efficient with highly abundant genomic targets, limiting its applicability. Here we develop an approach for identifying factors bound to the promoter region of the ribosomal RNA genes that we call end-targeting PICh (ePICh). Using ePICh, we could specifically enrich the RNA polymerase I pre-initiation complex, including the selectivity factor 1. The high purity of the ePICh material allowed the identification of ZFP106, a novel factor regulating transcription initiation by targeting RNA polymerase I to the promoter. Our results demonstrate that ePICh can uncover novel proteins controlling endogenous regulatory elements in mammals. PMID:25812914

  19. Analysis of the interaction between bovine mitochondrial 28 S ribosomal subunits and mRNA.

    PubMed

    Farwell, M A; Schirawski, J; Hager, P W; Spremulli, L L

    1996-11-11

    The small subunit of the bovine mitochondrial ribosome forms a tight complex with mRNAs. This [28 S:mRNA] complex forms as readily on circular mRNAs as on linear mRNAs indicating that a free 5' end on the mRNA is not required for the interaction observed. The effects of monovalent cations on the equilibrium association constant and on the forward and reverse rate constants governing this interaction have been determined. Monovalent cations have a strong effect on the forward rate constant. Increasing the KCl concentration from 1 mM to 100 mM reduces kon by nearly 100-fold. Monovalent cations have only a small effect on the reverse rate constant, koff'. Analysis of these data indicates that the rate laws governing the formation and dissociation of the [28 S:mRNA] complex cannot be deduced from the chemical equation. This observation suggests that there are "hidden intermediates' in the formation and dissociation of this complex. The implications of these observations are discussed in terms of a model for the interaction between the mitochondrial 28 S subunit and mRNAs.

  20. Structure of ERA in complex with the 3′ end of 16S rRNA: Implications for ribosome biogenesis

    SciTech Connect

    Tu, Chao; Zhou, Xiaomei; Tropea, Joseph E.; Austin, Brian P.; Waugh, David S.; Court, Donald L.; Ji, Xinhua

    2009-10-09

    ERA, composed of an N-terminal GTPase domain followed by an RNA-binding KH domain, is essential for bacterial cell viability. It binds to 16S rRNA and the 30S ribosomal subunit. However, its RNA-binding site, the functional relationship between the two domains, and its role in ribosome biogenesis remain unclear. We have determined two crystal structures of ERA, a binary complex with GDP and a ternary complex with a GTP-analog and the {sub 1531}AUCACCUCCUUA{sub 1542} sequence at the 3' end of 16S rRNA. In the ternary complex, the first nine of the 12 nucleotides are recognized by the protein. We show that GTP binding is a prerequisite for RNA recognition by ERA and that RNA recognition stimulates its GTP-hydrolyzing activity. Based on these and other data, we propose a functional cycle of ERA, suggesting that the protein serves as a chaperone for processing and maturation of 16S rRNA and a checkpoint for assembly of the 30S ribosomal subunit. The AUCA sequence is highly conserved among bacteria, archaea, and eukaryotes, whereas the CCUCC, known as the anti-Shine-Dalgarno sequence, is conserved in noneukaryotes only. Therefore, these data suggest a common mechanism for a highly conserved ERA function in all three kingdoms of life by recognizing the AUCA, with a 'twist' for noneukaryotic ERA proteins by also recognizing the CCUCC.

  1. Identification of characteristic oligonucleotides in the bacterial 16S ribosomal RNA sequence dataset

    NASA Technical Reports Server (NTRS)

    Zhang, Zhengdong; Willson, Richard C.; Fox, George E.

    2002-01-01

    MOTIVATION: The phylogenetic structure of the bacterial world has been intensively studied by comparing sequences of 16S ribosomal RNA (16S rRNA). This database of sequences is now widely used to design probes for the detection of specific bacteria or groups of bacteria one at a time. The success of such methods reflects the fact that there are local sequence segments that are highly characteristic of particular organisms or groups of organisms. It is not clear, however, the extent to which such signature sequences exist in the 16S rRNA dataset. A better understanding of the numbers and distribution of highly informative oligonucleotide sequences may facilitate the design of hybridization arrays that can characterize the phylogenetic position of an unknown organism or serve as the basis for the development of novel approaches for use in bacterial identification. RESULTS: A computer-based algorithm that characterizes the extent to which any individual oligonucleotide sequence in 16S rRNA is characteristic of any particular bacterial grouping was developed. A measure of signature quality, Q(s), was formulated and subsequently calculated for every individual oligonucleotide sequence in the size range of 5-11 nucleotides and for 15mers with reference to each cluster and subcluster in a 929 organism representative phylogenetic tree. Subsequently, the perfect signature sequences were compared to the full set of 7322 sequences to see how common false positives were. The work completed here establishes beyond any doubt that highly characteristic oligonucleotides exist in the bacterial 16S rRNA sequence dataset in large numbers. Over 16,000 15mers were identified that might be useful as signatures. Signature oligonucleotides are available for over 80% of the nodes in the representative tree.

  2. Characterization of mitochondrial ribosomal RNA genes in gadiformes: sequence variations, secondary structural features, and phylogenetic implications.

    PubMed

    Bakke, Ingrid; Johansen, Steinar

    2002-10-01

    Secondary structure features of mitochondrial ribosomal RNAs (mt-rRNAs) of bony fishes were investigated by a DNA sequence alignment approach. The small subunit (SSU) and large subunit (LSU) mt-rRNA genes were found to contain several additional variable regions compared to their mammalian counterparts. Fish mt-LSU rRNA genes were found to be longer than the mammalians due to increased length of some of the variable regions. The 5' and 3' ends of Atlantic cod mt-rRNAs were precisely mapped. The 3' ends of mt-SSU rRNAs were found to be homogenous and mono-adenylated, whereas that of the mt-LSU rRNAs were heterogenous and oligo-adenylated. The 5' ends of mt-SSU rRNAs appeared to be heterogenous, corresponding to the presumed first and second positions of the gene. Sequences of the central domain and the D-domain of the mt-SSU and mt-LSU rRNA genes, respectively, were determined and characterized for 11 gadiform species (representing the families Gadidae, Lotidae, Ranicipitidae, Merlucciidae, Phycidae, and Macrouridae) and one Lophiidae species. Detailed secondary structure models of the RNA regions are presented for the Atlantic cod (Gadus morhua) and Roundnose grenadier (Coryphaeonides rupestris). Saturation plots revealed that DNA nucleotide positions corresponding to unpaired RNA regions become saturated with transitions at sequence divergence levels about 0.15. Phylogenetic analyses revealed some aspects of gadiform relationships. Gadidae was identified as the most derived of the gadiform families. Lotidae was found to be the family closest related to Gadidae, and Ranicipitidae was also recognized as a derived gadiform taxon.

  3. [Characterization of Black and Dichothrix Cyanobacteria Based on the 16S Ribosomal RNA Gene Sequence

    NASA Technical Reports Server (NTRS)

    Ortega, Maya

    2010-01-01

    My project focuses on characterizing different cyanobacteria in thrombolitic mats found on the island of Highborn Cay, Bahamas. Thrombolites are interesting ecosystems because of the ability of bacteria in these mats to remove carbon dioxide from the atmosphere and mineralize it as calcium carbonate. In the future they may be used as models to develop carbon sequestration technologies, which could be used as part of regenerative life systems in space. These thrombolitic communities are also significant because of their similarities to early communities of life on Earth. I targeted two cyanobacteria in my research, Dichothrix spp. and whatever black is, since they are believed to be important to carbon sequestration in these thrombolitic mats. The goal of my summer research project was to molecularly identify these two cyanobacteria. DNA was isolated from each organism through mat dissections and DNA extractions. I ran Polymerase Chain Reactions (PCR) to amplify the 16S ribosomal RNA (rRNA) gene in each cyanobacteria. This specific gene is found in almost all bacteria and is highly conserved, meaning any changes in the sequence are most likely due to evolution. As a result, the 16S rRNA gene can be used for bacterial identification of different species based on the sequence of their 16S rRNA gene. Since the exact sequence of the Dichothrix gene was unknown, I designed different primers that flanked the gene based on the known sequences from other taxonomically similar cyanobacteria. Once the 16S rRNA gene was amplified, I cloned the gene into specialized Escherichia coli cells and sent the gene products for sequencing. Once the sequence is obtained, it will be added to a genetic database for future reference to and classification of other Dichothrix sp.

  4. Characterization and Physical Mapping of Ribosomal RNA Gene Families in Plantago

    PubMed Central

    DHAR, MANOJ K.; FRIEBE, BERND; KAUL, SANJANA; GILL, BIKRAM S.

    2006-01-01

    • Background and Aims The organization of rRNA genes in cultivated Plantago ovata Forsk. and several of its wild allies was analysed to gain insight into the phylogenetic relationships of these species in the genus which includes some 200 species. • Methods Specific primers were designed to amplify the internal transcribed spacer (ITS1 and ITS2) regions from seven Plantago species and the resulting fragments were cloned and sequenced. Similarly, using specific primers, the 5S rRNA genes from these species were amplified and subsequently cloned. Fluorescence in-situ hybridization (FISH) was used for physical mapping of 5S and 45S ribosomal RNA genes. • Results The ITS1 region is 19–29 bp longer than the ITS2 in different Plantago species. The 5S rRNA gene-repeating unit varies in length from 289 to 581 bp. Coding regions are highly conserved across species, but the non-transcribed spacers (NTS) do not match any database sequences. The clone from the cultivated species P. ovata was used for physical mapping of these genes by FISH. Four species have one FISH site while three have two FISH sites. In P. lanceolata and P. rhodosperma, the 5S and 45S (18S-5·8S-25S) sites are coupled. • Conclusions Characterization of 5S and 45S rRNA genes has indicated a possible origin of P. ovata, the only cultivated species of the genus and also the only species with x = 4, from a species belonging to subgenus Psyllium. Based on the studies reported here, P. ovata is closest to P. arenaria, although on the basis of other data the two species have been placed in different subgenera. FISH mapping can be used as an efficient tool to help determine phylogenetic relationships in the genus Plantago and show the interrelationship between P. lanceolata and P. lagopus. PMID:16481363

  5. Comparative analysis of Pseudomonas syringae pv. actinidiae and pv. phaseolicola based on phaseolotoxin-resistant ornithine carbamoyltransferase gene (argK) and 16S-23S rRNA intergenic spacer sequences.

    PubMed

    Sawada, H; Takeuchi, T; Matsuda, I

    1997-01-01

    Pseudomonas syringae pv. phaseolicola, which causes halo blight on various legumes, and pv. actinidiae, responsible for canker or leaf spot on actinidia plants, are known as phaseolotoxin producers, and the former possesses phaseolotoxin-resistant ornithine carbamoyltransferase (ROCT) which confers resistance to the toxin. We confirmed that the latter is also resistant to phaseolotoxin and possesses ROCT, and we compared the two pathovars by using sequence data of the ROCT gene and the intergenic spacer region located between the 16S and 23S rRNA genes (16S-23S spacer region) as an index. It was found that the identical ROCT gene (argK) is contained not only in bean isolates of P. syringae pv. phaseolicola in Mexico and the United States but also in bean isolates in Japan and Canada, and that it is also distributed in the kudzu (Pueraria lobata) isolates of P. syringae pv. phaseolicola. Moreover, the kiwifruit and tara vine isolates of P. syringae pv. actinidiae were also found to possess the identical argK. On the contrary, the 16S-23S spacer regions showed a significant level of sequence variation between P. syringae pv. actinidiae and pv. phaseolicola, suggesting that these two pathovars evolved differently from each other in the phylogenetic development. The fact that even synonymous substitution has not occurred in argK among these strains despite their extreme differences in phylogenetic evolution and geographical distribution suggests that it was only recently in evolutionary time that argK was transferred from its origin to P. syringae pv. actinidiae and/or pv. phaseolicola.

  6. Comparative analysis of Pseudomonas syringae pv. actinidiae and pv. phaseolicola based on phaseolotoxin-resistant ornithine carbamoyltransferase gene (argK) and 16S-23S rRNA intergenic spacer sequences.

    PubMed Central

    Sawada, H; Takeuchi, T; Matsuda, I

    1997-01-01

    Pseudomonas syringae pv. phaseolicola, which causes halo blight on various legumes, and pv. actinidiae, responsible for canker or leaf spot on actinidia plants, are known as phaseolotoxin producers, and the former possesses phaseolotoxin-resistant ornithine carbamoyltransferase (ROCT) which confers resistance to the toxin. We confirmed that the latter is also resistant to phaseolotoxin and possesses ROCT, and we compared the two pathovars by using sequence data of the ROCT gene and the intergenic spacer region located between the 16S and 23S rRNA genes (16S-23S spacer region) as an index. It was found that the identical ROCT gene (argK) is contained not only in bean isolates of P. syringae pv. phaseolicola in Mexico and the United States but also in bean isolates in Japan and Canada, and that it is also distributed in the kudzu (Pueraria lobata) isolates of P. syringae pv. phaseolicola. Moreover, the kiwifruit and tara vine isolates of P. syringae pv. actinidiae were also found to possess the identical argK. On the contrary, the 16S-23S spacer regions showed a significant level of sequence variation between P. syringae pv. actinidiae and pv. phaseolicola, suggesting that these two pathovars evolved differently from each other in the phylogenetic development. The fact that even synonymous substitution has not occurred in argK among these strains despite their extreme differences in phylogenetic evolution and geographical distribution suggests that it was only recently in evolutionary time that argK was transferred from its origin to P. syringae pv. actinidiae and/or pv. phaseolicola. PMID:8979356

  7. Mapping translation 'hot-spots' in live cells by tracking single molecules of mRNA and ribosomes

    PubMed Central

    Katz, Zachary B; English, Brian P; Lionnet, Timothée; Yoon, Young J; Monnier, Nilah; Ovryn, Ben; Bathe, Mark; Singer, Robert H

    2016-01-01

    Messenger RNA localization is important for cell motility by local protein translation. However, while single mRNAs can be imaged and their movements tracked in single cells, it has not yet been possible to determine whether these mRNAs are actively translating. Therefore, we imaged single β-actin mRNAs tagged with MS2 stem loops colocalizing with labeled ribosomes to determine when polysomes formed. A dataset of tracking information consisting of thousands of trajectories per cell demonstrated that mRNAs co-moving with ribosomes have significantly different diffusion properties from non-translating mRNAs that were exposed to translation inhibitors. These data indicate that ribosome load changes mRNA movement and therefore highly translating mRNAs move slower. Importantly, β-actin mRNA near focal adhesions exhibited sub-diffusive corralled movement characteristic of increased translation. This method can identify where ribosomes become engaged for local protein production and how spatial regulation of mRNA-protein interactions mediates cell directionality. DOI: http://dx.doi.org/10.7554/eLife.10415.001 PMID:26760529

  8. Local structural and environmental factors define the efficiency of an RNA pseudoknot involved in programmed ribosomal frameshift process.

    PubMed

    Gupta, Asmita; Bansal, Manju

    2014-10-16

    In programmed -1 ribosomal frameshift, an RNA pseudoknot stalls the ribosome at specific sequence and restarts translation in a new reading frame. A precise understanding of structural characteristics of these pseudoknots and their PRF inducing ability has not been clear to date. To investigate this phenomenon, we have studied various structural aspects of a -1 PRF inducing RNA pseudoknot from BWYV using extensive molecular dynamics simulations. A set of functional and poorly functional forms, for which previous mutational data were available, were chosen for analysis. These structures differ from each other by either single base substitutions or base-pair replacements from the native structure. We have rationalized how certain mutations in RNA pseudoknot affect its function; e.g., a specific base substitution in loop 2 stabilizes the junction geometry by forming multiple noncanonical hydrogen bonds, leading to a highly rigid structure that could effectively resist ribosome-induced unfolding, thereby increasing efficiency. While, a CG to AU pair substitution in stem 1 leads to loss of noncanonical hydrogen bonds between stems and loop, resulting in a less stable structure and reduced PRF inducing ability, inversion of a pair in stem 2 alters specific base-pair geometry that might be required in ribosomal recognition of nucleobase groups, negatively affecting pseudoknot functioning. These observations illustrate that the ability of an RNA pseudoknot to induce -1 PRF with an optimal rate depends on several independent factors that contribute to either the local conformational variability or geometry.

  9. Mapping translation 'hot-spots' in live cells by tracking single molecules of mRNA and ribosomes.

    PubMed

    Katz, Zachary B; English, Brian P; Lionnet, Timothée; Yoon, Young J; Monnier, Nilah; Ovryn, Ben; Bathe, Mark; Singer, Robert H

    2016-01-13

    Messenger RNA localization is important for cell motility by local protein translation. However, while single mRNAs can be imaged and their movements tracked in single cells, it has not yet been possible to determine whether these mRNAs are actively translating. Therefore, we imaged single β-actin mRNAs tagged with MS2 stem loops colocalizing with labeled ribosomes to determine when polysomes formed. A dataset of tracking information consisting of thousands of trajectories per cell demonstrated that mRNAs co-moving with ribosomes have significantly different diffusion properties from non-translating mRNAs that were exposed to translation inhibitors. These data indicate that ribosome load changes mRNA movement and therefore highly translating mRNAs move slower. Importantly, β-actin mRNA near focal adhesions exhibited sub-diffusive corralled movement characteristic of increased translation. This method can identify where ribosomes become engaged for local protein production and how spatial regulation of mRNA-protein interactions mediates cell directionality.

  10. Fluctuations between multiple EF-G-induced chimeric tRNA states during translocation on the ribosome

    NASA Astrophysics Data System (ADS)

    Adio, Sarah; Senyushkina, Tamara; Peske, Frank; Fischer, Niels; Wintermeyer, Wolfgang; Rodnina, Marina V.

    2015-06-01

    The coupled translocation of transfer RNA and messenger RNA through the ribosome entails large-scale structural rearrangements, including step-wise movements of the tRNAs. Recent structural work has visualized intermediates of translocation induced by elongation factor G (EF-G) with tRNAs trapped in chimeric states with respect to 30S and 50S ribosomal subunits. The functional role of the chimeric states is not known. Here we follow the formation of translocation intermediates by single-molecule fluorescence resonance energy transfer. Using EF-G mutants, a non-hydrolysable GTP analogue, and fusidic acid, we interfere with either translocation or EF-G release from the ribosome and identify several rapidly interconverting chimeric tRNA states on the reaction pathway. EF-G engagement prevents backward transitions early in translocation and increases the fraction of ribosomes that rapidly fluctuate between hybrid, chimeric and posttranslocation states. Thus, the engagement of EF-G alters the energetics of translocation towards a flat energy landscape, thereby promoting forward tRNA movement.

  11. Bacterial clade with the ribosomal RNA operon on a small plasmid rather than the chromosome.

    PubMed

    Anda, Mizue; Ohtsubo, Yoshiyuki; Okubo, Takashi; Sugawara, Masayuki; Nagata, Yuji; Tsuda, Masataka; Minamisawa, Kiwamu; Mitsui, Hisayuki

    2015-11-17

    rRNA is essential for life because of its functional importance in protein synthesis. The rRNA (rrn) operon encoding 16S, 23S, and 5S rRNAs is located on the "main" chromosome in all bacteria documented to date and is frequently used as a marker of chromosomes. Here, our genome analysis of a plant-associated alphaproteobacterium, Aureimonas sp. AU20, indicates that this strain has its sole rrn operon on a small (9.4 kb), high-copy-number replicon. We designated this unusual replicon carrying the rrn operon on the background of an rrn-lacking chromosome (RLC) as the rrn-plasmid. Four of 12 strains close to AU20 also had this RLC/rrn-plasmid organization. Phylogenetic analysis showed that those strains having the RLC/rrn-plasmid organization represented one clade within the genus Aureimonas. Our finding introduces a previously unaddressed viewpoint into studies of genetics, genomics, and evolution in microbiology and biology in general.

  12. Time-Dependent Decay of mRNA and Ribosomal RNA during Platelet Aging and Its Correlation with Translation Activity.

    PubMed

    Angénieux, Catherine; Maître, Blandine; Eckly, Anita; Lanza, François; Gachet, Christian; de la Salle, Henri

    2016-01-01

    Previous investigations have indicated that RNAs are mostly present in the minor population of the youngest platelets, whereas translation in platelets could be biologically important. To attempt to solve this paradox, we studied changes in the RNA content of reticulated platelets, i.e., young cells brightly stained by thiazole orange (TObright), a fluorescent probe for RNAs. We provoked in mice strong thrombocytopenia followed by dramatic thrombocytosis characterized by a short period with a vast majority of reticulated platelets. During thrombocytosis, the TObright platelet count rapidly reached a maximum, after which TOdim platelets accumulated, suggesting that most of the former were converted into the latter within 12 h. Experiments on platelets, freshly isolated or incubated ex vivo at 37°C, indicated that their "RNA content", here corresponding to the amounts of extracted RNA, and the percentage of TObright platelets were positively correlated. The "RNA Content" normalized to the number of platelets could be 20 to 40 fold higher when 80-90% of the cells were reticulated (20-40 fg/platelet), than when only 5-10% of control cells were TObright (less than 1fg/platelet). TObright platelets, incubated ex vivo at 37°C or transfused into mice, became TOdim within 24 h. Ex vivo at 37°C, platelets lost about half of their ribosomal and beta actin RNAs within 6 hours, and more than 98% of them after 24 hours. Accordingly, fluorescence in situ hybridization techniques confirmed the presence of beta actin mRNAs in most reticulated-enriched platelets, but detected them in only a minor subset of control platelets. In vitro, constitutive translation decreased considerably within less than 6 hours, questioning how protein synthesis in platelets, especially in non-reticulated ones, could have a biological function in vivo. Nevertheless, constitutive transient translation in young platelets under pathological conditions characterized by a dramatic increase in circulating

  13. How EF-Tu can contribute to efficient proofreading of aa-tRNA by the ribosome

    NASA Astrophysics Data System (ADS)

    Noel, Jeffrey K.; Whitford, Paul C.

    2016-10-01

    It has long been recognized that the thermodynamics of mRNA-tRNA base pairing is insufficient to explain the high fidelity and efficiency of aminoacyl-tRNA (aa-tRNA) selection by the ribosome. To rationalize this apparent inconsistency, Hopfield proposed that the ribosome may improve accuracy by utilizing a multi-step kinetic proofreading mechanism. While biochemical, structural and single-molecule studies have provided a detailed characterization of aa-tRNA selection, there is a limited understanding of how the physical-chemical properties of the ribosome enable proofreading. To this end, we probe the role of EF-Tu during aa-tRNA accommodation (the proofreading step) through the use of energy landscape principles, molecular dynamics simulations and kinetic models. We find that the steric composition of EF-Tu can reduce the free-energy barrier associated with the first step of accommodation: elbow accommodation. We interpret this effect within an extended kinetic model of accommodation and show how EF-Tu can contribute to efficient and accurate proofreading.

  14. Natural-abundance stable carbon isotopes of small-subunit ribosomal RNA (SSU rRNA) from Guaymas Basin (Mexico)

    NASA Astrophysics Data System (ADS)

    MacGregor, B. J.; Mendlovitz, H.; Albert, D.; Teske, A. P.

    2012-12-01

    Small-subunit ribosomal RNA (SSU rRNA) is a phylogenetically informative molecule found in all species. Because it is poorly preserved in most environments, it is a useful marker for active microbial populations. We are using the natural-abundance stable carbon isotopic composition of specific microbial groups to help identify the carbon substrates contributing to microbial biomass in a variety of marine environments. At Guaymas Basin, hydrothermal fluids interact with abundant sedimentary organic carbon to produce natural gas and petroleum. Where this reaches the sediment surface, it can support dense patches of seafloor life, including Beggiatoa mats. We report here on the stable carbon isotopic composition of SSU rRNA from a Beggiatoa mat transect, a cold background site, a warm site with high oil concentration, and a second Beggiatoa mat. The central part of the transect mat overlay the steepest temperature gradient, and was visually dominated by orange Beggiatoa. This was fringed by white Beggiatoa mat and bare, but still warm, sediment. Methane concentrations were saturating beneath the orange and white mats and at the oily site, lower beneath bare sediment, and below detection at the background site. Our initial hypotheses were that rRNA isotopic composition would be strongly influenced by methane supply, and that archaeal rRNA might be lighter than bacterial due to contributions from methanogens and anaerobic methane oxidizers. We used biotin-labeled oligonucleotides to capture Bacterial and Archaeal SSU rRNA for isotopic determination. Background-site rRNA was isotopically heaviest, and bacterial RNA from below 2 cm at the oily site was lightest, consistent with control by methane. Within the transect mat, however, the pattern was more complicated; at some sediment depths, rRNA from the mat periphery was isotopically lightest. Part of this may be due to the spatially and temporally variable paths followed by hydrothermal fluid, which can include horizontal

  15. 18S ribosomal RNA gene sequences of Cochliopodium (Himatismenida) and the phylogeny of Amoebozoa.

