Science.gov

Sample records for 23s ribosomal rna

  1. Sequence specific detection of bacterial 23S ribosomal RNA by TLR13

    PubMed Central

    Li, Xiao-Dong; Chen, Zhijian J

    2012-01-01

    Toll-like receptors (TLRs) detect microbial infections and trigger innate immune responses. Among vertebrate TLRs, the role of TLR13 and its ligand are unknown. Here we show that TLR13 detects the 23S ribosomal RNA of both gram-positive and gram-negative bacteria. A sequence containing 13 nucleotides near the active site of 23S rRNA ribozyme, which catalyzes peptide bond synthesis, was both necessary and sufficient to trigger TLR13-dependent interleukin-1β production. Single point mutations within this sequence destroyed the ability of the 23S rRNA to stimulate the TLR13 pathway. Knockout of TLR13 in mice abolished the induction of interleukin-1β and other cytokines by the 23S rRNA sequence. Thus, TLR13 detects bacterial RNA with exquisite sequence specificity. DOI: http://dx.doi.org/10.7554/eLife.00102.001 PMID:23110254

  2. Ribosome origins: The relative age of 23S rRNA Domains

    NASA Astrophysics Data System (ADS)

    Hury, James; Nagaswamy, Uma; Larios-Sanz, Maia; Fox, George E.

    2006-08-01

    The modern ribosome and its component RNAs are quite large and it is likely that at an earlier time they were much smaller. Hence, not all regions of the modern ribosomal RNAs (rRNA) are likely to be equally old. In the work described here, it is hypothesized that the oldest regions of the RNAs will usually be highly integrated into the machinery. When this is the case, an examination of the interconnectivity between local RNA regions can provide insight to the relative age of the various regions. Herein, we describe an analysis of all known long-range RNA/RNA interactions within the 23S rRNA and between the 23S rRNA and the 16S rRNA in order to assess the interconnectivity between the usual Domains as defined by secondary structure. Domain V, which contains the peptidyl transferase center is centrally located, extensively connected, and therefore likely to be the oldest region. Domain IV and Domain II are extensively interconnected with both themselves and Domain V. A portion of Domain IV is also extensively connected with the 30S subunit and hence Domain IV may be older than Domain II. These results are consistent with other evidence relating to the relative age of RNA regions. Although the relative time of addition of the GTPase center can not be reliably deduced it is pointed out that the development of this may have dramatically affected the progenotes that preceded the last common ancestor.

  3. Cloning, in vitro transcription, and biological activity of Escherichia coli 23S ribosomal RNA.

    PubMed

    Weitzmann, C J; Cunningham, P R; Ofengand, J

    1990-06-25

    The 23S rRNA gene was excised from the rrnB operon of pKK3535 and ligated into pUC19 behind the strong class III T7 promoter so that the correct 5' end of mature 23S RNA was produced upon transcription by T7 RNA polymerase. At the 3' end, generation of a restriction site for linearization required the addition of 2 adenosine residues to the mature 23S sequence. In vitro runoff transcripts were indistinguishable from natural 23S RNA in size on denaturing gels and in 5'-terminal sequence. The length and sequence of the 3' terminal T1 fragment was also as expected from the DNA sequence, except that an additional C, A, or U residue was added to 21%, 18%, or 5% of the molecules, respectively. Typical transcription reactions yielded 500-700 moles RNA per mole template. This transcript was used as a substrate for methyl transfer from S-adenosyl methionine catalyzed by Escherichia coli cell extracts. The majority (50-65%) of activity observed in a crude (S30) extract appeared in the post-ribosomal supernatant (S100). Activities catalyzing formation of m5C, m5U, m2G, and m6A residues in the synthetic transcript were observed. PMID:2194163