    PubMed

    Kudryavtsev, Alexander; Bernhard, Detlef; Schlegel, Martin; Chao, Ema E Y; Cavalier-Smith, Thomas

    2005-08-01

    Cochliopodium is a very distinctive genus of discoid amoebae covered by a dorsal tectum of carbohydrate microscales. Its phylogenetic position is unclear, since although sharing many features with naked "gymnamoebae", the tectum sets it apart. We sequenced 18S ribosomal RNA genes from three Cochliopodium species (minus, spiniferum and Cochliopodium sp., a new species resembling C. minutum). Phylogenetic analysis shows Cochliopodium as robustly holophyletic and within Amoebozoa, in full accord with morphological data. Cochliopodium is always one of the basal branches within Amoebozoa but its precise position is unstable. In Bayesian analysis it is sister to holophyletic Glycostylida, but distance trees mostly place it between Dermamoeba and a possibly artifactual long-branch cluster including Thecamoeba. These positions are poorly supported and basal amoebozoan branching ill-resolved, making it unclear whether Discosea (Glycostylida, Himatismenida, Dermamoebida) is holophyletic; however, Thecamoeba seems not specifically related to Dermamoeba. We also sequenced the small-subunit rRNA gene of Vannella persistens, which constantly grouped with other Vannella species, and two Hartmannella strains. Our trees suggest that Vexilliferidae, Variosea and Hartmannella are polyphyletic, confirming the existence of two very distinct Hartmannella clades: that comprising H. cantabrigiensis and another divergent species is sister to Glaeseria, whilst Hartmannella vermiformis branches more deeply.

  16. DNA structural variation affects complex formation and promoter melting in ribosomal RNA transcription.

    PubMed

    Marilley, M; Radebaugh, C A; Geiss, G K; Laybourn, P J; Paule, M R

    2002-08-01

    Eukaryotic ribosomal RNA promoters exhibit an unusual conservation of non-canonical DNA structure (curvature, twist angle and duplex stability) despite a lack of primary sequence conservation. This raises the possibility that rRNA transcription factors might utilize structural anomalies in their sequence recognition process. We have analyzed in detail the interaction of the polymerase I transcription factor TIF-IB from Acanthmoeba castellanii with the CORE promoter. TIF-IB interacts primarily with the minor groove of the promoter. By correlating the effects on transcription and on DNA structure of promoter point mutations, we show that the TIF-IB interaction is strongly inhibited by increases in minor groove width. This suggests that a particular DNA structure is required for interaction with the transcription factor. In addition, TIF-IB induces a small bend in the promoter upon binding. Modeling of this bend reveals that it requires an additional narrowing of the minor groove, which would favor binding to mutants with narrower grooves. We also discuss how this narrowing would induce a small destabilization of the helix upstream of the transcription start site. Telestability predicts this would result in destabilization of the sequence that melts during initiation, suggesting that TIF-IB may have a role in stimulating melting.

  17. Rapid identification of marine bioluminescent bacteria by amplified 16S ribosomal RNA gene restriction analysis.

    PubMed

    Kita-Tsukamoto, Kumiko; Wada, Minoru; Yao, Katomi; Kamiya, Akiko; Yoshizawa, Susumu; Uchiyama, Nami; Kogure, Kazuhiro

    2006-03-01

    To rapidly identify natural isolates of marine bioluminescent bacteria, we developed amplified ribosomal DNA restriction analysis (ARDRA) methods. ARDRA, which is based on the restriction patterns of 16S rRNA gene digested with five enzymes (EcoRI, DdeI, HhaI, HinfI, RsaI), clearly distinguished the 14 species of marine bioluminescent bacteria currently known, which belong to the genera Vibrio, Photobacterium, and Shewanella. When we applied ARDRA to 129 natural isolates from two cruises in Sagami Bay, Japan, 127 were grouped into six ARDRA types with distinctive restriction patterns; these isolates represented the bioluminescent species, P. angustum, P. leiognathi, P. phosphoreum, S. woodyi, V. fischeri, and V. harveyi. The other two isolates showing unexpected ARDRA patterns turned out to have 16S rRNA gene sequences similar to P. leiognathi and P. phosphoreum. Nevertheless, ARDRA provides a simple and fairly robust means for rapid identification of the natural isolates of marine bioluminescent bacteria, and is therefore useful in studying their diversity.

  18. Chromosomal organization of the ribosomal RNA genes in the genus Chironomus (Diptera, Chironomidae)

    PubMed Central

    Gunderina, Larisa; Golygina, Veronika; Broshkov, Andrey

    2015-01-01

    Abstract Chromosomal localization of ribosomal RNA coding genes has been studied by using FISH (fluorescence in situ hybridization) in 21 species from the genus Chironomus Meigen, 1803. Analysis of the data has shown intra- and interspecific variation in number and location of 5.8S rDNA hybridization sites in 17 species from the subgenus Chironomus and 4 species from the subgenus Camptochironomus Kieffer, 1914. In the majority of studied species the location of rDNA sites coincided with the sites where active NORs (nucleolus organizer regions) were found. The number of hybridization sites in karyotypes of studied chironomids varied from 1 to 6. More than half of the species possessed only one NOR (12 out of 21). Two rDNA hybridization sites were found in karyotypes of five species, three – in two species, and five and six sites – in one species each. NORs were found in all chromosomal arms of species from the subgenus Chironomus with one of them always located on arm G. On the other hand, no hybridization sites were found on arm G in four studied species from the subgenus Camptochironomus. Two species from the subgenus Chironomus – Chironomus balatonicus Devai, Wuelker & Scholl, 1983 and Chironomus “annularius” sensu Strenzke, 1959 – showed intraspecific variability in the number of hybridization signals. Possible mechanisms of origin of variability in number and location of rRNA genes in the karyotypes of species from the genus Chironomus are discussed. PMID:26140162

  19. High frequency of the 23S rRNA A2058G mutation of Treponema pallidum in Shanghai is associated with a current strategy for the treatment of syphilis.

    PubMed

    Lu, Haikong; Li, Kang; Gong, Weimin; Yan, Limeng; Gu, Xin; Chai, Ze; Guan, Zhifang; Zhou, Pingyu

    2015-02-01

    The preferred drugs for the treatment of syphilis, benzathine and procaine penicillin, have not been available in Shanghai for many years, and currently, the incidence of syphilis is increasing. Alternative antibiotics for patients with syphilis during the benzathine and procaine penicillin shortage include macrolides. The failure of macrolide treatment in syphilis patients has been reported in Shanghai, but the reason for this treatment failure remains unclear. We used polymerase chain reaction technology to detect a 23S rRNA A2058G mutation in Treponema pallidum in 109 specimens from syphilis patients. The use of azithromycin/erythromycin in the syphilis patients and the physicians' prescription habits were also assessed based on two questionnaires regarding the use of macrolides. A total of 104 specimens (95.4%) were positive for the A2058G mutation in both copies of the 23S rRNA gene, indicating macrolide resistance. A questionnaire provided to 122 dermatologists showed that during the penicillin shortage, they prescribed erythromycin and azithromycin for 8.24±13.95% and 3.21±6.37% of their patients, respectively, and in the case of penicillin allergy, erythromycin and azithromycin were prescribed 15.24±22.89% and 7.23±16.60% of the time, respectively. A second questionnaire provided to the syphilis patients showed that 150 (33.7%), 106 (23.8%) and 34 (7.6%) individuals had used azithromycin, erythromycin or both, respectively, although the majority did not use the drugs for syphilis treatment. Our findings suggest that macrolide resistance in Treponema pallidum is widespread in Shanghai. More than half of the syphilis patients had a history of macrolide use for other treatment purposes, which may have led to the high prevalence of macrolide resistance. Physicians in China are advised to not use azithromycin for early syphilis.

  20. Functional variants of 5S rRNA in the ribosomes of common sea urchin Paracentrotus lividus.

    PubMed

    Dimarco, Eufrosina; Cascone, Eleonora; Bellavia, Daniele; Caradonna, Fabio

    2012-10-15

    We have previously reported a molecular and cytogenetic characterization of three different 5S rDNA clusters in the sea urchin Paracentrotus lividus; this study, performed at DNA level only, lends itself as starting point to verify that these clusters could contain transcribed genes, then, to demonstrate the presence of heterogeneity at functional RNA level, also. In the present work we report in P. lividus ribosomes the existence of several transcribed variants of the 5S rRNA and we associate all transcribed variants to the cluster to which belong. Our finding is the first demonstration of the presence of high heterogeneity in functional 5S rRNA molecules in animal ribosomes, a feature that had been considered a peculiarity of some plants.

  1. The nucleotide sequence of the putative transcription initiation site of a cloned ribosomal RNA gene of the mouse.

    PubMed Central

    Urano, Y; Kominami, R; Mishima, Y; Muramatsu, M

    1980-01-01

    Approximately one kilobase pairs surrounding and upstream the transcription initiation site of a cloned ribosomal DNA (rDNA) of the mouse were sequenced. The putative transcription initiation site was determined by two independent methods: one nuclease S1 protection and the other reverse transcriptase elongation mapping using isolated 45S ribosomal RNA precursor (45S RNA) and appropriate restriction fragments of rDNA. Both methods gave an identical result; 45S RNA had a structure starting from ACTCTTAG---. Characteristically, mouse rDNA had many T clusters (greater than or equal to 5) upstream the initiation site, the longest being 21 consecutive T's. A pentadecanucleotide, TGCCTCCCGAGTGCA, appeared twice within 260 nucleotides upstream the putative initiation site. No such characteristic sequences were found downstream this site. Little similarity was found in the upstream of the transcription initiation site between the mouse, Xenopus laevis and Saccharomyces cerevisiae rDNA. Images PMID:6162156

  2. How EF-Tu can contribute to efficient proofreading of aa-tRNA by the ribosome

    PubMed Central

    Noel, Jeffrey K.; Whitford, Paul C.

    2016-01-01

    It has long been recognized that the thermodynamics of mRNA–tRNA base pairing is insufficient to explain the high fidelity and efficiency of aminoacyl-tRNA (aa-tRNA) selection by the ribosome. To rationalize this apparent inconsistency, Hopfield proposed that the ribosome may improve accuracy by utilizing a multi-step kinetic proofreading mechanism. While biochemical, structural and single-molecule studies have provided a detailed characterization of aa-tRNA selection, there is a limited understanding of how the physical–chemical properties of the ribosome enable proofreading. To this end, we probe the role of EF-Tu during aa-tRNA accommodation (the proofreading step) through the use of energy landscape principles, molecular dynamics simulations and kinetic models. We find that the steric composition of EF-Tu can reduce the free-energy barrier associated with the first step of accommodation: elbow accommodation. We interpret this effect within an extended kinetic model of accommodation and show how EF-Tu can contribute to efficient and accurate proofreading. PMID:27796304

  3. Dissociation rates of peptidyl-tRNA from the P-site of E.coli ribosomes.

    PubMed

    Karimi, R; Ehrenberg, M

    1996-03-01

    We studied the dissociation rates of peptidyl-tRNA from the P-site of poly(U)-programmed wild-type Escherichia coli ribosomes, hyperaccurate variants altered in S12 (SmD, SmP) and error-prone variants (Ram) altered in S4 or S5. The experiments were carried out in the presence and absence of streptomycin, and the effects of neomycin were tested in the wild-type ribosomes. Binding of peptidyl-tRNA to the P-site of wild-type ribosomes is much stronger than to their A-site. Addition of streptomycin dramatically reduces its affinity for the P-site. The S12 alternations make the P-site binding of peptidyl-tRNA much tighter, and the S4, S5 alterations make it weaker than in the case of the wild-type. We find that when binding of peptidyl-tRNA to the A-site is weak, then the affinity for the P-site is stronger, and vice versa. From these results, we formulate a hypothesis for the actions of streptomycin and neomycin based on deformations of the 16S rRNA tertiary structure. The results are also used to interpret some in vivo experiments on translational processivity.

  4. Universal bacterial identification by mass spectrometry of 16S ribosomal RNA cleavage products

    NASA Astrophysics Data System (ADS)

    Jackson, George W.; McNichols, Roger J.; Fox, George E.; Willson, Richard C.

    2007-03-01

    The public availability of over 180,000 bacterial 16S ribosomal RNA (rRNA) sequences has facilitated microbial identification and classification using nucleic acid hybridization and other molecular approaches. Species-specific PCR, microarrays, and in situ hybridization are based on the presence of unique subsequences in the target sequence and therefore require prior knowledge of what organisms are likely to be present in a sample. Mass spectrometry is not limited by a pre-synthesized inventory of probe/primer sequences. It has already been demonstrated that organism identification can be recovered from mass spectra using various methods including base-specific cleavage of nucleic acids. The feasibility of broad bacterial identification by comparing such mass spectral patterns to predictive databases derived from virtually all previously sequenced strains has yet to be demonstrated, however. Herein, we present universal bacterial identification by base-specific cleavage, mass spectrometry, and an efficient coincidence function for rapid spectral scoring against a large database of predicted "mass catalogs". Using this approach in conjunction with universal PCR of the 16S rDNA gene, four bacterial isolates and an uncultured clone were successfully identified against a database of predicted cleavage products derived 6rom over 47,000 16S rRNA sequences representing all major bacterial taxaE At present, the conventional DNA isolation and PCR steps require approximately 2 h, while subsequent transcription, enzymatic cleavage, mass spectrometric analysis, and database comparison require less than 45 min. All steps are amenable to high-throughput implementation.

  5. Paenibacillus larvae 16S-23S rDNA intergenic transcribed spacer (ITS) regions: DNA fingerprinting and characterization.

    PubMed

    Dingman, Douglas W

    2012-07-01

    Paenibacillus larvae is the causative agent of American foulbrood in honey bee (Apis mellifera) larvae. PCR amplification of the 16S-23S ribosomal DNA (rDNA) intergenic transcribed spacer (ITS) regions, and agarose gel electrophoresis of the amplified DNA, was performed using genomic DNA collected from 134 P. larvae strains isolated in Connecticut, six Northern Regional Research Laboratory stock strains, four strains isolated in Argentina, and one strain isolated in Chile. Following electrophoresis of amplified DNA, all isolates exhibited a common migratory profile (i.e., ITS-PCR fingerprint pattern) of six DNA bands. This profile represented a unique ITS-PCR DNA fingerprint that was useful as a fast, simple, and accurate procedure for identification of P. larvae. Digestion of ITS-PCR amplified DNA, using mung bean nuclease prior to electrophoresis, characterized only three of the six electrophoresis bands as homoduplex DNA and indicating three true ITS regions. These three ITS regions, DNA migratory band sizes of 915, 1010, and 1474 bp, signify a minimum of three types of rrn operons within P. larvae. DNA sequence analysis of ITS region DNA, using P. larvae NRRL B-3553, identified the 3' terminal nucleotides of the 16S rRNA gene, 5' terminal nucleotides of the 23S rRNA gene, and the complete DNA sequences of the 5S rRNA, tRNA(ala), and tRNA(ile) genes. Gene organization within the three rrn operon types was 16S-23S, 16S-tRNA(ala)-23S, and l6S-5S-tRNA(ile)-tRNA(ala)-23S and these operons were named rrnA, rrnF, and rrnG, respectively. The 23S rRNA gene was shown by I-CeuI digestion and pulsed-field gel electrophoresis of genomic DNA to be present as seven copies. This was suggestive of seven rrn operon copies within the P. larvae genome. Investigation of the 16S-23S rDNA regions of this bacterium has aided the development of a diagnostic procedure and has helped genomic mapping investigations via characterization of the ITS regions.

  6. When stable RNA becomes unstable: the degradation of ribosomes in bacteria and beyond.

    PubMed

    Maiväli, Ülo; Paier, Anton; Tenson, Tanel

    2013-07-01

    This review takes a comparative look at the various scenarios where ribosomes are degraded in bacteria and eukaryotes with emphasis on studies involving Escherichia coli and Saccharomyces cerevisiae. While the molecular mechanisms of degradation in bacteria and yeast appear somewhat different, we argue that the underlying causes of ribosome degradation are remarkably similar. In both model organisms during ribosomal assembly, partially formed pre-ribosomal particles can be degraded by at least two different sequentially-acting quality control pathways and fully assembled but functionally faulty ribosomes can be degraded in a separate quality control pathway. In addition, ribosomes that are both structurally- and functionally-sound can be degraded as an adaptive measure to stress.

  7. RdRP-synthesized antisense ribosomal siRNAs silence pre-rRNA via the nuclear RNAi pathway.

    PubMed

    Zhou, Xufei; Feng, Xuezhu; Mao, Hui; Li, Mu; Xu, Fei; Hu, Kai; Guang, Shouhong

    2017-03-01

    Expression of rRNA affects cell growth and proliferation, but mechanisms that modulate rRNA levels are poorly understood. We conducted a genetic screen for factors that negatively regulate generation of endogenous short interfering RNA (endo-siRNA) in Caenorhabditis elegans and identified a suppressor of siRNA (susi-1) and antisense ribosomal siRNAs (risiRNAs). risiRNAs show sequence complementary to 18S and 26S rRNAs and require RNA-dependent RNA polymerases (RdRPs) for their production. They act through the nuclear RNA interference (RNAi) pathway to downregulate pre-rRNA. Stress stimuli, including low temperature and UV irradiation, induced the accumulation of risiRNAs. SUSI-1 is a homolog of the human DIS3L2 exonuclease involved in 3'-5' degradation of oligouridylated RNAs. In susi-1 mutant and in low temperature-treated animals, 3'-tail oligouridylated 26S rRNA accumulated. The injection of oligouridylated rRNA elicited nuclear accumulation of NRDE-3. Our findings identify a new subset of 22G-RNAs that regulate pre-rRNA expression and a mechanism to maintain rRNA homeostasis.

  8. Steric interactions lead to collective tilting motion in the ribosome during mRNA–tRNA translocation

    PubMed Central

    Nguyen, Kien; Whitford, Paul C.

    2016-01-01

    Translocation of mRNA and tRNA through the ribosome is associated with large-scale rearrangements of the head domain in the 30S ribosomal subunit. To elucidate the relationship between 30S head dynamics and mRNA–tRNA displacement, we apply molecular dynamics simulations using an all-atom structure-based model. Here we provide a statistical analysis of 250 spontaneous transitions between the A/P–P/E and P/P–E/E ensembles. Consistent with structural studies, the ribosome samples a chimeric ap/P–pe/E intermediate, where the 30S head is rotated ∼18°. It then transiently populates a previously unreported intermediate ensemble, which is characterized by a ∼10° tilt of the head. To identify the origins of head tilting, we analyse 781 additional simulations in which specific steric features are perturbed. These calculations show that head tilting may be attributed to specific steric interactions between tRNA and the 30S subunit (PE loop and protein S13). Taken together, this study demonstrates how molecular structure can give rise to large-scale collective rearrangements. PMID:26838673

  9. Erythromycin, carbomycin, and spiramycin inhibit protein synthesis by stimulating the dissociation of peptidyl-tRNA from ribosomes.

    PubMed

    Menninger, J R; Otto, D P

    1982-05-01

    In mutant Escherichia coli with temperature-sensitive peptidyl-tRNA hydrolase (aminoacyl-tRNA hydrolase; EC 3.1.1.29), peptidyl-tRNA accumulates at the nonpermissive temperature (40 degrees C), and the cells die. These consequences of high temperature were enhanced if the cells were first treated with erythromycin, carbomycin, or spiramycin at doses sufficient to inhibit protein synthesis in wild-type cells but not sufficient to kill either mutant or wild-type cells at the permissive temperature (30 degrees C). Since peptidyl-tRNA hydrolase in he mutant cells is inactivated rapidly and irreversibly at 40 degrees C, the enhanced accumulation of peptidyl-tRNA and killing were the result of enhanced dissociation, stimulated by the antibiotics, of peptidyl-tRNA from ribosomes. The implications of these findings for inhibition of cell growth and protein synthesis are discussed. Certain alternative interpretations are shown to be inconsistent with the relevant data. Previous conflicting observations on the effects of macrolide antibiotics are explained in terms of our observations. We conclude that erythromycin, carbomycin, and spiramycin (and probably all macrolides) have as a primary mechanism of action the stimulation of dissociation of peptidyl-tRNA from ribosomes, probably during translocation.

  10. Replicative age induces mitotic recombination in the ribosomal RNA gene cluster of Saccharomyces cerevisiae.

    PubMed

    Lindstrom, Derek L; Leverich, Christina K; Henderson, Kiersten A; Gottschling, Daniel E

    2011-03-01

    Somatic mutations contribute to the development of age-associated disease. In earlier work, we found that, at high frequency, aging Saccharomyces cerevisiae diploid cells produce daughters without mitochondrial DNA, leading to loss of respiration competence and increased loss of heterozygosity (LOH) in the nuclear genome. Here we used the recently developed Mother Enrichment Program to ask whether aging cells that maintain the ability to produce respiration-competent daughters also experience increased genomic instability. We discovered that this population exhibits a distinct genomic instability phenotype that primarily affects the repeated ribosomal RNA gene array (rDNA array). As diploid cells passed their median replicative life span, recombination rates between rDNA arrays on homologous chromosomes progressively increased, resulting in mutational events that generated LOH at >300 contiguous open reading frames on the right arm of chromosome XII. We show that, while these recombination events were dependent on the replication fork block protein Fob1, the aging process that underlies this phenotype is Fob1-independent. Furthermore, we provide evidence that this aging process is not driven by mechanisms that modulate rDNA recombination in young cells, including loss of cohesion within the rDNA array or loss of Sir2 function. Instead, we suggest that the age-associated increase in rDNA recombination is a response to increasing DNA replication stress generated in aging cells.

  11. Phylogeny of chloromonas (chlorophyceae): A study of 18S ribosomal RNA gene sequences

    SciTech Connect

    Buchheim, M.A.; Buchheim, J.A.; Chapman, R.L.

    1997-04-01

    The unicellular, biflagellate genus Chloromonas differs from its ally, Chlamydomonas, primarily by the absence of pyrenoids in the vegetative stage of the former. As with most green flagellate genera, little is known about phylogenetic affinities within and among Chloromonas species. Phylogenetic analyses of nuclear-encoded small-subunit ribosomal RNA gene sequences demonstrate that a sampling of five Chloromonas taxa, obtained from major culture collections, do not form a monophyletic group. However, only three of these isolates, Chloromonas clathrata, Chloromonas serbinowi, and Chloromonas rosae, are diagnosable morphologically as Chloromonas species by the absence of a pyrenoid in the vegetative stage. The three diagnosable Chloromonas taxa form an alliance with two pyrenoid-bearing chlamydomonads, Chlamydomonas augustae and Chlamydomonas macrostellata. With the exception of Chloromonas serbinowi, which represents the basal lineage within the clade, each of the diagnosable Chloromonas taxa and their pyrenoid-bearing Chlamydomonas allies were isolated originally from mountain soils, snow, or cold peat. These observations suggest that hibitat, independent of pyrenoid status, may be most closely linked to the natural history of this clade of chlamydomonad flagellates. 51 refs., 3 figs., 3 tabs.

  12. Determination of the promoter region of mouse ribosomal RNA gene by an in vitro transcription system.

    PubMed Central

    Yamamoto, O; Takakusa, N; Mishima, Y; Kominami, R; Muramatsu, M

    1984-01-01

    Sequences required for a faithful and efficient transcription of a cloned mouse ribosomal RNA gene (rDNA) are determined by testing a series of deletion mutants in an in vitro transcription system utilizing two kinds of mouse cellular extract. Deletion of sequences upstream of -40 or downstream of +52 causes only slight reduction in promoter activity as compared with the "wild-type" template. For upstream deletion mutants, the removal of a sequence between -40 and -35 causes a significant decrease in the capacity to direct efficient initiation. This decrease becomes more pronounced when the deletion reaches -32 and the sequence A-T-C-T-T-T, conserved among mouse, rat, and human rDNAs, is lost. Residual template activity is further reduced as more upstream sequence is deleted and finally becomes undetectable when the deletion is extended from -22 down to -17, corresponding to the loss of the conserved sequence T-A-T-T-G. As for downstream deletion mutants, the removal of the sequence downstream of +23 causes some (and further deletions up to +11 cause a more) serious decrease in template activity in vitro. These deletions involve other conserved sequences downstream of the transcription start site. However, the removal of the original transcription start site does not abolish the transcription initiation completely, provided that the whole upstream sequence is intact. Images PMID:6320178

  13. Integrin binding and mechanical tension induce movement of mRNA and ribosomes to focal adhesions

    NASA Technical Reports Server (NTRS)

    Chicurel, M. E.; Singer, R. H.; Meyer, C. J.; Ingber, D. E.

    1998-01-01

    The extracellular matrix (ECM) activates signalling pathways that control cell behaviour by binding to cell-surface integrin receptors and inducing the formation of focal adhesion complexes (FACs). In addition to clustered integrins, FACs contain proteins that mechanically couple the integrins to the cytoskeleton and to immobilized signal-transducing molecules. Cell adhesion to the ECM also induces a rapid increase in the translation of preexisting messenger RNAs. Gene expression can be controlled locally by targeting mRNAs to specialized cytoskeletal domains. Here we investigate whether cell binding to the ECM promotes formation of a cytoskeletal microcompartment specialized for translational control at the site of integrin binding. High-resolution in situ hybridization revealed that mRNA and ribosomes rapidly and specifically localized to FACs that form when cells bind to ECM-coated microbeads. Relocation of these protein synthesis components to the FAC depended on the ability of integrins to mechanically couple the ECM to the contractile cytoskeleton and on associated tension-moulding of the actin lattice. Our results suggest a new type of gene regulation by integrins and by mechanical stress which may involve translation of mRNAs into proteins near the sites of signal reception.

  14. Combined large and small subunit ribosomal RNA phylogenies support a basal position of the acoelomorph flatworms.

    PubMed Central

    Telford, Maximilian J; Lockyer, Anne E; Cartwright-Finch, Chloë; Littlewood, D Timothy J

    2003-01-01

    The phylogenetic position of the phylum Platyhelminthes has been re-evaluated in the past decade by analysis of diverse molecular datasets. The consensus is that the Rhabditophora + Catenulida, which includes most of the flatworm taxa, are not primitively simple basal bilaterians but are related to coelomate phyla such as molluscs. The status of two other groups of acoelomate worms, Acoela and Nemertodermatida, is less clear. Although many characteristics unite these two groups, initial molecular phylogenetic studies placed the Nemertodermatida within the Rhabditophora, but placed the Acoela at the base of the Bilateria, distant from other flatworms. This contradiction resulted in scepticism about the basal position of acoels and led to calls for further data. We have sequenced large subunit ribosomal RNA genes from 13 rhabditophorans + catenulids, three acoels and one nemertodermatid, tripling the available data. Our analyses strongly support a basal position of both acoels and nemertodermatids. Alternative hypotheses are significantly less well supported by the data. We conclude that the Nemertodermatida and Acoela are basal bilaterians and, owing to their unique body plan and embryogenesis, should be recognized as a separate phylum, the Acoelomorpha. PMID:12803898

  15. Rejection of tmRNA·SmpB after GTP hydrolysis by EF-Tu on ribosomes stalled on intact mRNA.

    PubMed

    Kurita, Daisuke; Miller, Mickey R; Muto, Akira; Buskirk, Allen R; Himeno, Hyouta

    2014-11-01

    Messenger RNAs lacking a stop codon trap ribosomes at their 3' ends, depleting the pool of ribosomes available for protein synthesis. In bacteria, a remarkable quality control system rescues and recycles stalled ribosomes in a process known as trans-translation. Acting as a tRNA, transfer-messenger RNA (tmRNA) is aminoacylated, delivered by EF-Tu to the ribosomal A site, and accepts the nascent polypeptide. Translation then resumes on a reading frame within tmRNA, encoding a short peptide tag that targets the nascent peptide for degradation by proteases. One unsolved issue in trans-translation is how tmRNA and its protein partner SmpB preferentially recognize stalled ribosomes and not actively translating ones. Here, we examine the effect of the length of the 3' extension of mRNA on each step of trans-translation by pre-steady-state kinetic methods and fluorescence polarization binding assays. Unexpectedly, EF-Tu activation and GTP hydrolysis occur rapidly regardless of the length of the mRNA, although the peptidyl transfer to tmRNA decreases as the mRNA 3' extension increases and the tmRNA·SmpB binds less tightly to the ribosome with an mRNA having a long 3' extension. From these results, we conclude that the tmRNA·SmpB complex dissociates during accommodation due to competition between the downstream mRNA and the C-terminal tail for the mRNA channel. Rejection of the tmRNA·SmpB complex during accommodation is reminiscent of the rejection of near-cognate tRNA from the ribosome in canonical translation.

  16. Ribosomal protein S6 is highly expressed in non-Hodgkin lymphoma and associates with mRNA containing a 5' terminal oligopyrimidine tract.

    PubMed

    Hagner, P R; Mazan-Mamczarz, K; Dai, B; Balzer, E M; Corl, S; Martin, S S; Zhao, X F; Gartenhaus, R B

    2011-03-31

    The molecular mechanism(s) linking tumorigenesis and morphological alterations in the nucleolus are presently coming into focus. The nucleolus is the cellular organelle in which the formation of ribosomal subunits occurs. Ribosomal biogenesis occurs through the transcription of ribosomal RNA (rRNA), rRNA processing and production of ribosomal proteins. An error in any of these processes may lead to deregulated cellular translation, evident in multiple cancers and 'ribosomopathies'. Deregulated protein synthesis may be achieved through the overexpression of ribosomal proteins as seen in primary leukemic blasts with elevated levels of ribosomal proteins S11 and S14. In this study, we demonstrate that ribosomal protein S6 (RPS6) is highly expressed in primary diffuse large B-cell lymphoma (DLBCL) samples. Genetic modulation of RPS6 protein levels with specifically targeted short hairpin RNA (shRNA) lentiviruses led to a decrease in the actively proliferating population of cells compared with control shRNA. Low-dose rapamycin treatments have been shown to affect the translation of 5' terminal oligopyrimidine (5' TOP) tract mRNA, which encodes the translational machinery, implicating RPS6 in 5' TOP translation. Recently, it was shown that disruption of 40S ribosomal biogenesis through specific small inhibitory RNA knockdown of RPS6 defined RPS6 as a critical regulator of 5' TOP translation. For the first time, we show that RPS6 associates with multiple mRNAs containing a 5' TOP tract. These findings expand our understanding of the mechanism(s) involved in ribosomal biogenesis and deregulated protein synthesis in DLBCL.

  17. The influence of ignoring secondary structure on divergence time estimates from ribosomal RNA genes.

    PubMed

    Dohrmann, Martin

    2014-02-01

    Genes coding for ribosomal RNA molecules (rDNA) are among the most popular markers in molecular phylogenetics and evolution. However, coevolution of sites that code for pairing regions (stems) in the RNA secondary structure can make it challenging to obtain accurate results from such loci. While the influence of ignoring secondary structure on multiple sequence alignment and tree topology has been investigated in numerous studies, its effect on molecular divergence time estimates is still poorly known. Here, I investigate this issue in Bayesian Markov Chain Monte Carlo (BMCMC) and penalized likelihood (PL) frameworks, using empirical datasets from dragonflies (Odonata: Anisoptera) and glass sponges (Porifera: Hexactinellida). My results indicate that highly biased inferences under substitution models that ignore secondary structure only occur if maximum-likelihood estimates of branch lengths are used as input to PL dating, whereas in a BMCMC framework and in PL dating based on Bayesian consensus branch lengths, the effect is far less severe. I conclude that accounting for coevolution of paired sites in molecular dating studies is not as important as previously suggested, as long as the estimates are based on Bayesian consensus branch lengths instead of ML point estimates. This finding is especially relevant for studies where computational limitations do not allow the use of secondary-structure specific substitution models, or where accurate consensus structures cannot be predicted. I also found that the magnitude and direction (over- vs. underestimating node ages) of bias in age estimates when secondary structure is ignored was not distributed randomly across the nodes of the phylogenies, a phenomenon that requires further investigation.

  18. Ultraviolet damage and nucleosome folding of the 5S ribosomal RNA gene.

    SciTech Connect

    Liu, X; Mann, David B.; Suquet, C; Springer, David L. ); Smerdon, Michael J.

    2000-01-25

    The Xenopus borealis somatic 5S ribosomal RNA gene was used as a model system to determine the mutual effects of nucleosome folding and formation of ultraviolet (UV) photoproducts (primarily cis-syn cyclobutane pyrimidine dimers, or CPDs) in chromatin. We analyzed the preferred rotational and translational settings of 5S rDNA on the histone octamer surface after induction of up to 0.8 CPD/nucleosome core (2.5 kJ/m(2) UV dose). DNase I and hydroxyl radical footprints indicate that UV damage at these levels does not affect the average rotational setting of the 5S rDNA molecules. Moreover, a combination of nuclease trimming and restriction enzyme digestion indicates the preferred translational positions of the histone octamer are not affected by this level of UV damage. We also did not observe differences in the UV damage patterns of irradiated 5S rDNA before or after nucleosome formation, indicating there is little difference in the inhibition of nucleosome folding by specific CPD sites in the 5S rRNA gene. Conversely, nucleosome folding significantly restricts CPD formation at all sites in the three helical turns of the nontranscribed strand located in the dyad axis region of the nucleosome, where DNA is bound exclusively by the histone H3-H4 tetramer. Finally, modulation of the CPD distribution in a 14 nt long pyrimidine tract correlates with its rotational setting on the histone surface, when the strong sequence bias for CPD formation in this tract is minimized by normalization. These results help establish the mutual roles of histone binding and UV photoproducts on their formation in chromatin.

  19. Cyanobacterial ribosomal RNA genes with multiple, endonuclease-encoding group I introns

    PubMed Central

    Haugen, Peik; Bhattacharya, Debashish; Palmer, Jeffrey D; Turner, Seán; Lewis, Louise A; Pryer, Kathleen M

    2007-01-01

    Background Group I introns are one of the four major classes of introns as defined by their distinct splicing mechanisms. Because they catalyze their own removal from precursor transcripts, group I introns are referred to as autocatalytic introns. Group I introns are common in fungal and protist nuclear ribosomal RNA genes and in organellar genomes. In contrast, they are rare in all other organisms and genomes, including bacteria. Results Here we report five group I introns, each containing a LAGLIDADG homing endonuclease gene (HEG), in large subunit (LSU) rRNA genes of cyanobacteria. Three of the introns are located in the LSU gene of Synechococcus sp. C9, and the other two are in the LSU gene of Synechococcus lividus strain C1. Phylogenetic analyses show that these introns and their HEGs are closely related to introns and HEGs located at homologous insertion sites in organellar and bacterial rDNA genes. We also present a compilation of group I introns with homing endonuclease genes in bacteria. Conclusion We have discovered multiple HEG-containing group I introns in a single bacterial gene. To our knowledge, these are the first cases of multiple group I introns in the same bacterial gene (multiple group I introns have been reported in at least one phage gene and one prophage gene). The HEGs each contain one copy of the LAGLIDADG motif and presumably function as homodimers. Phylogenetic analysis, in conjunction with their patchy taxonomic distribution, suggests that these intron-HEG elements have been transferred horizontally among organelles and bacteria. However, the mode of transfer and the nature of the biological connections among the intron-containing organisms are unknown. PMID:17825109

  20. Different organisms associated with heartwater as shown by analysis of 16S ribosomal RNA gene sequences.

    PubMed

    Allsopp, M; Visser, E S; du Plessis, J L; Vogel, S W; Allsopp, B A

    1997-08-01

    Cowdria ruminantium is a rickettsial parasite which causes heartwater, a economically important disease of domestic and wild ruminants in tropical and subtropical Africa and parts of the Caribbean. Because existing diagnostic methods are unreliable, we investigated the small-subunit ribosomal RNA (srRNA) gene from heartwater-infected material to characterise the organisms present and to develop specific oligonucleotide probes for polymerase chain reaction (PCR) based diagnosis. DNA was obtained from ticks and ruminants from heartwater-free and heartwater-endemic areas from Cowdria in tissue culture. PCR was carried out using primers designed to amplify only rickettsial srRNA genes, the target region being the highly variable V1 loop. Amplicons were cloned and sequenced; 51% were C. ruminantium sequences corresponding to four genotypes, two of which were identical to previously reported C. ruminantium sequences while the other two were new. The four different Cowdria genotypes can be correlated with different phenotypes. Tissue-culture samples yielded only Cowdria genotype sequences, but an extraordinary heterogeneity of 16S sequences was obtained from field samples. In addition to Cowdria genotypes we found sequences from previously unknown Ehrlichia spp., sequences showing homology to other Rickettsiales and a variety of Pseudomonadaceae. One Ehrlichia sequence was phylogenetically closely related to Ehrlichia platys (Group II Ehrlichia) and one to Ehrlichia canis (Group III Ehrlichia). This latter sequence was from an isolate (Germishuys) made from a naturally infected sheep which, from brain smear examination and pathology, appeared to be suffering from heartwater; nevertheless no Cowdria genotype sequences were found in this isolate. In addition no Cowdria sequences were obtained from uninfected ticks. Complete 16S rRNA gene sequences were determined for two C. ruminantium genotypes and for two previously uncharacterised heartwater-associated Ehrlichia spp

  1. Thio-Modification of tRNA at the Wobble Position as Regulator of the Kinetics of Decoding and Translocation on the Ribosome.

    PubMed

    Ranjan, Namit; Rodnina, Marina V

    2017-04-13

    Uridine 34 (U34) at the wobble position of the tRNA anticodon is post-transcriptionally modified, usually to mcm(5)s(2), mcm(5), or mnm(5). The lack of the mcm(5) or s(2) modification at U34 of tRNA(Lys), tRNA(Glu), and tRNA(Gln) causes ribosome pausing at the respective codons in yeast. The pauses occur during the elongation step, but the mechanism that triggers ribosome pausing is not known. Here, we show how the s(2) modification in yeast tRNA(Lys) affects mRNA decoding and tRNA-mRNA translocation. Using real-time kinetic analysis we show that mcm(5)-modified tRNA(Lys) lacking the s(2) group has a lower affinity of binding to the cognate codon and is more efficiently rejected than the fully modified tRNA(Lys). The lack of the s(2) modification also slows down the rearrangements in the ribosome-EF-Tu-GDP-Pi-Lys-tRNA(Lys) complex following GTP hydrolysis by EF-Tu. Finally, tRNA-mRNA translocation is slower with the s(2)-deficient tRNA(Lys). These observations explain the observed ribosome pausing at AAA codons during translation and demonstrate how the s(2) modification helps to ensure the optimal translation rates that maintain proteome homeostasis of the cell.

  2. Expression of Variant Ribosomal RNA Genes in Mouse Oocytes and Preimplantation Embryos1

    PubMed Central

    Ihara, Motomasa; Tseng, Hung; Schultz, Richard M.

    2011-01-01

    Ribosomal DNA (rDNA) is not composed of multiple copies of identical transcription units, as commonly believed, but rather of at least seven rDNA variant subtypes that are expressed in somatic cells. This finding raises the possibility that ribosome function may be modulated as proposed by the ribosome filter hypothesis. We report here that mouse oocytes and preimplantation embryos express all the rDNA variants except variant V and that there is no marked developmental change in the qualitative pattern of variant expression. The maternal and embryonic ribosome pools are therefore quite similar, minimizing the likelihood that developmental changes in composition of the ribosome population are critical for preimplantation development. PMID:21209414

  3. EF4 disengages the peptidyl-tRNA CCA end and facilitates back-translocation on the 70S ribosome.

    PubMed

    Zhang, Dejiu; Yan, Kaige; Liu, Guangqiao; Song, Guangtao; Luo, Jiejian; Shi, Yi; Cheng, Erchao; Wu, Shan; Jiang, Taijiao; Lou, Jizhong; Gao, Ning; Qin, Yan

    2016-02-01

    EF4 catalyzes tRNA back-translocation through an unknown mechanism. We report cryo-EM structures of Escherichia coli EF4 in post- and pretranslocational ribosomes (Post- and Pre-EF4) at 3.7- and 3.2-Å resolution, respectively. In Post-EF4, peptidyl-tRNA occupies the peptidyl (P) site, but the interaction between its CCA end and the P loop is disrupted. In Pre-EF4, the peptidyl-tRNA assumes a unique position near the aminoacyl (A) site, denoted the A site/EF4 bound (A/4) site, with a large displacement at its acceptor arm. Mutagenesis analyses suggest that a specific region in the EF4 C-terminal domain (CTD) interferes with base-pairing between the peptidyl-tRNA 3'-CCA and the P loop, whereas the EF4 CTD enhances peptidyl-tRNA interaction at the A/4 site. Therefore, EF4 induces back-translocation by disengaging the tRNA's CCA end from the peptidyl transferase center of the translating ribosome.

  4. Inhibition by Siomycin and Thiostrepton of Both Aminoacyl-tRNA and Factor G Binding to Ribosomes

    PubMed Central

    Ll, Juan Modole; Cabrer, Bartolomé; Parmeggiani, Andrea; Azquez, David V

    1971-01-01

    Siomycin, a peptide antibiotic that interacts with the 50S ribosomal subunit and inhibits binding of factor G, is shown also to inhibit binding of aminoacyl-tRNA; however, it does not impair binding of fMet-tRNA and completion of the initiation complex. Moreover, unlike other inhibitors of aminoacyl-tRNA binding (tetracycline, sparsomycin, and streptogramin A), siomycin completely abolishes the GTPase activity associated with the binding of aminoacyl-tRNA catalyzed by factor Tu. A single-site interaction of siomycin appears to be responsible for its effect on both the binding of the aminoacyl-tRNA-Tu-GTP complex and that of factor G. PMID:4331558

  5. Improved ribosome-footprint and mRNA measurements provide insights into dynamics and regulation of yeast translation

    PubMed Central

    Weinberg, David E.; Shah, Premal; Eichhorn, Stephen W.; Hussmann, Jeffrey A.; Plotkin, Joshua B.; Bartel, David P.

    2016-01-01

    SUMMARY Ribosome-footprint profiling provides genome-wide snapshots of translation, but technical challenges can confound its analysis. Here, we use improved methods to obtain ribosome-footprint profiles and mRNA abundances that more faithfully reflect gene expression in Saccharomyces cerevisiae. Our results support proposals that both the beginning of coding regions and condos matching rare tRNAs are more slowly translated. They also indicate that emergent polypeptides with as few as three basic residues within a 10-residue window tend to slow translation. With the improved mRNA measurements, the variation attributable to translational control in exponentially growing yeast was less than previously reported, and most of this variation could be predicted with a simple model that considered mRNA abundance, upstream open reading frames, cap-proximal structure and nucleotide composition, and lengths of the coding and 5’-untranslated regions. Collectively, our results provide a framework for executing and interpreting ribosome-profiling studies and reveal key features of translational control in yeast. PMID:26876183

  6. Role of a short open reading frame in ribosome shunt on the cauliflower mosaic virus RNA leader.

    PubMed

    Pooggin, M M; Hohn, T; Fütterer, J

    2000-06-09

    The pregenomic 35 S RNA of cauliflower mosaic virus (CaMV) belongs to the growing number of mRNAs known to have a complex leader sequence. The 612-nucleotide leader contains several short open reading frames (sORFs) and forms an extended hairpin structure. Downstream translation of 35 S RNA is nevertheless possible due to the ribosome shunt mechanism, by which ribosomes are directly transferred from a take-off site near the capped 5' end of the leader to a landing site near its 3' end. There they resume scanning and reach the first long open reading frame. We investigated in detail how the multiple sORFs influence ribosome migration either via shunting or linear scanning along the CaMV leader. The sORFs together constituted a major barrier for the linear ribosome migration, whereas the most 5'-proximal sORF, sORF A, in combination with sORFs B and C, played a positive role in translation downstream of the leader by diverting scanning ribosomes to the shunt route. A simplified, shunt-competent leader was constructed with the most part of the hairpin including all the sORFs except sORF A replaced by a scanning-inhibiting structure. In this leader as well as in the wild type leader, proper translation and termination of sORF A was required for efficient shunt and also for the level of shunt enhancement by a CaMV-encoded translation transactivator. sORF A could be replaced by heterologous sORFs, but a one-codon (start/stop) sORF was not functional. The results implicate that in CaMV, shunt-mediated translation requires reinitiation. The efficiency of the shunt process is influenced by translational properties of the sORF.

  7. Touchdown Enzyme Time Release-PCR for Detection and Identification of Chlamydia trachomatis, C. pneumoniae, and C. psittaci Using the 16S and 16S-23S Spacer rRNA Genes

    PubMed Central

    Madico, Guillermo; Quinn, Thomas C.; Boman, Jens; Gaydos, Charlotte A.

    2000-01-01

    Three touchdown enzyme time release (TETR)-PCR assays were used to amplify different DNA sequences in the variable regions of the 16S and 16S-23S spacer rRNA genes specific for Chlamydia trachomatis, Chlamydia pneumoniae, and Chlamydia psittaci as improved tests for sensitive diagnosis and rapid species differentiation. The TETR-PCR protocol used 60 cycles of amplification, which provided improved analytical sensitivity (0.004 to 0.063 inclusion-forming unit of Chlamydia species per PCR). The sensitivity of TETR-PCR with primer set CTR 70-CTR 71 was 96.7%, and the specificity was 99.6%, compared to those of the AMPLICOR PCR for the detection of C. trachomatis in vaginal swab samples. TETR-PCR for C. pneumoniae with primer set CPN 90-CPN 91 was 90% sensitive and 93.3% specific compared with a nested PCR with primer set CP1/2-CPC/D for clinical respiratory samples. TETR-PCR for C. psittaci with primer set CPS 100-CPS 101 showed substantial agreement with cell culturing (κ, 0.78) for animal tissue samples. Primer sets were then combined into a single multiplex TETR-PCR test. The respective 315-, 195-, and 111-bp DNA target products were precisely amplified when DNA from each of the respective Chlamydia species or combinations of them was used. Multiplex chlamydia TETR-PCR correctly identified one strain of each of the 15 serovars of C. trachomatis, 22 isolates of C. pneumoniae, and 20 isolates of C. psittaci. The primer sets were specific for each species. No target products were amplified when DNA from C. pecorum or a variety of other microorganisms was tested for specificity. TETR-PCR with primers selected for specific sequences in the 16S and 16S-23S spacer rRNA genes is a valuable test that could be used either with individual primers or in a multiplex assay for the identification and differentiation of Chlamydia species from culture isolates or for the detection of chlamydiae in clinical samples. PMID:10699002

  8. Non-stop mRNA decay: a special attribute of trans-translation mediated ribosome rescue.

    PubMed

    Venkataraman, Krithika; Guja, Kip E; Garcia-Diaz, Miguel; Karzai, A Wali

    2014-01-01

    Decoding of aberrant mRNAs leads to unproductive ribosome stalling and sequestration of components of the translation machinery. Bacteria have evolved three seemingly independent pathways to resolve stalled translation complexes. The trans-translation process, orchestrated by the hybrid transfer-messenger RNA (tmRNA) and its essential protein co-factor, small protein B (SmpB), is the principal translation quality control system for rescuing unproductively stalled ribosomes. Two specialized alternative rescue pathways, coordinated by ArfA and ArfB, have been recently discovered. The SmpB-tmRNA mediated trans-translation pathway, in addition to re-mobilizing stalled translation complexes, co-translationally appends a degradation tag to the associated nascent polypeptides, marking them for proteolysis by various cellular proteases. Another unique feature of trans-translation, not shared by the alternative rescue pathways, is the facility to recruit ribonuclease R (RNase R) for targeted degradation of non-stop mRNAs, thus preventing further futile cycles of translation. The distinct C-terminal lysine-rich (K-rich) domain of RNase R is essential for its recruitment to stalled ribosomes. To gain new insights into the structure and function of RNase R, we investigated its global architecture, the spatial arrangement of its distinct domains, and the identities of key functional residues in its unique K-rich domain. Small-angle X-ray scattering models of RNase R reveal a tri-lobed structure with flexible N- and C-terminal domains, and suggest intimate contacts between the K-rich domain and the catalytic core of the enzyme. Alanine-scanning mutagenesis of the K-rich domain, in the region spanning residues 735 and 750, has uncovered the precise amino acid determinants required for the productive engagement of RNase R on tmRNA-rescued ribosomes. Theses analyses demonstrate that alanine substitution of conserved residues E740 and K741result in profound defects, not only in the

  9. Interaction of the cotranslational Hsp70 Ssb with ribosomal proteins and rRNA depends on its lid domain

    PubMed Central

    Gumiero, Andrea; Conz, Charlotte; Gesé, Genís Valentín; Zhang, Ying; Weyer, Felix Alexander; Lapouge, Karine; Kappes, Julia; von Plehwe, Ulrike; Schermann, Géza; Fitzke, Edith; Wölfle, Tina; Fischer, Tamás; Rospert, Sabine; Sinning, Irmgard

    2016-01-01

    Cotranslational chaperones assist in de novo folding of nascent polypeptides in all organisms. In yeast, the heterodimeric ribosome-associated complex (RAC) forms a unique chaperone triad with the Hsp70 homologue Ssb. We report the X-ray structure of full length Ssb in the ATP-bound open conformation at 2.6 Å resolution and identify a positively charged region in the α-helical lid domain (SBDα), which is present in all members of the Ssb-subfamily of Hsp70s. Mutational analysis demonstrates that this region is strictly required for ribosome binding. Crosslinking shows that Ssb binds close to the tunnel exit via contacts with both, ribosomal proteins and rRNA, and that specific contacts can be correlated with switching between the open (ATP-bound) and closed (ADP-bound) conformation. Taken together, our data reveal how Ssb dynamics on the ribosome allows for the efficient interaction with nascent chains upon RAC-mediated activation of ATP hydrolysis. PMID:27882919

  10. Interaction of the cotranslational Hsp70 Ssb with ribosomal proteins and rRNA depends on its lid domain.

    PubMed

    Gumiero, Andrea; Conz, Charlotte; Gesé, Genís Valentín; Zhang, Ying; Weyer, Felix Alexander; Lapouge, Karine; Kappes, Julia; von Plehwe, Ulrike; Schermann, Géza; Fitzke, Edith; Wölfle, Tina; Fischer, Tamás; Rospert, Sabine; Sinning, Irmgard

    2016-11-24

    Cotranslational chaperones assist in de novo folding of nascent polypeptides in all organisms. In yeast, the heterodimeric ribosome-associated complex (RAC) forms a unique chaperone triad with the Hsp70 homologue Ssb. We report the X-ray structure of full length Ssb in the ATP-bound open conformation at 2.6 Å resolution and identify a positively charged region in the α-helical lid domain (SBDα), which is present in all members of the Ssb-subfamily of Hsp70s. Mutational analysis demonstrates that this region is strictly required for ribosome binding. Crosslinking shows that Ssb binds close to the tunnel exit via contacts with both, ribosomal proteins and rRNA, and that specific contacts can be correlated with switching between the open (ATP-bound) and closed (ADP-bound) conformation. Taken together, our data reveal how Ssb dynamics on the ribosome allows for the efficient interaction with nascent chains upon RAC-mediated activation of ATP hydrolysis.