  4. Identification of the methyltransferase targeting C2499 in Deinococcus radiodurans 23S ribosomal RNA.

    PubMed

    Mundus, Julie; Flyvbjerg, Karen Freund; Kirpekar, Finn

    2016-01-01

    The bacterium Deinococcus radiodurans-like all other organisms-introduces nucleotide modifications into its ribosomal RNA. We have previously found that the bacterium contains a Carbon-5 methylation on cytidine 2499 of its 23S ribosomal RNA, which is so far the only modified version of cytidine 2499 reported. Using homology search, we identified the open reading frame DR_0049 as the primary candidate gene for the methyltransferase that modifies cytidine 2499. Mass spectrometric analysis demonstrated that recombinantly expressed DR0049 protein methylates E. coli cytidine 2499 both in vitro and in vivo. We also inactivated the DR_0049 gene in D. radiodurans through insertion of a chloramphenicol resistance cassette. This resulted in complete absence of the cytidine 2499 methylation, which all together demonstrates that DR_0049 encodes the methyltransferase producing m(5)C2499 in D. radiodurans 23S rRNA. Growth experiments disclosed that inactivation of DR_0049 is associated with a severe growth defect, but available ribosome structures show that cytidine 2499 is positioned very similar in D. radiodurans harbouring the modification and E. coli without the modification. Hence there is no obvious structure-based explanation for the requirement for the C2499 posttranscriptional modification in D. radiodurans.

  5. Evidence for functional interaction between domains II and V of 23S ribosomal RNA from an erythromycin-resistant mutant.

    PubMed Central

    Douthwaite, S; Prince, J B; Noller, H F

    1985-01-01

    A mutation affording low levels of erythromycin resistance has been obtained by in vitro hydroxylamine mutagenesis of a cloned ribosomal RNA operon from Escherichia coli. The site of the mutational event responsible for antibiotic resistance was localized to the gene region encoding domain II of 23S rRNA by replacement of restriction fragments in the wild-type plasmid by corresponding fragments from the mutant plasmid. DNA sequencing showed that positions 1219-1230 of the 23S rRNA gene are deleted in the mutant. Since all previously characterized rRNA mutations conferring resistance to erythromycin show changes exclusively in domain V, our present findings provide direct evidence for functional interaction between domains II and V of 23S rRNA. Images PMID:3909142

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

    PubMed Central

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

    2014-01-01

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

  7. Methylation sites in Escherichia coli ribosomal RNA: localization and identification of four new sites of methylation in 23S rRNA.

    PubMed

    Smith, J E; Cooperman, B S; Mitchell, P

    1992-11-10

    Four previously undetermined sites of methylation are mapped in Escherichia coli 23S rRNA employing a novel combination of methods. First, using a double-isotope approach, the total number of methyl groups in 23S rRNA was determined to be 14.9 +/- 1.6. Second, hybridization of methyl-labeled rRNA to complementary DNA restriction fragments and PAGE analysis were used to purify RNA-DNA heteroduplexes and to quantify methyl groups within specific 23S rRNA fragments. Third, the methylated nucleosides in these fragments were identified and quantified using HPLC, confirming the presence of 14 methylation sites in 23S rRNA, four more than had been previously identified. In contrast, a similar set of analyses conducted on 16S rRNA gave evidence for 10 sites of methylation, at all approximate locations consistent with published 16S methylated nucleoside identities and locations. Selected regions of the 23S rRNA molecule containing previously unidentified methylated nucleosides were released by site-directed cleavage with ribonuclease H and isolated by PAGE. Sites of methylation within the RNA fragments were determined by classical oligonucleotide analyses. The four newly identified methylation sites in 23S rRNA are m2G-1835, m5C-1962, m6A-2503, and m2G at one of positions 2445-2447. Together with previously described sites of modification, these new sites form a group that is clustered in a current model for the three-dimensional organization of the 23S rRNA in the 50S ribosomal subunit, at a locus congruent with nucleotides previously implicated in ribosomal function. PMID:1384701

  8. Single methylation of 23S rRNA triggers late steps of 50S ribosomal subunit assembly.

    PubMed

    Arai, Taiga; Ishiguro, Kensuke; Kimura, Satoshi; Sakaguchi, Yuriko; Suzuki, Takeo; Suzuki, Tsutomu

    2015-08-25

    Ribosome biogenesis requires multiple assembly factors. In Escherichia coli, deletion of RlmE, the methyltransferase responsible for the 2'-O-methyluridine modification at position 2552 (Um2552) in helix 92 of the 23S rRNA, results in slow growth and accumulation of the 45S particle. We demonstrate that the 45S particle that accumulates in ΔrlmE is a genuine precursor that can be assembled into the 50S subunit. Indeed, 50S formation from the 45S precursor could be promoted by RlmE-mediated Um2552 formation in vitro. Ribosomal protein L36 (encoded by rpmJ) was completely absent from the 45S precursor in ΔrlmE, and we observed a strong genetic interaction between rlmE and rpmJ. Structural probing of 23S rRNA and high-salt stripping of 45S components revealed that RlmE-mediated methylation promotes interdomain interactions via the association between helices 92 and 71, stabilized by the single 2'-O-methylation of Um2552, in concert with the incorporation of L36, triggering late steps of 50S subunit assembly. PMID:26261349