  11. Transcriptome-wide studies uncover the diversity of modes of mRNA recruitment to eukaryotic ribosomes.

    PubMed

    Shatsky, Ivan N; Dmitriev, Sergey E; Andreev, Dmitri E; Terenin, Ilya M

    2014-01-01

    The conventional paradigm of translation initiation in eukaryotes states that the cap-binding protein complex eIF4F (consisting of eIF4E, eIF4G and eIF4A) plays a central role in the recruitment of capped mRNAs to ribosomes. However, a growing body of evidence indicates that this paradigm should be revised. This review summarizes the data which have been mostly accumulated in a post-genomic era owing to revolutionary techniques of transcriptome-wide analysis. Unexpectedly, these techniques have uncovered remarkable diversity in the recruitment of cellular mRNAs to eukaryotic ribosomes. These data enable a preliminary classification of mRNAs into several groups based on their requirement for particular components of eIF4F. They challenge the widely accepted concept which relates eIF4E-dependence to the extent of secondary structure in the 5' untranslated regions of mRNAs. Moreover, some mRNA species presumably recruit ribosomes to their 5' ends without the involvement of either the 5' m(7)G-cap or eIF4F but instead utilize eIF4G or eIF4G-like auxiliary factors. The long-standing concept of internal ribosome entry site (IRES)-elements in cellular mRNAs is also discussed.

  12. Seasonal succession leads to habitat-dependent differentiation in ribosomal RNA:DNA ratios among freshwater lake bacteria

    DOE PAGES

    Denef, Vincent J.; Fujimoto, Masanori; Berry, Michelle A.; ...

    2016-04-29

    Relative abundance profiles of bacterial populations measured by sequencing DNA or RNA of marker genes can widely differ. These differences, made apparent when calculating ribosomal RNA:DNA ratios, have been interpreted as variable activities of bacterial populations. However, inconsistent correlations between ribosomal RNA:DNA ratios and metabolic activity or growth rates have led to a more conservative interpretation of this metric as the cellular protein synthesis potential (PSP). Little is known, particularly in freshwater systems, about how PSP varies for specific taxa across temporal and spatial environmental gradients and how conserved PSP is across bacterial phylogeny. Here, we generated 16S rRNA genemore » sequencing data using simultaneously extracted DNA and RNA from fractionated (free-living and particulate) water samples taken seasonally along a eutrophic freshwater estuary to oligotrophic pelagic transect in Lake Michigan. In contrast to previous reports, we observed frequent clustering of DNA and RNA data from the same sample. Analysis of the overlap in taxa detected at the RNA and DNA level indicated that microbial dormancy may be more common in the estuary, the particulate fraction, and during the stratified period. Across spatiotemporal gradients, PSP was often conserved at the phylum and class levels. PSPs for specific taxa were more similar across habitats in spring than in summer and fall. This was most notable for PSPs of the same taxa when located in the free-living or particulate fractions, but also when contrasting surface to deep, and estuary to Lake Michigan communities. Our results show that community composition assessed by RNA and DNA measurements are more similar than previously assumed in freshwater systems. Furthermore, the similarity between RNA and DNA measurements and taxa-specific PSPs that drive community-level similarities are conditional on spatiotemporal factors.« less

  13. Seasonal succession leads to habitat-dependent differentiation in ribosomal RNA:DNA ratios among freshwater lake bacteria

    SciTech Connect

    Denef, Vincent J.; Fujimoto, Masanori; Berry, Michelle A.; Schmidt, Marian L.

    2016-04-29

    Relative abundance profiles of bacterial populations measured by sequencing DNA or RNA of marker genes can widely differ. These differences, made apparent when calculating ribosomal RNA:DNA ratios, have been interpreted as variable activities of bacterial populations. However, inconsistent correlations between ribosomal RNA:DNA ratios and metabolic activity or growth rates have led to a more conservative interpretation of this metric as the cellular protein synthesis potential (PSP). Little is known, particularly in freshwater systems, about how PSP varies for specific taxa across temporal and spatial environmental gradients and how conserved PSP is across bacterial phylogeny. Here, we generated 16S rRNA gene sequencing data using simultaneously extracted DNA and RNA from fractionated (free-living and particulate) water samples taken seasonally along a eutrophic freshwater estuary to oligotrophic pelagic transect in Lake Michigan. In contrast to previous reports, we observed frequent clustering of DNA and RNA data from the same sample. Analysis of the overlap in taxa detected at the RNA and DNA level indicated that microbial dormancy may be more common in the estuary, the particulate fraction, and during the stratified period. Across spatiotemporal gradients, PSP was often conserved at the phylum and class levels. PSPs for specific taxa were more similar across habitats in spring than in summer and fall. This was most notable for PSPs of the same taxa when located in the free-living or particulate fractions, but also when contrasting surface to deep, and estuary to Lake Michigan communities. Our results show that community composition assessed by RNA and DNA measurements are more similar than previously assumed in freshwater systems. Furthermore, the similarity between RNA and DNA measurements and taxa-specific PSPs that drive community-level similarities are conditional on spatiotemporal factors.

  14. Mechanisms of the initiation of protein synthesis: in reading frame binding of ribosomes to mRNA.

    PubMed

    Nakamoto, Tokumasa

    2011-02-01

    The various mechanisms proposed to describe the initiation of protein synthesis are reviewed with a focus on their initiation signals. A characteristic feature of the various mechanisms is that each one of them postulates a distinct initiation signal. The signals of the Shine-Dalgarno (SD), the scanning and the internal ribosome entry site (IRES) mechanisms are all located exclusively in the 5' leader sequence, whereas, the signal of the cumulative specificity (CS) mechanism includes the entire initiation site (IS). Computer analysis of known E. coli IS sequences showed signal characteristics in the entire model IS consisting of 47 bases, in segments of the 5' leader and of the protein-coding regions. The proposal that eukaryotic translation actually occurs in two steps is scrutinized. In a first step, initiation factors (eIF4F) interact with the cap of the mRNA, thereby enhancing the accessibility of the IS. In the second step, initiation is by the conserved prokaryotic mechanism in which the ribosomes bind directly to the mRNA without ribosomal scanning. This binding occurs by the proposed process of in reading frame binding of ribosomes to mRNA, which is consistent with the CS mechanism. The basic CS mechanism is able to account for the initiation of translation of leaderless mRNAs, as well as for that of canonical mRNAs. The SD, the scanning and the IRES mechanisms, on the other hand, are inconsistent with the initiation of translation of leaderless mRNAs. Based on these and other observations, it is deemed that the CS mechanism is the universal initiation mechanism.

  15. Phylogenetic origins of the plant mitochondrion based on a comparative analysis of 5S ribosomal RNA sequences

    NASA Technical Reports Server (NTRS)

    Villanueva, E.; Delihas, N.; Luehrsen, K. R.; Fox, G. E.; Gibson, J.

    1985-01-01

    The complete nucleotide sequences of 5S ribosomal RNAs from Rhodocyclus gelatinosa, Rhodobacter sphaeroides, and Pseudomonas cepacia were determined. Comparisons of these 5S RNA sequences show that rather than being phylogenetically related to one another, the two photosynthetic bacterial 5S RNAs share more sequence and signature homology with the RNAs of two nonphotosynthetic strains. Rhodobacter sphaeroides is specifically related to Paracoccus denitrificans and Rc. gelatinosa is related to Ps. cepacia. These results support earlier 16S ribosomal RNA studies and add two important groups to the 5S RNA data base. Unique 5S RNA structural features previously found in P. denitrificans are present also in the 5S RNA of Rb. sphaeroides; these provide the basis for subdivisional signatures. The immediate consequence of obtaining these new sequences is that it is possible to clarify the phylogenetic origins of the plant mitochondrion. In particular, a close phylogenetic relationship is found between the plant mitochondria and members of the alpha subdivision of the purple photosynthetic bacteria, namely, Rb. sphaeroides, P. denitrificans, and Rhodospirillum rubrum.

  16. Updates on quick identification of acetic acid bacteria with a focus on the 16S-23S rRNA gene internal transcribed spacer and the analysis of cell proteins by MALDI-TOF mass spectrometry.

    PubMed

    Trček, Janja; Barja, François

    2015-03-02

    Acetic acid bacteria have attracted much attention over the past few years, due mainly to their metabolic traits that are of interest to the biotechnology industry. In addition, it turns out that their ecological habitats are almost unlimited since they have been found as symbionts in different insects and also as emerging opportunistic human pathogens. Very surprising is the finding that they colonize niches considered anaerobic, disproving the generalized statement that they are strict aerobes. Since they have taken on different biological roles in our environment, more and more people are charged with the task of identifying them. However, this turns out to be not always easy, especially if we are using phenotypic approaches for identification. A substantial step forward in making the identification of acetic acid bacteria easier was made possible using molecular biological methods, which have been extensively tested since 2000. However, some molecular methods require expensive machines and experienced staff, and moreover the level of their discrimination varies. All these factors must be considered when selecting the most appropriate approach for identifying acetic acid bacteria. With this objective in mind, this review article discusses the benefits and drawbacks of molecular biological methods for identification of acetic acid bacteria, with a focus on the 16S-23S rRNA gene ITS regions and the recently described alternative method for identification of acetic acid bacteria, MALDI-TOF MS.

  17. Stoichiometry and Change of the mRNA Closed-Loop Factors as Translating Ribosomes Transit from Initiation to Elongation.

    PubMed

    Wang, Xin; Xi, Wen; Toomey, Shaun; Chiang, Yueh-Chin; Hasek, Jiri; Laue, Thomas M; Denis, Clyde L

    2016-01-01

    Protein synthesis is a highly efficient process and is under exacting control. Yet, the actual abundance of translation factors present in translating complexes and how these abundances change during the transit of a ribosome across an mRNA remains unknown. Using analytical ultracentrifugation with fluorescent detection we have determined the stoichiometry of the closed-loop translation factors for translating ribosomes. A variety of pools of translating polysomes and monosomes were identified, each containing different abundances of the closed-loop factors eIF4E, eIF4G, and PAB1 and that of the translational repressor, SBP1. We establish that closed-loop factors eIF4E/eIF4G dissociated both as ribosomes transited polyadenylated mRNA from initiation to elongation and as translation changed from the polysomal to monosomal state prior to cessation of translation. eIF4G was found to particularly dissociate from polyadenylated mRNA as polysomes moved to the monosomal state, suggesting an active role for translational repressors in this process. Consistent with this suggestion, translating complexes generally did not simultaneously contain eIF4E/eIF4G and SBP1, implying mutual exclusivity in such complexes. For substantially deadenylated mRNA, however, a second type of closed-loop structure was identified that contained just eIF4E and eIF4G. More than one eIF4G molecule per polysome appeared to be present in these complexes, supporting the importance of eIF4G interactions with the mRNA independent of PAB1. These latter closed-loop structures, which were particularly stable in polysomes, may be playing specific roles in both normal and disease states for specific mRNA that are deadenylated and/or lacking PAB1. These analyses establish a dynamic snapshot of molecular abundance changes during ribosomal transit across an mRNA in what are likely to be critical targets of regulation.

  18. Selenocysteine insertion sequence (SECIS)-binding protein 2 alters conformational dynamics of residues involved in tRNA accommodation in 80 S ribosomes.

    PubMed

    Caban, Kelvin; Copeland, Paul R

    2012-03-23

    Sec-tRNA(Sec) is site-specifically delivered at defined UGA codons in selenoprotein mRNAs. This recoding event is specified by the selenocysteine insertion sequence (SECIS) element and requires the selenocysteine (Sec)-specific elongation factor, eEFSec, and the SECIS binding protein, SBP2. Sec-tRNA(Sec) is delivered to the ribosome by eEFSec-GTP, but this ternary complex is not sufficient for Sec incorporation, indicating that its access to the ribosomal A-site is regulated. SBP2 stably associates with ribosomes, and mutagenic analysis indicates that this interaction is essential for Sec incorporation. However, the ribosomal function of SBP2 has not been elucidated. To shed light on the functional relevance of the SBP2-ribosome interaction, we screened the functional centers of the 28 S rRNA in translationally competent 80 S ribosomes using selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE). We demonstrate that SBP2 specifically alters the reactivity of specific residues in Helix 89 (H89) and expansion segment 31 (ES31). These results are indicative of a conformational change in response to SBP2 binding. Based on the known functions of H89 during translation, we propose that SBP2 allows Sec incorporation by either promoting Sec-tRNA(Sec) accommodation into the peptidyltransferase center and/or by stimulating the ribosome-dependent GTPase activity of eEFSec.

  19. Ribosome-targeting antibiotics as inhibitors of oncogenic microRNAs biogenesis: Old scaffolds for new perspectives in RNA targeting.

    PubMed

    Tran, Thi Phuong Anh; Vo, Duc Duy; Di Giorgio, Audrey; Duca, Maria

    2015-09-01

    MicroRNAs (miRNAs) are non-coding RNAs that regulate gene expression at the post-transcriptional level. It is now well established that the overexpression of some miRNAs (oncogenic miRNAs) is responsible for initiation and progression of human cancers and the discovery of new molecules able to interfere with their production and/or function represents one of the most important challenges of current medicinal chemistry of RNA ligands. In this work, we studied the ability of 18 different antibiotics, known as prokaryotic ribosomal RNA, to bind to oncogenic miRNA precursors (stem-loop structured pre-miRNAs) in order to inhibit miRNAs production. In vitro inhibition, binding constants, thermodynamic parameters and binding sites were investigated and highlighted that aminoglycosides and tetracyclines represent interesting pre-miRNA ligands with the ability to inhibit Dicer processing.

  20. Analysis of a sequence region of 5S RNA from E. coli cross-linked in situ to the ribosomal protein L25.

    PubMed Central

    Szymkowiak, C; Wagner, R

    1985-01-01

    70S ribosomes from E. coli were chemically cross-linked under conditions of in vitro protein biosynthesis. The ribosomal RNAs were extracted from reacted ribosomes and separated on sucrose gradients. The 5S RNA was shown to contain the ribosomal protein L25 covalently bound. After total RNase T1 hydrolysis of the covalent RNA-protein complex several high molecular weight RNA fragments were obtained and identified by sequencing. One fragment, sequence region U103 to U120, was shown to be directly linked to the protein first by protein specific staining of the particular fragment and second by phosphor cellulose chromatography of the covalent RNA-protein complex. The other two fragments, U89 to G106 and A34 to G51, could not be shown to be directly linked to L25 but were only formed under cross-linking conditions. While the fragment U89 to G106 may be protected from RNase T1 digestion because of a strong interaction with the covalent RNA-protein complex, the formation of the fragment A34 to G51 is very likely the result of a double monovalent modification of two neighbouring guanosines in the 5S RNA. The RNA sequences U103 to U120 established to be in direct contact to the protein L25 within the ribosome falls into the sequence region previously proposed as L25 binding site from studies with isolated 5S RNA-protein complexes. Images PMID:3892485

  1. The histone demethylase JMJD2A/KDM4A links ribosomal RNA transcription to nutrients and growth factors availability.

    PubMed

    Salifou, Kader; Ray, Swagat; Verrier, Laure; Aguirrebengoa, Marion; Trouche, Didier; Panov, Konstantin I; Vandromme, Marie

    2016-01-05

    The interplay between methylation and demethylation of histone lysine residues is an essential component of gene expression regulation and there is considerable interest in elucidating the roles of proteins involved. Here we report that histone demethylase KDM4A/JMJD2A, which is involved in the regulation of cell proliferation and is overexpressed in some cancers, interacts with RNA Polymerase I, associates with active ribosomal RNA genes and is required for serum-induced activation of rDNA transcription. We propose that KDM4A controls the initial stages of transition from 'poised', non-transcribed rDNA chromatin into its active form. We show that PI3K, a major signalling transducer central for cell proliferation and survival, controls cellular localization of KDM4A and consequently its association with ribosomal DNA through the SGK1 downstream kinase. We propose that the interplay between PI3K/SGK1 signalling cascade and KDM4A constitutes a mechanism by which cells adapt ribosome biogenesis level to the availability of growth factors and nutrients.

  2. The histone demethylase JMJD2A/KDM4A links ribosomal RNA transcription to nutrients and growth factors availability

    PubMed Central

    Salifou, Kader; Ray, Swagat; Verrier, Laure; Aguirrebengoa, Marion; Trouche, Didier; Panov, Konstantin I.; Vandromme, Marie

    2016-01-01

    The interplay between methylation and demethylation of histone lysine residues is an essential component of gene expression regulation and there is considerable interest in elucidating the roles of proteins involved. Here we report that histone demethylase KDM4A/JMJD2A, which is involved in the regulation of cell proliferation and is overexpressed in some cancers, interacts with RNA Polymerase I, associates with active ribosomal RNA genes and is required for serum-induced activation of rDNA transcription. We propose that KDM4A controls the initial stages of transition from ‘poised', non-transcribed rDNA chromatin into its active form. We show that PI3K, a major signalling transducer central for cell proliferation and survival, controls cellular localization of KDM4A and consequently its association with ribosomal DNA through the SGK1 downstream kinase. We propose that the interplay between PI3K/SGK1 signalling cascade and KDM4A constitutes a mechanism by which cells adapt ribosome biogenesis level to the availability of growth factors and nutrients. PMID:26729372

  3. Ribosome profiling-guided depletion of an mRNA increases cell growth rate and protein secretion

    NASA Astrophysics Data System (ADS)

    Kallehauge, Thomas Beuchert; Li, Shangzhong; Pedersen, Lasse Ebdrup; Ha, Tae Kwang; Ley, Daniel; Andersen, Mikael Rørdam; Kildegaard, Helene Faustrup; Lee, Gyun Min; Lewis, Nathan E.

    2017-01-01

    Recombinant protein production coopts the host cell machinery to provide high protein yields of industrial enzymes or biotherapeutics. However, since protein translation is energetically expensive and tightly controlled, it is unclear if highly expressed recombinant genes are translated as efficiently as host genes. Furthermore, it is unclear how the high expression impacts global translation. Here, we present the first genome-wide view of protein translation in an IgG-producing CHO cell line, measured with ribosome profiling. Through this we found that our recombinant mRNAs were translated as efficiently as the host cell transcriptome, and sequestered up to 15% of the total ribosome occupancy. During cell culture, changes in recombinant mRNA translation were consistent with changes in transcription, demonstrating that transcript levels influence specific productivity. Using this information, we identified the unnecessary resistance marker NeoR to be a highly transcribed and translated gene. Through siRNA knock-down of NeoR, we improved the production- and growth capacity of the host cell. Thus, ribosomal profiling provides valuable insights into translation in CHO cells and can guide efforts to enhance protein production.

  4. Ribosome profiling-guided depletion of an mRNA increases cell growth rate and protein secretion

    PubMed Central

    Kallehauge, Thomas Beuchert; Li, Shangzhong; Pedersen, Lasse Ebdrup; Ha, Tae Kwang; Ley, Daniel; Andersen, Mikael Rørdam; Kildegaard, Helene Faustrup; Lee, Gyun Min; Lewis, Nathan E.

    2017-01-01

    Recombinant protein production coopts the host cell machinery to provide high protein yields of industrial enzymes or biotherapeutics. However, since protein translation is energetically expensive and tightly controlled, it is unclear if highly expressed recombinant genes are translated as efficiently as host genes. Furthermore, it is unclear how the high expression impacts global translation. Here, we present the first genome-wide view of protein translation in an IgG-producing CHO cell line, measured with ribosome profiling. Through this we found that our recombinant mRNAs were translated as efficiently as the host cell transcriptome, and sequestered up to 15% of the total ribosome occupancy. During cell culture, changes in recombinant mRNA translation were consistent with changes in transcription, demonstrating that transcript levels influence specific productivity. Using this information, we identified the unnecessary resistance marker NeoR to be a highly transcribed and translated gene. Through siRNA knock-down of NeoR, we improved the production- and growth capacity of the host cell. Thus, ribosomal profiling provides valuable insights into translation in CHO cells and can guide efforts to enhance protein production. PMID:28091612

  5. Knock-out of the plastid ribosomal protein L11 in Arabidopsis: effects on mRNA translation and photosynthesis.

    PubMed

    Pesaresi, P; Varotto, C; Meurer, J; Jahns, P; Salamini, F; Leister, D

    2001-08-01

    The prpl11-1 mutant of Arabidopsis thaliana was identified among a collection of T-DNA tagged lines on the basis of a decrease in the effective quantum yield of photosystem II. The mutation responsible was localized to Prpl11, a single-copy nuclear gene that encodes PRPL11, a component of the large subunit of the plastid ribosome. The amino acid sequence of Arabidopsis PRPL11 is very similar to those of L11 proteins from spinach and prokaryotes. In the prpl11-1 mutant, photosensitivity and chlorophyll fluorescence parameters are significantly altered owing to changes in the levels of thylakoid protein complexes and stromal proteins. The abundance of most plastome transcripts examined, such as those of genes coding for the photosystem II core complex and RbcL, is not decreased. Plastid ribosomal RNA accumulates in wild-type amounts, and the assembly of plastid polysomes on the transcripts of the rbcL, psbA and psbE genes remains mainly unchanged in mutant plants, indicating that lack of PRPL11 affects neither the abundance of plastid ribosomes nor their assembly into polysomes. However, in vivo translation assays demonstrate that the rate of translation of the large subunit of Rubisco (RbcL) is significantly reduced in prpl11-1 plastids. Our data suggest a major role for PRPL11 in plastid ribosome activity per se, consistent with its location near the GTPase-binding centre of the chloroplast 50S ribosomal subunit. Additional effects of the mutation, including the pale green colour of the leaves and a drastic reduction in growth rate under greenhouse conditions, are compatible with reduced levels of protein synthesis in plastids.

  6. Identification of Egyptian Fasciola species by PCR and restriction endonucleases digestion of the nuclear small subunit ribosomal RNA gene.

    PubMed

    El-Gozamy, Bothina R; Shoukry, Nahla M

    2009-08-01

    Fascioliasis is one of the familiar zoonotic health problems of worldwide distribution including Egypt. In this study, a simple and rapid polymerase chain reaction/restriction fragment length polymorphisms (PCR/RFLPs) assay, using the common restriction endonucleases Aval, EcoRI, Eael, Sac11 and Avail was applied to differentiate between both Fasciola gigantica and F. hepatica. The five restriction endonucleases were used to differentiate between the two species of Fasciola based on -1950 bp long sequence of the 18S nuclear small subunit ribosomal RNA gene. Aval and EcoRI restriction endonucleases failed to differentiate between the two Fasciola species when each restriction enzyme gave the same restriction patterns in both of them. However, F. gigantica and F. hepatica were well-differentiated when their small subunit ribosomal DNA were digested with Eael and Sac 11 restriction endonucleases.

  7. A novel mutation 3090 G>A of the mitochondrial 16S ribosomal RNA associated with myopathy.

    PubMed

    Coulbault, L; Deslandes, B; Herlicoviez, D; Read, M H; Leporrier, N; Schaeffer, S; Mouadil, A; Lombès, A; Chapon, F; Jauzac, P; Allouche, S

    2007-10-26

    We describe a young woman who presented with a progressive myopathy since the age of 9. Spectrophotometric analysis of the respiratory chain in muscle tissue revealed combined and profound complex I, III, II+III, and IV deficiency ranging from 60% to 95% associated with morphological and histochemical abnormalities of the muscle. An exhaustive screening of mitochondrial transfer and ribosomal RNAs showed a novel G>A substitution at nucleotide position 3090 which was detected only in urine sediment and muscle of the patient and was not found in her mother's blood cells and urine sample. We suggest that this novel de novo mutation in the 16S ribosomal RNA, a nucleotide which is highly conserved in different species, would impair mitochondrial protein synthesis and would cause a severe myopathy.

  8. Low-molecular-weight (4.5S) ribonucleic acid in higher-plant chloroplast ribosomes.

    PubMed Central

    Whitfeld, P R; Leaver, C J; Bottomley, W; Atchison, B

    1978-01-01

    A species of RNA that migrates on 10% (w/v) polyacrylamide gels between 5S and 4S RNA was detected in spinach chloroplasts. This RNA (referred to as 4.5 S RNA) was present in amounts equimolar to the 5S RNA and its molecular weight was estimated to be approx. 33 000. Fractionation of the chloroplast components showed that the 4.5S RNA was associated with the 50 S ribosomal subunit and that it could be removed by washing the ribosomes with a buffer containing 0.01 M-EDTA and 0.5 M-KCl. It did not appear to be a cleavage product of the labile 23 S RNA of spinach chloroplast ribosomes. When 125I-labelled 4.5 S RNA was hybridized to fragments of spinach chloroplast DNA produced by SmaI restriction endonuclease, a single fragment (mol.wt. 1.15 times 10(6)) became labelled. The same DNA fragment also hybridized to chloroplast 5 S RNA and part of the 23 S RNA. It was concluded that the coding sequence for 4.5 S RNA was part of, or immediately adjacent to, the rRNA-gene region in chloroplast DNA . A comparable RNA species was observed in chloroplasts of tobacco and pea leaves. Images Fig. 8. PMID:743229

  9. Posttranscriptional regulation of ribosomal protein S20 and stability of the S20 mRNA species.

    PubMed Central

    Mackie, G A

    1987-01-01

    I have tested whether selective degradation of mRNA for ribosomal protein S20 of Escherichia coli occurs under conditions for which the expression of S20 is regulated posttranscriptionally. Blot hybridization of total RNA extracted from cultures at different times after addition of rifampin has permitted the estimation of relative levels of the two S20 mRNA species and their half-lives. In a strain harboring a plasmid containing the complete gene for S20, including the transcriptional terminator, moderate posttranscriptional repression of S20 synthesis is accompanied by a substantial increase in the half-lives of both S20 mRNAs relative to those in the haploid parental strain. In an otherwise identical strain in which the transcriptional terminator is deleted from the plasmid-borne S20 genes, the half-life of total S20 mRNA declines more than twofold relative to that in the haploid parent. Thus accelerated decay of the mRNAs for ribosomal protein S20 appears to be an artifact of deletion of the transcriptional terminator, rather than a physiologically significant consequence of translational repression. Images PMID:2438268

  10. Overexpression of Ribosomal RNA in the Development of Human Cervical Cancer Is Associated with rDNA Promoter Hypomethylation

    PubMed Central

    Zhou, Hong; Wang, Yapei; Lv, Qiongying; Zhang, Juan; Wang, Qing; Gao, Fei; Hou, Haoli; Zhang, Hao; Zhang, Wei; Li, Lijia

    2016-01-01

    The ribosomal RNA (rRNA) gene encodes rRNA for protein synthesis. Aberrant expression of the rRNA gene has been generally observed in tumor cells and levels of its promoter methylation as an epigenetic regulator affect rRNA gene transcription. The possible relationship between expression and promoter methylation of rDNA has not been examined in human clinical cervical cancer. Here we investigate rRNA gene expression by quantitative real time PCR, and promoter methylation levels by HpaII/MspI digestion and sodium bisulfite sequencing in the development of human cervical cancer. We find that indeed rRNA levels are elevated in most of cervical intraepithelial neoplasia (CIN) specimens as compared with non-cancer tissues. The rDNA promoter region in cervical intraepithelial neoplasia (CIN) tissues reveals significant hypomethylation at cytosines in the context of CpG dinucleotides, accompanied with rDNA chromatin decondensation. Furthermore treatment of HeLa cells with the methylation inhibitor drug 5-aza-2’-deoxycytidine (DAC) demonstrates the negative correlation between the expression of 45S rDNA and the methylation level in the rDNA promoter region. These data suggest that a decrease in rDNA promoter methylation levels can result in an increase of rRNA synthesis in the development of human cervical cancer. PMID:27695092

  11. Linker 2 of the eukaryotic pre-ribosomal processing factor Mrd1p is an essential interdomain functionally coupled to upstream RNA Binding Domain 2 (RBD2).

    PubMed

    Lackmann, Fredrik; Belikov, Sergey; Wieslander, Lars

    2017-01-01

    Ribosome synthesis is an essential process in all cells. In Sacharomyces cerevisiae, the precursor rRNA, 35S pre-rRNA, is folded and assembled into a 90S pre-ribosomal complex. The 40S ribosomal subunit is processed from the pre-ribosomal complex. This requires concerted action of small nucleolar RNAs, such as U3 snoRNA, and a large number of trans-acting factors. Mrd1p, one of the essential small ribosomal subunit synthesis factors is required for cleavage of the 35S pre-rRNA to generate 18S rRNA of the small ribosomal subunit. Mrd1p is evolutionary conserved in all eukaryotes and in yeast it contains five RNA Binding Domains (RBDs) separated by linker regions. One of these linkers, Linker 2 between RBD2 and RBD3, is conserved in length, predicted to be structured and contains conserved clusters of amino acid residues. In this report, we have analysed Linker 2 mutations and demonstrate that it is essential for Mrd1p function during pre-ribosomal processing. Extensive changes of amino acid residues as well as specific changes of conserved clusters of amino acid residues were found to be incompatible with synthesis of pre-40S ribosomes and cell growth. In addition, gross changes in primary sequence of Linker 2 resulted in Mrd1p instability, leading to degradation of the N-terminal part of the protein. Our data indicates that Linker 2 is functionally coupled to RBD2 and argues for that these domains constitute a functional module in Mrd1p. We conclude that Linker 2 has an essential role for Mrd1p beyond just providing a defined length between RBD2 and RBD3.

  12. Ralstonia paucula (Formerly CDC Group IV c-2): Unsuccessful Strain Differentiation with PCR-Based Methods, Study of the 16S-23S Spacer of the rRNA Operon, and Comparison with Other Ralstonia Species (R. eutropha, R. pickettii, R. gilardii, and R. solanacearum)

    PubMed Central

    Moissenet, Didier; Bidet, Philippe; Garbarg-Chenon, Antoine; Arlet, Guillaume; Vu-Thien, Hoang

    2001-01-01

    Ralstonia paucula (formerly CDC group IV c-2) can cause serious human infections. Confronted in 1995 with five cases of nosocomial bacteremia, we found that pulsed-field gel electrophoresis could not distinguish between the isolates and that randomly amplified polymorphic DNA analysis was poorly discriminatory. In this study, we used PCR-ribotyping and PCR-restriction fragment length polymorphism analysis of the spacer 16S-23S ribosomal DNA (rDNA); both methods were unable to differentiate R. paucula isolates. Eighteen strains belonging to other Ralstonia species (one R. eutropha strain, six R. pickettii strains, three R. solanacearum strains, and eight R. gilardii strains) were also tested by PCR-ribotyping, which failed to distinguish between the four species. The 16S-23S rDNA intergenic spacer of R. paucula contains the tRNAIle and tRNAAla genes, which are identical to genes described for R. pickettii and R. solanacearum. PMID:11136807

  13. Ribosomal RNA depletion or exclusion has negligible effect on the detection of viruses in a pan viral microarray.

    PubMed

    McGowan, Sarah; Nunez-Garcia, Javier; Steinbach, Falko; La Rocca, Anna; Blake, Damer; Dastjerdi, Akbar

    2014-10-01

    Pan viral DNA microarrays, which can detect known, novel and multiple viral infections, are major laboratory assets contributing to the control of infectious diseases. The large quantity of ribosomal RNA (rRNA) found in tissue samples is thought to be a major factor contributing to the comparatively lower sensitivity of detecting RNA viruses, as a sequence-independent PCR is used to amplify unknown samples for microarray analysis. This study aimed to determine whether depletion or exclusion of rRNA can improve microarray detection and simplify its analysis. Therefore, two different rRNA depletion and exclusion protocols, RiboMinus™ technology and non-rRNA binding hexanucleotides, were applied to the microarray sample processing and the outcome was compared with those of the sequence-independent amplification protocol. This study concludes that the two procedures, described to deplete or exclude rRNA, have negligible effect on the microarrays detection and analysis and might only in combination with further techniques result in a significant enhancement of sensitivity. Currently, existing protocols of random amplification and background adjustment are pertinent for the purpose of sample processing for microarray analysis.

  14. LKB1 promotes cell survival by modulating TIF-IA-mediated pre-ribosomal RNA synthesis under uridine downregulated conditions.

    PubMed

    Liu, Fakeng; Jin, Rui; Liu, Xiuju; Huang, Henry; Wilkinson, Scott C; Zhong, Diansheng; Khuri, Fadlo R; Fu, Haian; Marcus, Adam; He, Yulong; Zhou, Wei

    2016-01-19

    We analyzed the mechanism underlying 5-aminoimidazole-4-carboxamide riboside (AICAR) mediated apoptosis in LKB1-null non-small cell lung cancer (NSCLC) cells. Metabolic profile analysis revealed depletion of the intracellular pyrimidine pool after AICAR treatment, but uridine was the only nucleotide precursor capable of rescuing this apoptosis, suggesting the involvement of RNA metabolism. Because half of RNA transcription in cancer is for pre-ribosomal RNA (rRNA) synthesis, which is suppressed by over 90% after AICAR treatment, we evaluated the role of TIF-IA-mediated rRNA synthesis. While the depletion of TIF-IA by RNAi alone promoted apoptosis in LKB1-null cells, the overexpression of a wild-type or a S636A TIF-IA mutant, but not a S636D mutant, attenuated AICAR-induced apoptosis. In LKB1-null H157 cells, pre-rRNA synthesis was not suppressed by AICAR when wild-type LKB1 was present, and cellular fractionation analysis indicated that TIF-IA quickly accumulated in the nucleus in the presence of a wild-type LKB1 but not a kinase-dead mutant. Furthermore, ectopic expression of LKB1 was capable of attenuating AICAR-induced death in AMPK-null cells. Because LKB1 promotes cell survival by modulating TIF-IA-mediated pre-rRNA synthesis, this discovery suggested that targeted depletion of uridine related metabolites may be exploited in the clinic to eliminate LKB1-null cancer cells.

  15. The PIN domain endonuclease Utp24 cleaves pre-ribosomal RNA at two coupled sites in yeast and humans

    PubMed Central

    Wells, Graeme R.; Weichmann, Franziska; Colvin, David; Sloan, Katherine E.; Kudla, Grzegorz; Tollervey, David; Watkins, Nicholas J.; Schneider, Claudia

    2016-01-01

    During ribosomal RNA (rRNA) maturation, cleavages at defined sites separate the mature rRNAs from spacer regions, but the identities of several enzymes required for 18S rRNA release remain unknown. PilT N-terminus (PIN) domain proteins are frequently endonucleases and the PIN domain protein Utp24 is essential for early cleavages at three pre-rRNA sites in yeast (A0, A1 and A2) and humans (A0, 1 and 2a). In yeast, A1 is cleaved prior to A2 and both cleavages require base-pairing by the U3 snoRNA to the central pseudoknot elements of the 18S rRNA. We found that yeast Utp24 UV-crosslinked in vivo to U3 and the pseudoknot, placing Utp24 close to cleavage at site A1. Yeast and human Utp24 proteins exhibited in vitro endonuclease activity on an RNA substrate containing yeast site A2. Moreover, an intact PIN domain in human UTP24 was required for accurate cleavages at sites 1 and 2a in vivo, whereas mutation of another potential site 2a endonuclease, RCL1, did not affect 18S production. We propose that Utp24 cleaves sites A1/1 and A2/2a in yeast and human cells. PMID:27034467

  16. Comparison of multiple genes and 16S-23S rRNA intergenic space region for their capacity in high resolution melt curve analysis to differentiate Mycoplasma gallisepticum vaccine strain ts-11 from field strains.

    PubMed

    Ghorashi, Seyed A; Bradbury, Janet M; Ferguson-Noel, Naola M; Noormohammadi, Amir H

    2013-12-27

    Mycoplasma gallisepticum (MG) is an important avian pathogen causing significant economic losses in the global poultry industry. In an attempt to compare and evaluate existing genotyping methods for differentiation of MG strains/isolates, high resolution melt (HRM) curve analysis was applied to 5 different PCR methods targeting vlhA, pvpA, gapA, mgc2 genes and 16S-23S rRNA intergenic space region (IGSR). To assess the discriminatory power of PCR-HRM of examined genes and IGSR, MG strains ts-11, F, 6/85 and S6, and, initially, 8 field isolates were tested. All MG strains/isolates were differentiated using PCR-HRM curve analysis and genotype confidence percentage (GCP) values of vlhA and pvpA genes, while only 0, 3 and 4 out of 12 MG strains/isolates were differentiated using gapA, mgc2 genes and IGSR, respectively. The HRM curve analysis of vlhA and pvpA genes was found to be highly correlated with the genetic diversity of the targeted genes confirmed by sequence analysis of amplicons generated from MG strains. The potential of the vlhA and pvpA genes was also demonstrated for genotyping of 12 additional MG strains from Europe and the USA. Results from this study provide a direct comparison between genes previously used in sequencing-based genotyping methods for MG strain identification and highlight the usefulness of vlhA and pvpA HRM curve analyses as rapid and reliable tools specially for diagnosis and differentiation of MG strains used here.

  17. A Nucleolar Protein, Ribosomal RNA Processing 1 Homolog B (RRP1B), Enhances the Recruitment of Cellular mRNA in Influenza Virus Transcription

    PubMed Central

    Su, Wen-Chi; Hsu, Shih-Feng; Lee, Yi-Yuan; Jeng, King-Song

    2015-01-01

    ABSTRACT Influenza A virus (IAV) undergoes RNA transcription by a unique capped-mRNA-dependent transcription, which is carried out by the viral RNA-dependent RNA polymerase (RdRp), consisting of the viral PA, PB1, and PB2 proteins. However, how the viral RdRp utilizes cellular factors for virus transcription is not clear. Previously, we conducted a genome-wide pooled short hairpin RNA (shRNA) screen to identify host factors important for influenza A virus replication. Ribosomal RNA processing 1 homolog B (RRP1B) was identified as one of the candidates. RRP1B is a nucleolar protein involved in ribosomal biogenesis. Upon IAV infection, part of RRP1B was translocated from the nucleolus to the nucleoplasm, where viral RNA synthesis likely takes place. The depletion of RRP1B significantly reduced IAV mRNA transcription in a minireplicon assay and in virus-infected cells. Furthermore, we showed that RRP1B interacted with PB1 and PB2 of the RdRp and formed a coimmunoprecipitable complex with RdRp. The depletion of RRP1B reduced the amount of capped mRNA in the RdRp complex. Taken together, these findings indicate that RRP1B is a host factor essential for IAV transcription and provide a target for new antivirals. IMPORTANCE Influenza virus is an important human pathogen that causes significant morbidity and mortality and threatens the human population with epidemics and pandemics every year. Due to the high mutation rate of the virus, antiviral drugs targeting viral proteins might ultimately lose their effectiveness. An alternative strategy that explores the genetic stability of host factors indispensable for influenza virus replication would thus be desirable. Here, we characterized the rRNA processing 1 homolog B (RRP1B) protein as an important cellular factor for influenza A virus transcription. We showed that silencing RRP1B hampered viral RNA-dependent RNA polymerase (RdRp) activity, which is responsible for virus transcription and replication. Furthermore, we

  18. Detection of Aspergillus fumigatus pulmonary fungal infections in mice with 99mTc-labeledMORF oligomers targeting ribosomal RNA

    PubMed Central

    Wang, Yuzhen; Chen, Ling; Liu, Xinrong; Cheng, Dengfeng; Liu, Guozheng; Liu, Yuxia; Dou, Shuping; Hnatowich, Donald J.; Rusckowski, Mary

    2012-01-01

    Purpose Invasive aspergillosis is a major cause of infectious morbidity and mortality in immunocompromised hosts. The fungus Aspergillus fumigatus (A. fumigatus) is the primary causative agent of invasive aspergillosis. However, A. fumigatus infections remain difficult to diagnose particularly in the early stages due to the lack of a rapid, sensitive and specific diagnostic approach. In this study, we investigated 99mTc labeled MORF oligomers targeting fungal ribosomal RNA (rRNA) for the imaging detection of fungal infections. Procedures Three phosphorodiamidate morpholino (MORF) oligomer (a DNA analogue) probes were designed: AGEN, complementary to a sequence of the fungal 28S ribosomal RNA (rRNA) of Aspergillus, as a genus-specific probe; AFUM, complementary to the 28S rRNA sequence of A. fumigatus, as a fungus species-specific probe; and cMORF, irrelevant to all fungi species, as a control probe. The probes were conjugated with Alexa Fluor 633 carboxylic acid succinimidyl ester (AF633) for fluorescence imaging or with NHS-mercaptoacetyl triglycine (NHS-MAG3) for nuclear imaging with 99mTc and then evaluated in vitro and in vivo. Results The specific binding of AGEN and AFUM to fungal total RNA was confirmed by dot blot hybridization while specific binding of AGEN and AFUM in fixed and live A. fumigatus was demonstrated by both fluorescent in situ hybridization (FISH) analysis and accumulation in live cells. SPECT imaging of BALB/c mice with pulmonary A. fumigatus infections and administered 99mTc labeled AGEN and AFUM showed immediate and obvious accumulation in the infected lungs, while no significant accumulation of the control 99mTc-cMORF in the infected lung was observed. Compared to non-infected mice, with sacrifice at 1 hour, the accumulation of 99mTc-AGEN and 99mTc-AFUM in the lungs of mice infected with A. fumigatus were 2 and 2.7 fold higher respectively. Conclusions In vivo targeting fungal ribosomal RNA with 99mTc labeled MORF probes AGEN and AFUM may

  19. Characterization of DbpA, an Escherichia coli DEAD box protein with ATP independent RNA unwinding activity.

    PubMed Central

    Böddeker, N; Stade, K; Franceschi, F

    1997-01-01

    DbpA is a putative Escherichia coli ATP dependent RNA helicase belonging to the family of DEAD box proteins. It hydrolyzes ATP in the presence of 23S ribosomal RNA and 93 bases in the peptidyl transferase center of 23S rRNA are sufficient to trigger 100% of the ATPase activity of DbpA. In the present study we characterized the ATPase and RNA unwinding activities of DbpA in more detail. We report that-in contrast to eIF-4A, the prototype of the DEAD box protein family-the ATPase and the helicase activities of DbpA are not coupled. Moreover, the RNA unwinding activity of DbpA is not specific for 23S rRNA, since DbpA is also able to unwind 16S rRNA hybrids. Furthermore, we determined that the ATPase activity of DbpA is triggered to a significant extent not only by the 93 bases of the 23S rRNA previously reported but also by other regions of the 23S rRNA molecule. Since all these regions of 23S rRNA are either part of the 'functional core' of the 50S ribosomal subunit or involved in the 50S assembly, DbpA may play an important role in the ribosomal assembly process. PMID:9016593

  20. Studies of the GTPase domain of archaebacterial ribosomes.

    PubMed

    Beauclerk, A A; Hummel, H; Holmes, D J; Böck, A; Cundliffe, E

    1985-09-02

    Ribosomes from the methanogens Methanococcus vannielii and Methanobacterium formicicum catalyse uncoupled hydrolysis of GTP in the presence of factor EF-2 from rat liver (but not factor EF-G from Escherichia coli). In this assay, and in poly(U)-dependent protein synthesis, they were sensitive to thiostrepton. In contrast, ribosomes from Sulfolobus solfataricus did not respond to factor EF-2 (or factor EF-G) but possessed endogenous GTPase activity, which was also sensitive to thiostrepton. Ribosomes from the methanogens did not support (p)ppGpp production, but did appear to possess the equivalent of protein L11, which in E. coli is normally required for guanosine polyphosphate synthesis. Protein L11 from E. coli bound well to 23S rRNA from all three archaebacteria (as did thiostrepton) and oligonucleotides protected by the protein were sequenced and compared with rRNA sequences from other sources.

  1. Direct Activation of Ribosome-Associated Double-Stranded RNA-Dependent Protein Kinase (PKR) by Deoxynivalenol, Anisomycin and Ricin: A New Model for Ribotoxic Stress Response Induction

    PubMed Central

    Zhou, Hui-Ren; He, Kaiyu; Landgraf, Jeff; Pan, Xiao; Pestka, James J.

    2014-01-01

    Double-stranded RNA (dsRNA)-activated protein kinase (PKR) is a critical upstream mediator of the ribotoxic stress response (RSR) to the trichothecene deoxynivalenol (DON) and other translational inhibitors. Here, we employed HeLa cell lysates to: (1) characterize PKR’s interactions with the ribosome and ribosomal RNA (rRNA); (2) demonstrate cell-free activation of ribosomal-associated PKR and (3) integrate these findings in a unified model for RSR. Robust PKR-dependent RSR was initially confirmed in intact cells. PKR basally associated with 40S, 60S, 80S and polysome fractions at molar ratios of 7, 2, 23 and 3, respectively. Treatment of ATP-containing HeLa lysates with DON or the ribotoxins anisomycin and ricin concentration-dependently elicited phosphorylation of PKR and its substrate eIF2α. These phosphorylations could be blocked by PKR inhibitors. rRNA immunoprecipitation (RNA-IP) of HeLa lysates with PKR-specific antibody and sequencing revealed that in the presence of DON or not, the kinase associated with numerous discrete sites on both the 18S and 28S rRNA molecules, a number of which contained double-stranded hairpins. These findings are consistent with a sentinel model whereby multiple PKR molecules basally associate with the ribosome positioning them to respond to ribotoxin-induced alterations in rRNA structure by dimerizing, autoactivating and, ultimately, evoking RSR. PMID:25521494

  2. Translation complex profile sequencing to study the in vivo dynamics of mRNA-ribosome interactions during translation initiation, elongation and termination.

    PubMed

    Shirokikh, Nikolay E; Archer, Stuart K; Beilharz, Traude H; Powell, David; Preiss, Thomas

    2017-04-01

    Messenger RNA (mRNA) translation is a tightly controlled process that is integral to gene expression. It features intricate and dynamic interactions of the small and large subunits of the ribosome with mRNAs, aided by multiple auxiliary factors during distinct initiation, elongation and termination phases. The recently developed ribosome profiling method can generate transcriptome-wide surveys of translation and its regulation. Ribosome profiling records the footprints of fully assembled ribosomes along mRNAs and thus primarily interrogates the elongation phase of translation. Importantly, it does not monitor multiple substeps of initiation and termination that involve complexes between the small ribosomal subunit (SSU) and mRNA. Here we describe a related method, termed 'translation complex profile sequencing' (TCP-seq), that is uniquely capable of recording positions of any type of ribosome-mRNA complex transcriptome-wide. It uses fast covalent fixation of translation complexes in live cells, followed by RNase footprinting of translation intermediates and their separation into complexes involving either the full ribosome or the SSU. The footprints derived from each type of complex are then deep-sequenced separately, generating native distribution profiles during the elongation, as well as the initiation and termination stages of translation. We provide the full TCP-seq protocol for Saccharomyces cerevisiae liquid suspension culture, including a data analysis pipeline. The protocol takes ∼3 weeks to complete by a researcher who is well acquainted with standard molecular biology techniques and who has some experience in ultracentrifugation and the preparation of RNA sequencing (RNA-seq) libraries. Basic Bash and UNIX/Linux command skills are required to use the bioinformatics tools provided.

  3. Human ribosomal RNA gene: nucleotide sequence of the transcription initiation region and comparison of three mammalian genes.

    PubMed Central

    Financsek, I; Mizumoto, K; Mishima, Y; Muramatsu, M

    1982-01-01

    The transcription initiation site of the human ribosomal RNA gene (rDNA) was located by using the single-strand specific nuclease protection method and by determining the first nucleotide of the in vitro capped 45S preribosomal RNA. The sequence of 1,211 nucleotides surrounding the initiation site was determined. The sequenced region was found to consist of 75% G and C and to contain a number of short direct and inverted repeats and palindromes. By comparison of the corresponding initiation regions of three mammalian species, several conserved sequences were found upstream and downstream from the transcription starting point. Two short A + T-rich sequences are present on human, mouse, and rat ribosomal RNA genes between the initiation site and 40 nucleotides upstream, and a C + T cluster is located at a position around -60. At and downstream from the initiation site, a common sequence, T-AG-C-T-G-A-C-A-C-G-C-T-G-T-C-C-T-CT-T, was found in the three genes from position -1 through +18. The strong conservation of these sequences suggests their functional significance in rDNA. The S1 nuclease protection experiments with cloned rDNA fragments indicated the presence in human 45S RNA of molecules several hundred nucleotides shorter than the supposed primary transcript. The first 19 nucleotides of these molecules appear identical--except for one mismatch--to the nucleotide sequence of the 5' end of a supposed early processing product of the mouse 45S RNA. Images PMID:6954460

  4. Protein synthesis. Rqc2p and 60S ribosomal subunits mediate mRNA-independent elongation of nascent chains.

    PubMed

    Shen, Peter S; Park, Joseph; Qin, Yidan; Li, Xueming; Parsawar, Krishna; Larson, Matthew H; Cox, James; Cheng, Yifan; Lambowitz, Alan M; Weissman, Jonathan S; Brandman, Onn; Frost, Adam

    2015-01-02

    In Eukarya, stalled translation induces 40S dissociation and recruitment of the ribosome quality control complex (RQC) to the 60S subunit, which mediates nascent chain degradation. Here we report cryo-electron microscopy structures revealing that the RQC components Rqc2p (YPL009C/Tae2) and Ltn1p (YMR247C/Rkr1) bind to the 60S subunit at sites exposed after 40S dissociation, placing the Ltn1p RING (Really Interesting New Gene) domain near the exit channel and Rqc2p over the P-site transfer RNA (tRNA). We further demonstrate that Rqc2p recruits alanine- and threonine-charged tRNA to the A site and directs the elongation of nascent chains independently of mRNA or 40S subunits. Our work uncovers an unexpected mechanism of protein synthesis, in which a protein--not an mRNA--determines tRNA recruitment and the tagging of nascent chains with carboxy-terminal Ala and Thr extensions ("CAT tails").

  5. Slip of grip of a molecular motor on a crowded track: Modeling shift of reading frame of ribosome on RNA template

    NASA Astrophysics Data System (ADS)

    Mishra, Bhavya; Schütz, Gunter M.; Chowdhury, Debashish

    2016-06-01

    We develop a stochastic model for the programmed frameshift of ribosomes synthesizing a protein while moving along a mRNA template. Normally the reading frame of a ribosome decodes successive triplets of nucleotides on the mRNA in a step-by-step manner. We focus on the programmed shift of the ribosomal reading frame, forward or backward, by only one nucleotide which results in a fusion protein; it occurs when a ribosome temporarily loses its grip to its mRNA track. Special “slippery” sequences of nucleotides and also downstream secondary structures of the mRNA strand are believed to play key roles in programmed frameshift. Here we explore the role of an hitherto neglected parameter in regulating -1 programmed frameshift. Specifically, we demonstrate that the frameshift frequency can be strongly regulated also by the density of the ribosomes, all of which are engaged in simultaneous translation of the same mRNA, at and around the slippery sequence. Monte Carlo simulations support the analytical predictions obtained from a mean-field analysis of the stochastic dynamics.

  6. Mechanistic Insight into the Reactivation of BCAII Enzyme from Denatured and Molten Globule States by Eukaryotic Ribosomes and Domain V rRNAs

    PubMed Central

    Chakraborty, Biprashekhar; Bhakta, Sayan; Sengupta, Jayati

    2016-01-01

    In all life forms, decoding of messenger-RNA into polypeptide chain is accomplished by the ribosome. Several protein chaperones are known to bind at the exit of ribosomal tunnel to ensure proper folding of the nascent chain by inhibiting their premature folding in the densely crowded environment of the cell. However, accumulating evidence suggests that ribosome may play a chaperone role in protein folding events in vitro. Ribosome-mediated folding of denatured proteins by prokaryotic ribosomes has been studied extensively. The RNA-assisted chaperone activity of the prokaryotic ribosome has been attributed to the domain V, a span of 23S rRNA at the intersubunit side of the large subunit encompassing the Peptidyl Transferase Centre. Evidently, this functional property of ribosome is unrelated to the nascent chain protein folding at the exit of the ribosomal tunnel. Here, we seek to scrutinize whether this unique function is conserved in a primitive kinetoplastid group of eukaryotic species Leishmania donovani where the ribosome structure possesses distinct additional features and appears markedly different compared to other higher eukaryotic ribosomes. Bovine Carbonic Anhydrase II (BCAII) enzyme was considered as the model protein. Our results manifest that domain V of the large subunit rRNA of Leishmania ribosomes preserves chaperone activity suggesting that ribosome-mediated protein folding is, indeed, a conserved phenomenon. Further, we aimed to investigate the mechanism underpinning the ribosome-assisted protein reactivation process. Interestingly, the surface plasmon resonance binding analyses exhibit that rRNA guides productive folding by directly interacting with molten globule-like states of the protein. In contrast, native protein shows no notable affinity to the rRNA. Thus, our study not only confirms conserved, RNA-mediated chaperoning role of ribosome but also provides crucial insight into the mechanism of the process. PMID:27099964

  7. Mechanistic Insight into the Reactivation of BCAII Enzyme from Denatured and Molten Globule States by Eukaryotic Ribosomes and Domain V rRNAs.

    PubMed

    Chakraborty, Biprashekhar; Bhakta, Sayan; Sengupta, Jayati

    2016-01-01

    In all life forms, decoding of messenger-RNA into polypeptide chain is accomplished by the ribosome. Several protein chaperones are known to bind at the exit of ribosomal tunnel to ensure proper folding of the nascent chain by inhibiting their premature folding in the densely crowded environment of the cell. However, accumulating evidence suggests that ribosome may play a chaperone role in protein folding events in vitro. Ribosome-mediated folding of denatured proteins by prokaryotic ribosomes has been studied extensively. The RNA-assisted chaperone activity of the prokaryotic ribosome has been attributed to the domain V, a span of 23S rRNA at the intersubunit side of the large subunit encompassing the Peptidyl Transferase Centre. Evidently, this functional property of ribosome is unrelated to the nascent chain protein folding at the exit of the ribosomal tunnel. Here, we seek to scrutinize whether this unique function is conserved in a primitive kinetoplastid group of eukaryotic species Leishmania donovani where the ribosome structure possesses distinct additional features and appears markedly different compared to other higher eukaryotic ribosomes. Bovine Carbonic Anhydrase II (BCAII) enzyme was considered as the model protein. Our results manifest that domain V of the large subunit rRNA of Leishmania ribosomes preserves chaperone activity suggesting that ribosome-mediated protein folding is, indeed, a conserved phenomenon. Further, we aimed to investigate the mechanism underpinning the ribosome-assisted protein reactivation process. Interestingly, the surface plasmon resonance binding analyses exhibit that rRNA guides productive folding by directly interacting with molten globule-like states of the protein. In contrast, native protein shows no notable affinity to the rRNA. Thus, our study not only confirms conserved, RNA-mediated chaperoning role of ribosome but also provides crucial insight into the mechanism of the process.

  8. A tRNA body with high affinity for EF-Tu hastens ribosomal incorporation of unnatural amino acids.

    PubMed

    Ieong, Ka-Weng; Pavlov, Michael Y; Kwiatkowski, Marek; Ehrenberg, Måns; Forster, Anthony C

    2014-05-01

    There is evidence that tRNA bodies have evolved to reduce differences between aminoacyl-tRNAs in their affinity to EF-Tu. Here, we study the kinetics of incorporation of L-amino acids (AAs) Phe, Ala allyl-glycine (aG), methyl-serine (mS), and biotinyl-lysine (bK) using a tRNA(Ala)-based body (tRNA(AlaB)) with a high affinity for EF-Tu. Results are compared with previous data on the kinetics of incorporation of the same AAs using a tRNA(PheB) body with a comparatively low affinity for EF-Tu. All incorporations exhibited fast and slow phases, reflecting the equilibrium fraction of AA-tRNA in active ternary complex with EF-Tu:GTP before the incorporation reaction. Increasing the concentration of EF-Tu increased the amplitude of the fast phase and left its rate unaltered. This allowed estimation of the affinity of each AA-tRNA to EF-Tu:GTP during translation, showing about a 10-fold higher EF-Tu affinity for AA-tRNAs formed from the tRNA(AlaB) body than from the tRNA(PheB) body. At ∼1 µM EF-Tu, tRNA(AlaB) conferred considerably faster incorporation kinetics than tRNA(PheB), especially in the case of the bulky bK. In contrast, the swap to the tRNA(AlaB) body did not increase the fast phase fraction of N-methyl-Phe incorporation, suggesting that the slow incorporation of N-methyl-Phe had a different cause than low EF-Tu:GTP affinity. The total time for AA-tRNA release from EF-Tu:GDP, accommodation, and peptidyl transfer on the ribosome was similar for the tRNA(AlaB) and tRNA(PheB) bodies. We conclude that a tRNA body with high EF-Tu affinity can greatly improve incorporation of unnatural AAs in a potentially generalizable manner.

  9. Dissociation rate of cognate peptidyl-tRNA from the A-site of hyper-accurate and error-prone ribosomes.

    PubMed

    Karimi, R; Ehrenberg, M

    1994-12-01

    The binding stability of the aminoacyl-tRNA site (A-site), estimated from the dissociation rate constant kd, of AcPhe-Phe-tRNA(Phe) has been studied for wild-type (wt), for hyperaccurate ribosomes altered in S12 [streptomycin-dependent (SmD) and streptomycin-pseudodependent (SmP) phenotypes], for error-prone ribosomes altered in S4 (Ram phenotype), and for ribosomes in complex with the error-inducing aminoglycosides streptomycin and neomycin. The AcPhe2-tRNA stability is slightly and identically reduced for SmD and SmP phenotypes in relation to wt ribosomes. The stability is increased (kd is reduced) for Ram ribosomes to about the same extent as the proof-reading accuracy is decreased for this phenotype. kd is also reduced by the action of streptomycin and neomycin, but much less than the reduction in proof-reading accuracy induced by streptomycin. Similar kd values for SmD and SmP ribosomes indicate that the cause of streptomycin dependence is not excessive drop-off of peptidyl-tRNAs from the A-site.

  10. A genome-wide analysis of RNA pseudoknots that stimulate efficient -1 ribosomal frameshifting or readthrough in animal viruses.

    PubMed

    Huang, Xiaolan; Cheng, Qiang; Du, Zhihua

    2013-01-01

    Programmed -1 ribosomal frameshifting (PRF) and stop codon readthrough are two translational recoding mechanisms utilized by some RNA viruses to express their structural and enzymatic proteins at a defined ratio. Efficient recoding usually requires an RNA pseudoknot located several nucleotides downstream from the recoding site. To assess the strategic importance of the recoding pseudoknots, we have carried out a large scale genome-wide analysis in which we used an in-house developed program to detect all possible H-type pseudoknots within the genomic mRNAs of 81 animal viruses. Pseudoknots are detected downstream from ~85% of the recoding sites, including many previously unknown pseudoknots. ~78% of the recoding pseudoknots are the most stable pseudoknot within the viral genomes. However, they are not as strong as some designed pseudoknots that exhibit roadblocking effect on the translating ribosome. Strong roadblocking pseudoknots are not detected within the viral genomes. These results indicate that the decoding pseudoknots have evolved to possess optimal stability for efficient recoding. We also found that the sequence at the gag-pol frameshift junction of HIV1 harbors potential elaborated pseudoknots encompassing the frameshift site. A novel mechanism is proposed for possible involvement of the elaborated pseudoknots in the HIV1 PRF event.

  11. miRNA repression of translation in vitro takes place during 43S ribosomal scanning

    PubMed Central

    Ricci, Emiliano P.; Limousin, Taran; Soto-Rifo, Ricardo; Rubilar, Paulina S.; Decimo, Didier; Ohlmann, Théophile

    2013-01-01

    microRNAs (miRNAs) regulate gene expression at multiple levels by repressing translation, stimulating deadenylation and inducing the premature decay of target messenger RNAs (mRNAs). Although the mechanism by which miRNAs repress translation has been widely studied, the precise step targeted and the molecular insights of such repression are still evasive. Here, we have used our newly designed in vitro system, which allows to study miRNA effect on translation independently of deadenylation. By using specific inhibitors of various stages of protein synthesis, we first show that miRNAs target exclusively the early steps of translation with no effect on 60S ribosomal subunit joining, elongation or termination. Then, by using viral proteases and IRES-driven mRNA constructs, we found that translational inhibition takes place during 43S ribosomal scanning and requires both the poly(A) binding protein and eIF4G independently from their physical interaction. PMID:23161679

  12. The nuclear 18S ribosomal RNA gene as a source of phylogenetic information in the genus Taenia.

    PubMed

    Yan, Hongbin; Lou, Zhongzi; Li, Li; Ni, Xingwei; Guo, Aijiang; Li, Hongmin; Zheng, Yadong; Dyachenko, Viktor; Jia, Wanzhong

    2013-03-01

    Most species of the genus Taenia are of considerable medical and veterinary significance. In this study, complete nuclear 18S rRNA gene sequences were obtained from seven members of genus Taenia [Taenia multiceps, Taenia saginata, Taenia asiatica, Taenia solium, Taenia pisiformis, Taenia hydatigena, and Taenia taeniaeformis] and a phylogeny inferred using these sequences. Most of the variable sites fall within the variable regions, V1-V5. We show that sequences from the nuclear 18S ribosomal RNA gene have considerable promise as sources of phylogenetic information within the genus Taenia. Furthermore, given that almost all the variable sites lie within defined variable portions of that gene, it will be appropriate and economical to sequence only those regions for additional species of Taenia.

  13. Molecular analysis of lungworms from European bison (Bison bonasus) on the basis of small subunit ribosomal RNA gene (SSU).

    PubMed

    Pyziel, Anna M

    2014-03-01

    Dictyocaulosis (Nematoda: Trichostrongyloidea) is a widespread parasitosis of the European bison (Bison bonasus) inhabiting Bialowieza Primeval Forest. Bearing in mind the current coexistence of bison with wild cervids, and with domestic ruminants in the 19th and 20th century, the need arose for molecular identification of lungworm species. Molecular analysis was done on adult lungworms that were obtained from the respiratory track of four free-roaming bison euthanized as a part of the population health control program. As the result of the study four identical small subunit-ribosomal RNA gene sequences from the lungworms were obtained and deposited in GenBank as sequence, 1708 bp long (GenBank KC771250). Comparative analysis of the SSU rRNA sequences revealed the European bison to be a host for the bovine lungworm Dictyocaulus viviparus.

  14. Expression of ribosomal RNA genes in lines of barley with a standard karyotype and with a translocated nucleolar organizer

    SciTech Connect

    Karag'ozov, L.K.; Ananiev, E.D.; Mateeva, Z.E.; Khadzhiolov, A.A.

    1986-10-01

    The authors have investigated the rRNA synthesis and the sensitivity of rRNA genes to the action of DNAase I in developing embryos of two forms of barley. The Frigga variety has a standard karyotype and the T/sub 506/ line is characterized by translocation of the nucleolar organizer, which leads to a reduction in the number of nucleoli observed in the telophase. The results of the investigation of rRNA synthesis in vivo and of the activity of RNA polymerase I in isolated nuclei revealed the absence of differences between the two barley forms. They have established that the genes of ribosomal RNAs possess greater sensitivity to digestion by DNAase the authors compared to that of the total nuclear DNA. They conclude that the translocation of one of the nucleolar organizers causes a delay in the appearance of its activity during the telophase, this not changing the expression of the rRNA genes in the subsequent stages of cell development.

  15. A pseudouridylation switch in rRNA is implicated in ribosome function during the life cycle of Trypanosoma brucei

    PubMed Central

    Chikne, Vaibhav; Doniger, Tirza; Rajan, K. Shanmugha; Bartok, Osnat; Eliaz, Dror; Cohen-Chalamish, Smadar; Tschudi, Christian; Unger, Ron; Hashem, Yaser; Kadener, Sebastian; Michaeli, Shulamit

    2016-01-01

    The protozoan parasite Trypanosoma brucei, which causes devastating diseases in humans and animals in sub-Saharan Africa, undergoes a complex life cycle between the mammalian host and the blood-feeding tsetse fly vector. However, little is known about how the parasite performs most molecular functions in such different environments. Here, we provide evidence for the intriguing possibility that pseudouridylation of rRNA plays an important role in the capacity of the parasite to transit between the insect midgut and the mammalian bloodstream. Briefly, we mapped pseudouridines (Ψ) on rRNA by Ψ-seq in procyclic form (PCF) and bloodstream form (BSF) trypanosomes. We detected 68 Ψs on rRNA, which are guided by H/ACA small nucleolar RNAs (snoRNA). The small RNome of both life cycle stages was determined by HiSeq and 83 H/ACAs were identified. We observed an elevation of 21 Ψs modifications in BSF as a result of increased levels of the guiding snoRNAs. Overexpression of snoRNAs guiding modification on H69 provided a slight growth advantage to PCF parasites at 30 °C. Interestingly, these modifications are predicted to significantly alter the secondary structure of the large subunit (LSU) rRNA suggesting that hypermodified positions may contribute to the adaption of ribosome function during cycling between the two hosts. PMID:27142987

  16. RRP1, a Saccharomyces cerevisiae gene affecting rRNA processing and production of mature ribosomal subunits.

    PubMed Central

    Fabian, G R; Hopper, A K

    1987-01-01

    The Saccharomyces cerevisiae mutant ts351 had been shown to affect processing of 27S pre-rRNA to mature 25S and 5.8S rRNAs (C. Andrew, A. K. Hopper, and B. D. Hall, Mol. Gen. Genet. 144:29-37, 1976). We showed that this strain contains two mutations leading to temperature-sensitive lethality. The rRNA-processing defect, however, is a result of only one of the two mutations. We designated the lesion responsible for the rRNA-processing defect rrp1 and showed that it is located on the right arm of chromosome IV either allelic to or tightly linked to mak21. This rrp1 lesion also results in hypersensitivity to aminoglycoside antibiotics and a reduced 25S/18S rRNA ratio at semipermissive temperatures. We cloned the RRP1 gene and provide evidence that it encodes a moderately abundant mRNA which is in lower abundance and larger than most mRNAs encoding ribosomal proteins. Images PMID:3549696

  17. Symportin 1 chaperones 5S RNP assembly during ribosome biogenesis by occupying an essential rRNA-binding site.

    PubMed

    Calviño, Fabiola R; Kharde, Satyavati; Ori, Alessandro; Hendricks, Astrid; Wild, Klemens; Kressler, Dieter; Bange, Gert; Hurt, Ed; Beck, Martin; Sinning, Irmgard

    2015-04-07

    During 60S biogenesis, mature 5S RNP consisting of 5S RNA, RpL5 and RpL11, assembles into a pre-60S particle, where docking relies on RpL11 interacting with helix 84 (H84) of the 25S RNA. How 5S RNP is assembled for recruitment into the pre-60S is not known. Here we report the crystal structure of a ternary symportin Syo1-RpL5-N-RpL11 complex and provide biochemical and structural insights into 5S RNP assembly. Syo1 guards the 25S RNA-binding surface on RpL11 and competes with H84 for binding. Pull-down experiments show that H84 releases RpL11 from the ternary complex, but not in the presence of 5S RNA. Crosslinking mass spectrometry visualizes structural rearrangements on incorporation of 5S RNA into the Syo1-RpL5-RpL11 complex supporting the formation of a pre-5S RNP. Our data underline the dual role of Syo1 in ribosomal protein transport and as an assembly platform for 5S RNP.

  18. A 5.8S nuclear ribosomal RNA gene sequence database: applications to ecology and evolution

    NASA Technical Reports Server (NTRS)

    Cullings, K. W.; Vogler, D. R.

    1998-01-01

    We complied a 5.8S nuclear ribosomal gene sequence database for animals, plants, and fungi using both newly generated and GenBank sequences. We demonstrate the utility of this database as an internal check to determine whether the target organism and not a contaminant has been sequenced, as a diagnostic tool for ecologists and evolutionary biologists to determine the placement of asexual fungi within larger taxonomic groups, and as a tool to help identify fungi that form ectomycorrhizae.

  19. Ribosome maturation in E. coli.

    PubMed

    Silengo, L; Altruda, F; Dotto, G P; Lacquaniti, F; Perlo, C; Turco, E; Mangiarotti, G

    1977-01-01

    In vivo and in vitro experiments have shown that processing of ribosomal RNA is a late event in ribosome biogenesis. The precursor form of RNA is probably necessary to speed up the assembly of ribomal proteins. Newly formed ribosomal particles which have already entered polyribosomes differ from mature ribosomes not only in their RNA content but also in their susceptibility to unfolding in low Mg concentration and to RNase attack. Final maturation of new ribosomes is probably dependent on their functioning in protein synthesis. Thus only those ribosomes which have proven to be functional may be converted into stable cellular structures.

  20. Spb4p, an essential putative RNA helicase, is required for a late step in the assembly of 60S ribosomal subunits in Saccharomyces cerevisiae.

    PubMed Central

    de la Cruz, J; Kressler, D; Rojo, M; Tollervey, D; Linder, P

    1998-01-01

    Spb4p is a putative ATP-dependent RNA helicase that is required for synthesis of 60S ribosomal subunits. Polysome analyses of strains genetically depleted of Spb4p or carrying the cold-sensitive spb4-1 mutation revealed an underaccumulation of 60S ribosomal subunits. Analysis of pre-rRNA processing by pulse-chase labeling, northern hybridization, and primer extension indicated that these strains exhibited a reduced synthesis of the 25S/5.8S rRNAs, due to inhibition of processing of the 27SB pre-rRNAs. At later times of depletion of Spb4p or following transfer of the spb4-1 strain to more restrictive temperatures, the early pre-rRNA processing steps at sites A0, Al, and A2 were also inhibited. Sucrose gradient fractionation showed that the accumulated 27SB pre-rRNAs are associated with a high-molecular-weight complex, most likely the 66S pre-ribosomal particle. An HA epitope-tagged Spb4p is localized to the nucleolus and the adjacent nucleoplasmic area. On sucrose gradients, HA-Spb4p was found almost exclusively in rapidly sedimenting complexes and showed a peak in the fractions containing the 66S pre-ribosomes. We propose that Spb4p is involved directly in a late and essential step during assembly of 60S ribosomal subunits, presumably by acting as an rRNA helicase. PMID:9769101

  1. An RNA pseudoknot and an optimal heptameric shift site are required for highly efficient ribosomal frameshifting on a retroviral messenger RNA.

    PubMed Central

    Chamorro, M; Parkin, N; Varmus, H E

    1992-01-01

    Synthesis of the pol gene products of most retroviruses requires ribosomes to shift frame once or twice in the -1 direction while translating gag-pol mRNA. The viral signals for frameshifting include a heptanucleotide sequence on which the shift occurs and higher-order RNA structure just downstream of the shift site. We have made site-directed mutations in two stems (S1 and S2) of a putative RNA pseudoknot that begins 7 nucleotides 3' of the previously identified shift site (A AAA AAC) in the gag-pro region of mouse mammary tumor virus (MMTV) RNA. The mutants confirm the predicted structure, show that loss of either S1 or S2 impairs frameshifting, and exclude alternative RNA structures as significant for frameshifting. The importance of the MMTV pseudoknot has been further demonstrated by showing that shift sites from two other retroviruses function more efficiently in the position of the MMTV site than in their native contexts. However, the MMTV pseudoknot cannot promote detectable frameshifting in the absence of a recognizable upstream shift site. In addition, the species of tRNA that reads the second codon in the shift site appears to be a critical determinant, since changing the 7th nucleotide in the MMTV gag-pro shift site from C to A, U, or G severely impairs frameshifting. Images PMID:1309954

  2. The binding site for ribosomal protein S8 in 16S rRNA and spc mRNA from Escherichia coli: minimum structural requirements and the effects of single bulged bases on S8-RNA interaction.

    PubMed Central

    Wu, H; Jiang, L; Zimmermann, R A

    1994-01-01

    Through specific interactions with rRNA and mRNA, ribosomal protein S8 of Escherichia coli plays a central role in both assembly of the 30S ribosomal subunit and translational regulation of spc operon expression. To better understand S8-RNA association, we have measured the affinity of S8 for a number of variants of its rRNA and mRNA binding sites prepared by in vitro transcription or chemical synthesis. With the aid of site-directed deletions, we demonstrate that an imperfect, 33-nucleotide helical stem encompassing nucleotides 588-603 and 635-651 possesses all of the structural information necessary for specific binding of S8 to the 16S rRNA. This segment consists of two short duplexes that enclose a conserved, asymmetric internal loop which contains features crucial for protein recognition. The S8 binding site in spc operon mRNA is very similar in both primary and secondary structure to that in 16S rRNA except for the presence of two single bulged bases in one of the duplex segments. In addition, the apparent association constant for the S8-mRNA interaction is approximately fivefold less than that for the S8-rRNA interaction. We show that the difference in affinity can be attributed to the effects of the bulged bases. Deletion of the bulged bases from the mRNA site increases its affinity for S8 to a level similar to that of the rRNA, whereas insertion of single-base bulges at equivalent positions within the rRNA site reduces its affinity for S8 to a value typical of the mRNA. Single-base bulges in the proximity of essential recognition features are therefore capable of modulating the strength of protein-RNA interactions. PMID:7515489

  3. Dbp7p, a putative ATP-dependent RNA helicase from Saccharomyces cerevisiae, is required for 60S ribosomal subunit assembly.

    PubMed Central

    Daugeron, M C; Linder, P

    1998-01-01

    Putative ATP-dependent RNA helicases are ubiquitous, highly conserved proteins that are found in most organisms and they are implicated in all aspects of cellular RNA metabolism. Here we present the functional characterization of the Dbp7 protein, a putative ATP-dependent RNA helicase of the DEAD-box protein family from Saccharomyces cerevisiae. The complete deletion of the DBP7 ORF causes a severe slow-growth phenotype. In addition, the absence of Dbp7p results in a reduced amount of 60S ribosomal subunits and an accumulation of halfmer polysomes. Subsequent analysis of pre-rRNA processing indicates that this 60S ribosomal subunit deficit is due to a strong decrease in the production of 27S and 7S precursor rRNAs, which leads to reduced levels of the mature 25S and 5.8S rRNAs. Noticeably, the overall decrease of the 27S pre-rRNA species is neither associated with the accumulation of preceding precursors nor with the emergence of abnormal processing intermediates, suggesting that these 27S pre-rRNA species are degraded rapidly in the absence of Dbp7p. Finally, an HA epitope-tagged Dbp7 protein is localized in the nucleolus. We propose that Dbp7p is involved in the assembly of the pre-ribosomal particle during the biogenesis of the 60S ribosomal subunit. PMID:9582098

  4. Transcriptomic profiling of Ichthyophthirius multifiliis reveals polyadenylation of the large subunit ribosomal RNA

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Polyadenylation of eukaryotic transcripts is usually restricted to mRNA, whereby providing transcripts with stability from degradation by nucleases. Conversely, an RNA degradation pathway can be signaled through poly (A) tailing in prokaryotic, archeal, and organeller biology. Recently polyadenyla...

  5. The majority of total nuclear-encoded non-ribosomal RNA in a human cell is 'dark matter' un-annotated RNA

    PubMed Central

    2010-01-01

    Background Discovery that the transcriptional output of the human genome is far more complex than predicted by the current set of protein-coding annotations and that most RNAs produced do not appear to encode proteins has transformed our understanding of genome complexity and suggests new paradigms of genome regulation. However, the fraction of all cellular RNA whose function we do not understand and the fraction of the genome that is utilized to produce that RNA remain controversial. This is not simply a bookkeeping issue because the degree to which this un-annotated transcription is present has important implications with respect to its biologic function and to the general architecture of genome regulation. For example, efforts to elucidate how non-coding RNAs (ncRNAs) regulate genome function will be compromised if that class of RNAs is dismissed as simply 'transcriptional noise'. Results We show that the relative mass of RNA whose function and/or structure we do not understand (the so called 'dark matter' RNAs), as a proportion of all non-ribosomal, non-mitochondrial human RNA (mt-RNA), can be greater than that of protein-encoding transcripts. This observation is obscured in studies that focus only on polyA-selected RNA, a method that enriches for protein coding RNAs and at the same time discards the vast majority of RNA prior to analysis. We further show the presence of a large number of very long, abundantly-transcribed regions (100's of kb) in intergenic space and further show that expression of these regions is associated with neoplastic transformation. These overlap some regions found previously in normal human embryonic tissues and raises an interesting hypothesis as to the function of these ncRNAs in both early development and neoplastic transformation. Conclusions We conclude that 'dark matter' RNA can constitute the majority of non-ribosomal, non-mitochondrial-RNA and a significant fraction arises from numerous very long, intergenic transcribed regions

  6. An automated procedure for covariation-based detection of RNA structure

    SciTech Connect

    Winker, S.; Overbeek, R.; Woese, C.R.; Olsen, G.J.; Pfluger, N.

    1989-12-01

    This paper summarizes our investigations into the computational detection of secondary and tertiary structure of ribosomal RNA. We have developed a new automated procedure that not only identifies potential bondings of secondary and tertiary structure, but also provides the covariation evidence that supports the proposed bondings, and any counter-evidence that can be detected in the known sequences. A small number of previously unknown bondings have been detected in individual RNA molecules (16S rRNA and 7S RNA) through the use of our automated procedure. Currently, we are systematically studying mitochondrial rRNA. Our goal is to detect tertiary structure within 16S rRNA and quaternary structure between 16S and 23S rRNA. Our ultimate hope is that automated covariation analysis will contribute significantly to a refined picture of ribosome structure. Our colleagues in biology have begun experiments to test certain hypotheses suggested by an examination of our program's output. These experiments involve sequencing key portions of the 23S ribosomal RNA for species in which the known 16S ribosomal RNA exhibits variation (from the dominant pattern) at the site of a proposed bonding. The hope is that the 23S ribosomal RNA of these species will exhibit corresponding complementary variation or generalized covariation. 24 refs.

  7. Insects' RNA Profiling Reveals Absence of "Hidden Break" in 28S Ribosomal RNA Molecule of Onion Thrips, Thrips tabaci.

    PubMed

    Macharia, Rosaline Wanjiru; Ombura, Fidelis Levi; Aroko, Erick Onyango

    2015-01-01

    With an exception of aphids, insects' 28S rRNA is thought to harbor a "hidden break" which cleaves under denaturing conditions to comigrate with 18S rRNA band to exhibit a degraded appearance on native agarose gels. The degraded appearance confounds determination of RNA integrity in laboratories that rely on gel electrophoresis. To provide guidelines for RNA profiles, RNA from five major insect orders, namely, Diptera, Hemiptera, Thysanoptera, Hymenoptera, and Lepidoptera, was compared under denaturing and nondenaturing conditions. This study confirmed that although present in most of insect's RNA, the "hidden break" is absent in the 28S rRNA of onion thrips, Thrips tabaci. On the other hand, presence of "hidden break" was depicted in whiteflies' 28S rRNA despite their evolutionary grouping under same order with aphids. Divergence of 28S rRNA sequences confirms variation of both size and composition of gap region among insect species. However, phylogeny reconstruction does not support speciation as a possible source of the hidden break in insect's 28S rRNA. In conclusion, we show that RNA from a given insect order does not conform to a particular banding profile and therefore this approach cannot be reliably used to characterize newly discovered species.

  8. p73 expression is regulated by ribosomal protein RPL26 through mRNA translation and protein stability

    PubMed Central

    Yan, Wensheng; Chen, Xinbin

    2016-01-01

    p73, a p53 family tumor suppressor, is regulated by multiple mechanisms, including transcription and mRNA and protein stability. However, whether p73 expression is regulated via mRNA translation has not been explored. To test this, we examined whether ribosomal protein 26 (RPL26) plays a role in p73 expression. Here, we showed that p73 expression is controlled by RPL26 via protein stability and mRNA translation. To examine whether MDM2 mediates RPL26 to regulate p73 protein stability, we generated multiple MDM2-knockout cell lines by CRISPR-cas9. We found that in the absence of MDM2, the half-life of p73 protein is markedly increased. Interestingly, we also found that RPL26 is still capable of regulating p73 expression, albeit to a lesser extent, in MDM2-KO cells compared to that in isogenic control cells, suggesting that RPL26 regulates p73 expression via multiple mechanisms. Indeed, we found that RPL26 is necessary for efficient assembly of polysomes on p73 mRNA and de novo synthesis of p73 protein. Consistently, we found that RPL26 directly binds to p73 3′ untranslated region (3′UTR) and that RPL26 is necessary for efficient expression of an eGFP reporter that carries p73 3′UTR. We also found that RPL26 interacts with cap-binding protein eIF4E and enhances the association of eIF4E with p73 mRNA, leading to increased p73 mRNA translation. Finally, we showed that knockdown of RPL26 promotes, whereas ectopic expression of RPL26 inhibits, cell growth in a TAp73-dependent manner. Together, our data indicate that RPL26 regulates p73 expression via two distinct mechanisms: protein stability and mRNA translation. PMID:27825141

  9. Design and properties of efficient tRNA:EF-Tu FRET system for studies of ribosomal translation.

    PubMed

    Chudaev, Maxim; Poruri, Kiran; Goldman, Emanuel; Jakubowski, Hieronim; Jain, Mohit Raja; Chen, Wei; Li, Hong; Tyagi, Sanjay; Mandecki, Wlodek

    2013-05-01

    Formation of the ternary complex between GTP-bound form of elongation factor Tu (EF-Tu) and aminoacylated transfer RNA (aa-tRNA) is a key event in protein biosynthesis. Here we show that fluorescently modified Escherichia coli EF-Tu carrying three mutations, C137A, C255V and E348C, and fluorescently modified Phe-tRNA(Phe) form functionally active ternary complex that has properties similar to those of the naturally occurring (unmodified) complex. Similarities include the binding and binding rate constants, behavior in gel retardation assay, as well as activities in tRNA protection and in vitro translation assays. Proper labeling of EF-Tu was demonstrated in MALDI mass spectroscopy experiments. To generate the mutant EF-Tu, a series of genetic constructions were performed. Two native cysteine residues in the wild-type EF-Tu at positions 137 and 255 were replaced by Ala and Val, respectively, and an additional cysteine was introduced either in position 324 or 348. The assembly FRET assay showed a 5- to 7-fold increase of Cy5-labeled EF-Tu E348C mutant fluorescence upon formation of ternary complex with charged tRNA(Phe)(Cy3-labeled) when the complex was excited at 532 nm and monitored at 665 nm. In a control experiment, we did not observe FRET using uncharged tRNA(Phe)(Cy3), nor with wild-type EF-Tu preparation that was allowed to react with Cy5 maleimide, nor in the absence of GTP. The results obtained demonstrate that the EF-Tu:tRNA FRET system described can be used for investigations of ribosomal translation in many types of experiments.

  10. LKB1 promotes cell survival by modulating TIF-IA-mediated pre-ribosomal RNA synthesis under uridine downregulated conditions

    PubMed Central

    Liu, Xiuju; Huang, Henry; Wilkinson, Scott C.; Zhong, Diansheng; Khuri, Fadlo R.; Fu, Haian; Marcus, Adam; He, Yulong; Zhou, Wei

    2016-01-01

    We analyzed the mechanism underlying 5-aminoimidazole-4-carboxamide riboside (AICAR) mediated apoptosis in LKB1-null non-small cell lung cancer (NSCLC) cells. Metabolic profile analysis revealed depletion of the intracellular pyrimidine pool after AICAR treatment, but uridine was the only nucleotide precursor capable of rescuing this apoptosis, suggesting the involvement of RNA metabolism. Because half of RNA transcription in cancer is for pre-ribosomal RNA (rRNA) synthesis, which is suppressed by over 90% after AICAR treatment, we evaluated the role of TIF-IA-mediated rRNA synthesis. While the depletion of TIF-IA by RNAi alone promoted apoptosis in LKB1-null cells, the overexpression of a wild-type or a S636A TIF-IA mutant, but not a S636D mutant, attenuated AICAR-induced apoptosis. In LKB1-null H157 cells, pre-rRNA synthesis was not suppressed by AICAR when wild-type LKB1 was present, and cellular fractionation analysis indicated that TIF-IA quickly accumulated in the nucleus in the presence of a wild-type LKB1 but not a kinase-dead mutant. Furthermore, ectopic expression of LKB1 was capable of attenuating AICAR-induced death in AMPK-null cells. Because LKB1 promotes cell survival by modulating TIF-IA-mediated pre-rRNA synthesis, this discovery suggested that targeted depletion of uridine related metabolites may be exploited in the clinic to eliminate LKB1-null cancer cells. PMID:26506235

  11. T7 Early RNAs and Escherichia coli Ribosomal RNAs are Cut from Large Precursor RNAs In Vivo by Ribonuclease III

    PubMed Central

    Dunn, John J.; Studier, F. William

    1973-01-01

    The early region of T7 DNA is transcribed as a single unit in a Ribonuclease III-deficient E. coli strain to produce large molecules essentially identical to those produced in vitro by E. coli RNA polymerase. As with the in vitro RNAs, these molecules are cut by purified RNase III in vitro to produce the messenger RNAs normally observed in vivo. Thus, the normal pathway for producing the T7 early messenger RNAs in vivo appears to involve endonucleolytic cleavage by RNase III. The uninfected RNase III-deficient strain contains several RNAs not observed in the parent strain. Patterns of labeling in vivo suggest that the largest of these RNAs, about 1.8 × 106 daltons, may be a precursor to the 16S and 23S ribosomal RNAs. When this large molecule is treated in vitro with purified RNase III, molecules the size of precursor 16S and 23S ribosomal RNAs are released; hybridization competition experiments also indicate that the 1.8 × 106 dalton RNA does indeed represent ribosomal RNA. Thus, RNase III cleavage seems to be part of the normal pathway for producing at least the 16S and 23S ribosomal RNAs in vivo. Several smaller molecules are also released from the 1.8 × 106 dalton RNA by RNase III, but it is not yet established whether any of these contain 5S RNA sequences. Images PMID:4587248

  12. Engineered ribosomal RNA operon copy-number variants of E. coli reveal the evolutionary trade-offs shaping rRNA operon number.

    PubMed

    Gyorfy, Zsuzsanna; Draskovits, Gabor; Vernyik, Viktor; Blattner, Frederick F; Gaal, Tamas; Posfai, Gyorgy

    2015-02-18

    Ribosomal RNA (rrn) operons, characteristically present in several copies in bacterial genomes (7 in E. coli), play a central role in cellular physiology. We investigated the factors determining the optimal number of rrn operons in E. coli by constructing isogenic variants with 5-10 operons. We found that the total RNA and protein content, as well as the size of the cells reflected the number of rrn operons. While growth parameters showed only minor differences, competition experiments revealed a clear pattern: 7-8 copies were optimal under conditions of fluctuating, occasionally rich nutrient influx and lower numbers were favored in stable, nutrient-limited environments. We found that the advantages of quick adjustment to nutrient availability, rapid growth and economic regulation of ribosome number all contribute to the selection of the optimal rrn operon number. Our results suggest that the wt rrn operon number of E. coli reflects the natural, 'feast and famine' life-style of the bacterium, however, different copy numbers might be beneficial under different environmental conditions. Understanding the impact of the copy number of rrn operons on the fitness of the cell is an important step towards the creation of functional and robust genomes, the ultimate goal of synthetic biology.

  13. Rqc2p and 60S ribosomal subunits mediate mRNA-independent elongation of nascent chains

    PubMed Central

    Shen, Peter S.; Park, Joseph; Qin, Yidan; Li, Xueming; Parsawar, Krishna; Larson, Matthew H.; Cox, James; Cheng, Yifan; Lambowitz, Alan M.; Weissman, Jonathan S.; Brandman, Onn; Frost, Adam

    2015-01-01

    In Eukarya, stalled translation induces 40S dissociation and recruitment of the Ribosome Quality control Complex (RQC) to the 60S subunit, which mediates nascent chain degradation. Here, we report cryoEM structures revealing that the RQC components Rqc2p (YPL009C/Tae2) and Ltn1p (YMR247C/Rkr1) bind to the 60S at sites exposed after 40S dissociation, placing the Ltn1p RING domain near the exit channel and Rqc2p over the P-site tRNA. We further demonstrate that Rqc2p recruits alanine and threonine charged tRNA to the A-site and directs elongation of nascent chains independently of mRNA or 40S subunits. Our work uncovers an unexpected mechanism of protein synthesis in which a protein—not an mRNA—determines tRNA recruitment and the tagging of nascent chains with Carboxy-terminal Ala and Thr extensions (“CAT tails”). PMID:25554787

  14. Distinct tRNA Accommodation Intermediates Observed on the Ribosome with the Antibiotics Hygromycin A and A201A.

    PubMed

    Polikanov, Yury S; Starosta, Agata L; Juette, Manuel F; Altman, Roger B; Terry, Daniel S; Lu, Wanli; Burnett, Benjamin J; Dinos, George; Reynolds, Kevin A; Blanchard, Scott C; Steitz, Thomas A; Wilson, Daniel N

    2015-06-04

    The increase in multi-drug-resistant bacteria is limiting the effectiveness of currently approved antibiotics, leading to a renewed interest in antibiotics with distinct chemical scaffolds. We have solved the structures of the Thermus thermophilus 70S ribosome with A-, P-, and E-site tRNAs bound and in complex with either the aminocyclitol-containing antibiotic hygromycin A (HygA) or the nucleoside antibiotic A201A. Both antibiotics bind at the peptidyl transferase center and sterically occlude the CCA-end of the A-tRNA from entering the A site of the peptidyl transferase center. Single-molecule Förster resonance energy transfer (smFRET) experiments reveal that HygA and A201A specifically interfere with full accommodation of the A-tRNA, leading to the presence of tRNA accommodation intermediates and thereby inhibiting peptide bond formation. Thus, our results provide not only insight into the mechanism of action of HygA and A201A, but also into the fundamental process of tRNA accommodation during protein synthesis.

  15. Accuracy of initial codon selection by aminoacyl-tRNAs on the mRNA-programmed bacterial ribosome.

    PubMed

    Zhang, Jingji; Ieong, Ka-Weng; Johansson, Magnus; Ehrenberg, Måns

    2015-08-04

    We used a cell-free system with pure Escherichia coli components to study initial codon selection of aminoacyl-tRNAs in ternary complex with elongation factor Tu and GTP on messenger RNA-programmed ribosomes. We took advantage of the universal rate-accuracy trade-off for all enzymatic selections to determine how the efficiency of initial codon readings decreased linearly toward zero as the accuracy of discrimination against near-cognate and wobble codon readings increased toward the maximal asymptote, the d value. We report data on the rate-accuracy variation for 7 cognate, 7 wobble, and 56 near-cognate codon readings comprising about 15% of the genetic code. Their d values varied about 400-fold in the 200-80,000 range depending on type of mismatch, mismatch position in the codon, and tRNA isoacceptor type. We identified error hot spots (d = 200) for U:G misreading in second and U:U or G:A misreading in third codon position by His-tRNA(His) and, as also seen in vivo, Glu-tRNA(Glu). We suggest that the proofreading mechanism has evolved to attenuate error hot spots in initial selection such as those found here.

  16. Coupled transcription and processing of mouse ribosomal RNA in a cell-free system.

    PubMed Central

    Mishima, Y; Mitsuma, T; Ogata, K

    1985-01-01

    An in vitro processing system of mouse rRNA was achieved using an RNA polymerase I-specific transcription system, (S100) and recombinant plasmids consisting of mouse rRNA gene (rDNA) segments containing the transcription initiation and 5'-terminal region of 18S (or 41S) rRNA. Pulse-chase experiments showed that a specific processing occurred with transcripts of the plasmid DNAs when the direction of transcription was the correct orientation relative to the 18S rRNA coding sequence, but not with transcripts of the DNA templates in which this coding sequence was in the opposite orientation. From the S1 nuclease protection analyses, we concluded that there are several steps of endonucleolytic cleavage including one 105 nucleotides upstream from the 5' end of 18S rRNA. Intermediates cleaved at this site were identified in in vivo processing of rRNA. This result indicates that endonucleolytic cleavage takes place 105 nucleotides upstream from the 5' terminus of 18S rRNA prior to the formation of mature 18S rRNA. Trimming or cleavage of the 105 nucleotides may be involved in the formation of the 5' terminus of mature 18S rRNA. Images Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. PMID:3004977

  17. Paralog-Specific Functions of RPL7A and RPL7B Mediated by Ribosomal Protein or snoRNA Dosage in Saccharomyces cerevisiae

    PubMed Central

    Palumbo, Ryan J.; Fuchs, Gabriele; Lutz, Sheila; Curcio, M. Joan

    2016-01-01

    Most ribosomal proteins in Saccharomyces cerevisiae are encoded by two paralogs that additively produce the optimal protein level for cell growth. Nonetheless, deleting one paralog of most ribosomal protein gene pairs results in a variety of phenotypes not observed when the other paralog is deleted. To determine whether paralog-specific phenotypes associated with deleting RPL7A or RPL7B stem from distinct functions or different levels of the encoded isoforms, the coding region and introns of one paralog, including an intron-embedded snoRNA (small nucleolar RNA) gene, were exchanged with that of the other paralog. Among mutants harboring a single native or chimeric RPL7 allele, expression from the RPL7A locus exceeded that from the RPL7B locus, and more Rpl7a was expressed from either locus than Rpl7b. Phenotypic differences in tunicamycin sensitivity, ASH1 mRNA localization, and mobility of the Ty1 retrotransposon were strongly correlated with Rpl7 and ribosome levels, but not with the Rpl7 or snoRNA isoform expressed. Although Ty1 RNA is cotranslationally localized, depletion of Rpl7 minimally affected synthesis of Ty1 Gag protein, but strongly influenced Ty1 RNA localization. Unlike the other processes studied, Ty1 cDNA accumulation was influenced by both the level and isoform of Rpl7 or snoRNA expressed. These cellular processes had different minimal threshold values for Rpl7 and ribosome levels, but all were functional when isoforms of either paralog were expressed from the RPL7A locus or both RPL7 loci. This study illustrates the broad range of phenotypes that can result from depleting ribosomes to different levels. PMID:28007835

  18. Structural insights into ribosome translocation

    PubMed Central

    Ling, Clarence

    2016-01-01

    During protein synthesis, tRNA and mRNA are translocated from the A to P to E sites of the ribosome thus enabling the ribosome to translate one codon of mRNA after the other. Ribosome translocation along mRNA is induced by the universally conserved ribosome GTPase, elongation factor G (EF‐G) in bacteria and elongation factor 2 (EF‐2) in eukaryotes. Recent structural and single‐molecule studies revealed that tRNA and mRNA translocation within the ribosome is accompanied by cyclic forward and reverse rotations between the large and small ribosomal subunits parallel to the plane of the intersubunit interface. In addition, during ribosome translocation, the ‘head’ domain of small ribosomal subunit undergoes forward‐ and back‐swiveling motions relative to the rest of the small ribosomal subunit around the axis that is orthogonal to the axis of intersubunit rotation. tRNA/mRNA translocation is also coupled to the docking of domain IV of EF‐G into the A site of the small ribosomal subunit that converts the thermally driven motions of the ribosome and tRNA into the forward translocation of tRNA/mRNA inside the ribosome. Despite recent and enormous progress made in the understanding of the molecular mechanism of ribosome translocation, the sequence of structural rearrangements of the ribosome, EF‐G and tRNA during translocation is still not fully established and awaits further investigation. WIREs RNA 2016, 7:620–636. doi: 10.1002/wrna.1354 For further resources related to this article, please visit the WIREs website. PMID:27117863

  19. RNA polymerase beta subunit (rpoB) gene and the 16S-23S rRNA intergenic transcribed spacer region (ITS) as complementary molecular markers in addition to the 16S rRNA gene for phylogenetic analysis and identification of the species of the family Mycoplasmataceae.

    PubMed

    Volokhov, Dmitriy V; Simonyan, Vahan; Davidson, Maureen K; Chizhikov, Vladimir E

    2012-01-01

    Conventional classification of the species in the family Mycoplasmataceae is mainly based on phenotypic criteria, which are complicated, can be difficult to measure, and have the potential to be hampered by phenotypic deviations among the isolates. The number of biochemical reactions suitable for phenotypic characterization of the Mycoplasmataceae is also very limited and therefore the strategy for the final identification of the Mycoplasmataceae species is based on comparative serological results. However, serological testing of the Mycoplasmataceae species requires a performance panel of hyperimmune sera which contains anti-serum to each known species of the family, a high level of technical expertise, and can only be properly performed by mycoplasma-reference laboratories. In addition, the existence of uncultivated and fastidious Mycoplasmataceae species/isolates in clinical materials significantly complicates, or even makes impossible, the application of conventional bacteriological tests. The analysis of available genetic markers is an additional approach for the primary identification and phylogenetic classification of cultivable species and uncultivable or fastidious organisms in standard microbiological laboratories. The partial nucleotide sequences of the RNA polymerase β-subunit gene (rpoB) and the 16S-23S rRNA intergenic transcribed spacer (ITS) were determined for all known type strains and the available non-type strains of the Mycoplasmataceae species. In addition to the available 16S rRNA gene data, the ITS and rpoB sequences were used to infer phylogenetic relationships among these species and to enable identification of the Mycoplasmataceae isolates to the species level. The comparison of the ITS and rpoB phylogenetic trees with the 16S rRNA reference phylogenetic tree revealed a similar clustering patterns for the Mycoplasmataceae species, with minor discrepancies for a few species that demonstrated higher divergence of their ITS and rpoB in

  20. A new fungal large subunit ribosomal RNA primer for high throughput sequencing surveys

    DOE PAGES

    Mueller, Rebecca C.; Gallegos-Graves, La Verne; Kuske, Cheryl R.

    2015-12-09

    The inclusion of phylogenetic metrics in community ecology has provided insights into important ecological processes, particularly when combined with high-throughput sequencing methods; however, these approaches have not been widely used in studies of fungal communities relative to other microbial groups. Two obstacles have been considered: (1) the internal transcribed spacer (ITS) region has limited utility for constructing phylogenies and (2) most PCR primers that target the large subunit (LSU) ribosomal unit generate amplicons that exceed current limits of high-throughput sequencing platforms. We designed and tested a PCR primer (LR22R) to target approximately 300–400 bp region of the D2 hypervariable regionmore » of the fungal LSU for use with the Illumina MiSeq platform. Both in silico and empirical analyses showed that the LR22R–LR3 pair captured a broad range of fungal taxonomic groups with a small fraction of non-fungal groups. Phylogenetic placement of publically available LSU D2 sequences showed broad agreement with taxonomic classification. Comparisons of the LSU D2 and the ITS2 ribosomal regions from environmental samples and known communities showed similar discriminatory abilities of the two primer sets. Altogether, these findings show that the LR22R–LR3 primer pair has utility for phylogenetic analyses of fungal communities using high-throughput sequencing methods.« less

  1. A new fungal large subunit ribosomal RNA primer for high throughput sequencing surveys

    SciTech Connect

    Mueller, Rebecca C.; Gallegos-Graves, La Verne; Kuske, Cheryl R.

    2015-12-09

    The inclusion of phylogenetic metrics in community ecology has provided insights into important ecological processes, particularly when combined with high-throughput sequencing methods; however, these approaches have not been widely used in studies of fungal communities relative to other microbial groups. Two obstacles have been considered: (1) the internal transcribed spacer (ITS) region has limited utility for constructing phylogenies and (2) most PCR primers that target the large subunit (LSU) ribosomal unit generate amplicons that exceed current limits of high-throughput sequencing platforms. We designed and tested a PCR primer (LR22R) to target approximately 300–400 bp region of the D2 hypervariable region of the fungal LSU for use with the Illumina MiSeq platform. Both in silico and empirical analyses showed that the LR22R–LR3 pair captured a broad range of fungal taxonomic groups with a small fraction of non-fungal groups. Phylogenetic placement of publically available LSU D2 sequences showed broad agreement with taxonomic classification. Comparisons of the LSU D2 and the ITS2 ribosomal regions from environmental samples and known communities showed similar discriminatory abilities of the two primer sets. Altogether, these findings show that the LR22R–LR3 primer pair has utility for phylogenetic analyses of fungal communities using high-throughput sequencing methods.

  2. Intra-Genomic Ribosomal RNA Polymorphism and Morphological Variation in Elphidium macellum Suggests Inter-Specific Hybridization in Foraminifera

    PubMed Central

    Pillet, Loïc; Fontaine, Delia; Pawlowski, Jan

    2012-01-01

    Elphidium macellum is a benthic foraminifer commonly found in the Patagonian fjords. To test whether its highly variable morphotypes are ecophenotypes or different genotypes, we analysed 70 sequences of the SSU rRNA gene from 25 specimens. Unexpectedly, we identified 11 distinct ribotypes, with up to 5 ribotypes co-occurring within the same specimen. The ribotypes differ by varying blocks of sequence located at the end of stem-loop motifs in the three expansion segments specific to foraminifera. These changes, distinct from typical SNPs and indels, directly affect the structure of the expansion segments. Their mosaic distribution suggests that ribotypes originated by recombination of two or more clusters of ribosomal genes. We propose that this expansion segment polymorphism (ESP) could originate from hybridization of morphologically different populations of Patagonian Elphidium. We speculate that the complex geological history of Patagonia enhanced divergence of coastal foraminiferal species and contributed to increasing genetic and morphological variation. PMID:22393402

  3. Salmonella detection using 16S ribosomal DNA/RNA probe-gold nanoparticles and lateral flow immunoassay.

    PubMed

    Liu, Cheng-Che; Yeung, Chun-Yan; Chen, Po-Hao; Yeh, Ming-Kung; Hou, Shao-Yi

    2013-12-01

    An ultrasensitive, simple, and fast lateral flow immunoassay for Salmonella detection using gold nanoparticles conjugated with a DNA probe, which is complementary to the 16S ribosomal RNA and DNA of Salmonella, has been developed. The detection limit is 5 fmol for the synthetic single-stranded DNA. For the Salmonella cultured samples, the nucleic acids from 10(7) bacteria were rapidly detected in 30 min. After silver enhancement, the detection limit was as low as 10(4) cells which is lower than 10(5) bacteria cells, the human infective dose of food-borne Salmonella. Furthermore, the probes used in this study are specific to Salmonella compared to several other Enterobacteriaceae. This approach would be a useful tool for microbial detection regarding food safety or clinical diagnosis. It is also suitable for large-scale screening in developing countries because it is low-cost, sensitive, specific and convenient.

  4. Stb3 binds to ribosomal RNA processing element motifs that control transcriptional responses to growth in Saccharomyces cerevisiae.

    PubMed

    Liko, Dritan; Slattery, Matthew G; Heideman, Warren

    2007-09-07

    Transfer of quiescent Saccharomyces cerevisiae cells to fresh medium rapidly induces hundreds of genes needed for growth. A large subset of these genes is regulated via a DNA sequence motif known as the ribosomal RNA processing element (RRPE). However, no RRPE-binding proteins have been identified. We screened a panel of 6144 glutathione S-transferase-open reading frame fusions for RRPE-binding proteins and identified Stb3 as a specific RRPE-binding protein, both in vitro and in vivo. Chromatin immunoprecipitation experiments showed that glucose increases Stb3 binding to RRPE-containing promoters. Microarray experiments demonstrated that the loss of Stb3 inhibits the transcriptional response to fresh glucose, especially for genes with RRPE motifs. However, these experiments also showed that not all genes containing RRPEs were dependent on Stb3 for expression. Overall our data support a model in which Stb3 plays an important but not exclusive role in the transcriptional response to growth conditions.

  5. Comparison of the small 16S to 23S intergenic spacer region (ISR) of the rRNA operons of some Escherichia coli strains of the ECOR collection and E. coli K-12.

    PubMed

    García-Martínez, J; Martínez-Murcia, A; Antón, A I; Rodríguez-Valera, F

    1996-11-01

    Several 16S to 23S spacers of 354 bp have been sequenced from six Escherichia coli strains belonging to the ECOR collection. Four phylogenetically informative variable sites were identified. The results of their comparison confirm the existence of two major phylogenetic branches in this species, as previously reported. Remarkable intercistronic heterogeneity was found in strain ECOR35 and its closest relatives, in which at least one of the operons has suffered a major mutagenic event or has an independent phylogenetic origin.

  6. Secondary structure and molecular evolution of the mitochondrial small subunit ribosomal RNA in Agaricales (Euagarics clade, Homobasidiomycota).

    PubMed

    Barroso, Gérard; Sirand-Pugnet, Pascal; Mouhamadou, Bello; Labarère, Jacques

    2003-10-01

    The complete sequences and secondary structures of the mitochondrial small subunit (SSU) ribosomal RNAs of both mostly cultivated mushrooms Agaricus bisporus (1930 nt) and Lentinula edodes (2164 nt) were achieved. These secondary structures and that of Schizophyllum commune (1872 nt) were compared to that previously established for Agrocybe aegerita. The four structures are near the model established for Archae, Bacteria, plastids, and mitochondria; particularly the helices 23 and 37, described as specific to bacteria, are present. Within the four Agaricales (Homobasidiomycota), the SSU-rRNA "core" is conserved in size (966 to 1009 nt) with the exception of an unusual extension of 40 nt in the H17 helix of S. commune. The four core sequences possess 76% of conserved positions and a cluster of C in their 3' end, which could constitute a signal involved in the RNA maturation process. Among the nine putative variable domains, three (V3, V5, V7) do not show significant length variations and possess similar percentages of conserved positions (69%) than the core. The other six variable domains show important length variations, due to independent large size inserted/deleted sequences, and higher rates of nucleotide substitutions than the core (only 31% of conserved positions between the four species). Interestingly, the inserted/deleted sequences are located in few preferential sites (hot spots for insertion/deletion) where they seem to arise or disappear haphazardly during evolution. These sites are located on the surface of the tertiary structure of the 30S ribosomal subunit, at the beginning of hairpin loops; the insertions lead to a lengthening of existing hairpins or to branching loops bearing up to five additional helices.

  7. Rapid identification of Helicoverpa armigera and Helicoverpa zea (Lepidoptera: Noctuidae) using ribosomal RNA internal transcribed spacer 1.

    PubMed

    Perera, Omaththage P; Allen, Kerry C; Jain, Devendra; Purcell, Matthew; Little, Nathan S; Luttrell, Randall G

    2015-01-01

    Rapid identification of invasive species is crucial for deploying management strategies to prevent establishment. Recent Helicoverpa armigera (Hübner) invasions and subsequent establishment in South America has increased the risk of this species invading North America. Morphological similarities make differentiation of H. armigera from the native Helicoverpa zea (Boddie) difficult. Characteristics of adult male genitalia and nucleotide sequence differences in mitochondrial DNA are two of the currently available methods to differentiate these two species. However, current methods are likely too slow to be employed as rapid detection methods. In this study, conserved differences in the internal transcribed spacer 1 (ITS1) of the ribosomal RNA genes were used to develop species-specific oligonucleotide primers that amplified ITS1 fragments of 147 and 334 bp from H. armigera and H. zea, respectively. An amplicon (83 bp) from a conserved region of 18S ribosomal RNA subunit served as a positive control. Melting temperature differences in ITS1 amplicons yielded species-specific dissociation curves that could be used in high resolution melt analysis to differentiate the two Helicoverpa species. In addition, a rapid and inexpensive procedure for obtaining amplifiable genomic DNA from a small amount of tissue was identified. Under optimal conditions, the process was able to detect DNA from one H. armigera leg in a pool of 25 legs. The high resolution melt analysis combined with rapid DNA extraction could be used as an inexpensive method to genetically differentiate large numbers of H. armigera and H. zea using readily available reagents.

  8. Import of desired nucleic acid sequences using addressing motif of mitochondrial ribosomal 5S-rRNA for fluorescent in vivo hybridization of mitochondrial DNA and RNA.

    PubMed

    Zelenka, Jaroslav; Alán, Lukáš; Jabůrek, Martin; Ježek, Petr

    2014-04-01

    Based on the matrix-addressing sequence of mitochondrial ribosomal 5S-rRNA (termed MAM), which is naturally imported into mitochondria, we have constructed an import system for in vivo targeting of mitochondrial DNA (mtDNA) or mt-mRNA, in order to provide fluorescence hybridization of the desired sequences. Thus DNA oligonucleotides were constructed, containing the 5'-flanked T7 RNA polymerase promoter. After in vitro transcription and fluorescent labeling with Alexa Fluor(®) 488 or 647 dye, we obtained the fluorescent "L-ND5 probe" containing MAM and exemplar cargo, i.e., annealing sequence to a short portion of ND5 mRNA and to the light-strand mtDNA complementary to the heavy strand nd5 mt gene (5'-end 21 base pair sequence). For mitochondrial in vivo fluorescent hybridization, HepG2 cells were treated with dequalinium micelles, containing the fluorescent probes, bringing the probes proximally to the mitochondrial outer membrane and to the natural import system. A verification of import into the mitochondrial matrix of cultured HepG2 cells was provided by confocal microscopy colocalizations. Transfections using lipofectamine or probes without 5S-rRNA addressing MAM sequence or with MAM only were ineffective. Alternatively, the same DNA oligonucleotides with 5'-CACC overhang (substituting T7 promoter) were transcribed from the tetracycline-inducible pENTRH1/TO vector in human embryonic kidney T-REx®-293 cells, while mitochondrial matrix localization after import of the resulting unlabeled RNA was detected by PCR. The MAM-containing probe was then enriched by three-order of magnitude over the natural ND5 mRNA in the mitochondrial matrix. In conclusion, we present a proof-of-principle for mitochondrial in vivo hybridization and mitochondrial nucleic acid import.

  9. Ribosomes in a stacked array: elucidation of the step in translation elongation at which they are stalled during S-adenosyl-L-methionine-induced translation arrest of CGS1 mRNA.

    PubMed

    Yamashita, Yui; Kadokura, Yoshitomo; Sotta, Naoyuki; Fujiwara, Toru; Takigawa, Ichigaku; Satake, Akiko; Onouchi, Hitoshi; Naito, Satoshi

    2014-05-02

    Expression of CGS1, which codes for an enzyme of methionine biosynthesis, is feedback-regulated by mRNA degradation in response to S-adenosyl-L-methionine (AdoMet). In vitro studies revealed that AdoMet induces translation arrest at Ser-94, upon which several ribosomes stack behind the arrested one, and mRNA degradation occurs at multiple sites that presumably correspond to individual ribosomes in a stacked array. Despite the significant contribution of stacked ribosomes to inducing mRNA degradation, little is known about the ribosomes in the stacked array. Here, we assigned the peptidyl-tRNA species of the stacked second and third ribosomes to their respective codons and showed that they are arranged at nine-codon intervals behind the Ser-94 codon, indicating tight stacking. Puromycin reacts with peptidyl-tRNA in the P-site, releasing the nascent peptide as peptidyl-puromycin. This reaction is used to monitor the activity of the peptidyltransferase center (PTC) in arrested ribosomes. Puromycin reaction of peptidyl-tRNA on the AdoMet-arrested ribosome, which is stalled at the pre-translocation step, was slow. This limited reactivity can be attributed to the peptidyl-tRNA occupying the A-site at this step rather than to suppression of PTC activity. In contrast, puromycin reactions of peptidyl-tRNA with the stacked second and third ribosomes were slow but were not as slow as pre-translocation step ribosomes. We propose that the anticodon end of peptidyl-tRNA resides in the A-site of the stacked ribosomes and that the stacked ribosomes are stalled at an early step of translocation, possibly at the P/E hybrid state.

  10. Early diagnosis of Exophiala CAPD peritonitis by 18S ribosomal RNA gene sequencing and its clinical significance.

    PubMed

    Lau, Susanna K P; Woo, Patrick C Y; Chiu, Siu-kau; Leung, Kit-wah; Yung, Raymond W H; Yuen, Kwok-yung

    2003-06-01

    Phenotypic identification of fungi in clinical microbiology laboratories is often difficult and late, especially for slow growing and rarely encountered fungi. We describe the application of 18S ribosomal RNA (rRNA) gene sequencing in the early diagnosis of a case of Exophiala peritonitis. A yeast-like fungus was isolated from the dialysate fluid of a 66-year-old man undergoing continuous ambulatory peritoneal dialysis. It grew slowly after 12 days of incubation to yield mature cultures to permit recognition of microscopic features resembling those of Exophiala, a dematiacerous mold. 18S rRNA gene sequencing provided results 12 days earlier than phenotypic identification and revealed 15 base difference (0.9%) between the isolate and Exophiala sp. strain GHP 1205 (GenBank Accession no. AJ232954), indicating that the isolate most closely resembles a strain of Exophiala species. The patient responded to 4 weeks of intravenous amphotericin B therapy. Early identification of the fungus was important for the choice of anti-fungal regimen. As opportunistic fungal infections in immunocompromised patients are globally emerging problems, the development of molecular techniques for fungal identification is crucial for early diagnosis and appropriate treatment.

  11. A possible mechanism for the inhibition of ribosomal RNA gene transcription during mitosis.

    PubMed

    Weisenberger, D; Scheer, U

    1995-05-01

    When cells enter mitosis, RNA synthesis ceases. Yet the RNA polymerase I (pol I) transcription machinery involved in the production of pre-rRNA remains bound to the nucleolus organizing region (NOR), the chromosome site harboring the tandemly repeated rRNA genes. Here we examine whether rDNA transcription units are transiently blocked or "frozen" during mitosis. By using fluorescent in situ hybridization we were unable to detect nascent pre-rRNA chains on the NORs of mouse 3T3 and rat kangaroo PtK2 cells. Appropriate controls showed that our approach was sensitive enough to visualize, at the light microscopic level, individual transcriptionally active rRNA genes both in situ after experimental unfolding of nucleoli and in chromatin spreads ("Miller spreads"). Analysis of the cell cycle-dependent redistribution of transcript-associated components also revealed that most transcripts are released from the rDNA at mitosis. Upon disintegration of the nucleolus during mitosis, U3 small nucleolar RNA (snoRNA) and the nucleolar proteins fibrillarin and nucleolin became dispersed throughout the cytoplasm and were excluded from the NORs. Together, our data rule out the presence of "frozen Christmas-trees" at the mitotic NORs but are compatible with the view that inactive pol I remains on the rDNA. We propose that expression of the rRNA genes is regulated during mitosis at the level of transcription elongation, similarly to what is known for a number of genes transcribed by pol II. Such a mechanism may explain the decondensed state of the NOR chromatin and the immediate transcriptional reactivation of the rRNA genes following mitosis.

  12. Site-specific fluorescence dynamics in an RNA ‘thermometer’ reveals the role of ribosome binding in its temperature-sensitive switch function

    PubMed Central

    Narayan, Satya; Kombrabail, Mamta H.; Das, Sudipta; Singh, Himanshu; Chary, Kandala V. R.; Rao, Basuthkar J.; Krishnamoorthy, Guruswamy

    2015-01-01

    RNA thermometers control the translation of several heat shock and virulence genes by their temperature-sensitive structural transitions. Changes in the structure and dynamics of MiniROSE RNA, which regulates translation in the temperature range of 20–45°C, were studied by site specifically replacing seven adenine residues with the fluorescent analog, 2-aminopurine (2-AP), one at a time. Dynamic fluorescence observables of 2-AP-labeled RNAs were compared in their free versus ribosome-bound states for the first time. Noticeably, position dependence of fluorescence observables, which was prominent at 20°C, was persistent even at 45ºC, suggesting the persistence of structural integrity up to 45ºC. Interestingly, position-dependent dispersion of fluorescence lifetime and quenching constant at 45°C was ablated in ribosome-bound state, when compared to those at 20°C, underscoring loss of structural integrity at 45°C, in ribosome-bound RNA. Significant increase in the value of mean lifetime for 2-AP corresponding to Shine–Dalgarno sequences, when the temperature was raised from 20 to 45°C, to values seen in the presence of urea at 45°C was a strong indicator of melting of the 3D structure of MiniROSE RNA at 45°C, only when it was ribosome bound. Taken all together, we propose a model where we invoke that ribosome binding of the RNA thermometer critically regulates temperature sensing functions in MiniROSE RNA. PMID:25477380

  13. A mutant RNA pseudoknot that promotes ribosomal frameshifting in mouse mammary tumor virus.

    PubMed

    Kang, H; Tinoco, I

    1997-05-15

    A single A-->G mutation that changes a potential A.U base pair to a G.U pair at the junction of the stems and loops of a non-frameshifting pseudoknot dramatically increases its frameshifting efficiency in mouse mammary tumor virus. The structure of the non-frameshifting pseudoknot APK has been found to be very different from that of pseudoknots that cause efficient frameshifting [Kang,H., Hines,J.V. and Tinoco,I. (1995) J. Mol. Biol. , 259, 135-147]. The 3-dimensional structure of the mutant pseudoknot was determined by restrained molecular dynamics based on NMR-derived interproton distance and torsion angle constraints. One striking feature of the mutant pseudoknot compared with the parent pseudoknot is that a G.U base pair forms at the top of stem 2, thus leaving only 1 nt at the junction of the two stems. The conformation is very different from that of the previously determined non-frameshifting parent pseudoknot, which lacks the A.U base pair at the top of the stem and has 2 nt between the stems. However, the conformation is quite similar to that of efficient frameshifting pseudoknots whose structures were previously determined by NMR. A single adenylate residue intervenes between the two stems and interrupts their coaxial stacking. This unpaired nucleotide produces a bent structure. The structural similarity among the efficient frameshifting pseudoknots indicates that a specific conformation is required for ribosomal frameshifting, further implying a specific interaction of the pseudoknot with the ribosome.

  14. Comment on ``Length-dependent translation of messenger RNA by ribosomes''

    NASA Astrophysics Data System (ADS)

    Zhang, Yunxin

    2012-02-01

    In a recent paper by Valleriani [Phys. Rev. EPLEEE81539-375510.1103/PhysRevE.83.042903 83, 042903 (2011)], a simple model for the translation of messenger RNA (mRNA) is presented. Using this model, the protein translational ratio r, defined as the ratio of protein translation rate ωtl from mRNA to protein degradation rate ωp, is obtained. The key point in obtaining the translational ratio r is to get the protein translation rate ωtl. In Valleriani 's paper, ωtl is obtained as the mean value of the measured translation rate, which is the ratio of the synthesized protein number to the mRNA lifetime. However, in experiments, different methods might be used to obtain the value of ωtl. Therefore, to apply Valleriani 's model to more general experiments, in this Comment three methods to obtain the translation rate ωtl, and consequently the translational ratio r, are presented. Based on one of the methods which might be employed in most of the experiments, we find that the translational ratio r decays exponentially with mRNA length in prokaryotic cells, and decays reciprocally with mRNA length in eukaryotic cells. This result is slight different from that which was obtained in Valleriani 's paper.

  15. Structures of the orthosomycin antibiotics avilamycin and evernimicin in complex with the bacterial 70S ribosome

    PubMed Central

    Arenz, Stefan; Graf, Michael; Nguyen, Fabian; Huter, Paul; Polikanov, Yury S.; Blanchard, Scott C.; Wilson, Daniel N.

    2016-01-01

    The ribosome is one of the major targets for therapeutic antibiotics; however, the rise in multidrug resistance is a growing threat to the utility of our current arsenal. The orthosomycin antibiotics evernimicin (EVN) and avilamycin (AVI) target the ribosome and do not display cross-resistance with any other classes of antibiotics, suggesting that they bind to a unique site on the ribosome and may therefore represent an avenue for development of new antimicrobial agents. Here we present cryo-EM structures of EVN and AVI in complex with the Escherichia coli ribosome at 3.6- to 3.9-Å resolution. The structures reveal that EVN and AVI bind to a single site on the large subunit that is distinct from other known antibiotic binding sites on the ribosome. Both antibiotics adopt an extended conformation spanning the minor grooves of helices 89 and 91 of the 23S rRNA and interacting with arginine residues of ribosomal protein L16. This binding site overlaps with the elbow region of A-site bound tRNA. Consistent with this finding, single-molecule FRET (smFRET) experiments show that both antibiotics interfere with late steps in the accommodation process, wherein aminoacyl-tRNA enters the peptidyltransferase center of the large ribosomal subunit. These data provide a structural and mechanistic