  9. Single methylation of 23S rRNA triggers late steps of 50S ribosomal subunit assembly

    PubMed Central

    Arai, Taiga; Ishiguro, Kensuke; Kimura, Satoshi; Sakaguchi, Yuriko; Suzuki, Takeo; Suzuki, Tsutomu

    2015-01-01

    Ribosome biogenesis requires multiple assembly factors. In Escherichia coli, deletion of RlmE, the methyltransferase responsible for the 2′-O-methyluridine modification at position 2552 (Um2552) in helix 92 of the 23S rRNA, results in slow growth and accumulation of the 45S particle. We demonstrate that the 45S particle that accumulates in ΔrlmE is a genuine precursor that can be assembled into the 50S subunit. Indeed, 50S formation from the 45S precursor could be promoted by RlmE-mediated Um2552 formation in vitro. Ribosomal protein L36 (encoded by rpmJ) was completely absent from the 45S precursor in ΔrlmE, and we observed a strong genetic interaction between rlmE and rpmJ. Structural probing of 23S rRNA and high-salt stripping of 45S components revealed that RlmE-mediated methylation promotes interdomain interactions via the association between helices 92 and 71, stabilized by the single 2′-O-methylation of Um2552, in concert with the incorporation of L36, triggering late steps of 50S subunit assembly. PMID:26261349

  10. Mycobacterial toxin MazF-mt6 inhibits translation through cleavage of 23S rRNA at the ribosomal A site.

    PubMed

    Schifano, Jason M; Edifor, Regina; Sharp, Jared D; Ouyang, Ming; Konkimalla, Arvind; Husson, Robert N; Woychik, Nancy A

    2013-05-21

    The Mycobacterium tuberculosis genome contains an unusually high number of toxin-antitoxin modules, some of which have been suggested to play a role in the establishment and maintenance of latent tuberculosis. Nine of these toxin-antitoxin loci belong to the mazEF family, encoding the intracellular toxin MazF and its antitoxin inhibitor MazE. Nearly every MazF ortholog recognizes a unique three- or five-base RNA sequence and cleaves mRNA. As a result, these toxins selectively target a subset of the transcriptome for degradation and are known as "mRNA interferases." Here we demonstrate that a MazF family member from M. tuberculosis, MazF-mt6, has an additional role--inhibiting translation through targeted cleavage of 23S rRNA in the evolutionarily conserved helix/loop 70. We first determined that MazF-mt6 cleaves mRNA at (5')UU↓CCU(3') sequences. We then discovered that MazF-mt6 also cleaves M. tuberculosis 23S rRNA at a single UUCCU in the ribosomal A site that contacts tRNA and ribosome recycling factor. To gain further mechanistic insight, we demonstrated that MazF-mt6-mediated cleavage of rRNA can inhibit protein synthesis in the absence of mRNA cleavage. Finally, consistent with the position of 23S rRNA cleavage, MazF-mt6 destabilized 50S-30S ribosomal subunit association. Collectively, these results show that MazF toxins do not universally act as mRNA interferases, because MazF-mt6 inhibits protein synthesis by cleaving 23S rRNA in the ribosome active center. PMID:23650345

  11. Initiation factor IF 2 binds to the alpha-sarcin loop and helix 89 of Escherichia coli 23S ribosomal RNA.

    PubMed Central

    La Teana, A; Gualerzi, C O; Dahlberg, A E

    2001-01-01

    During initiation of protein synthesis in bacteria, translation initiation factor IF2 is responsible for the recognition of the initiator tRNA (fMet-tRNA). To perform this function, IF2 binds to the ribosome interacting with both 30S and 50S ribosomal subunits. Here we report the topographical localization of translation initiation factor IF2 on the 70S ribosome determined by base-specific chemical probing. Our results indicate that IF2 specifically protects from chemical modification two sites in domain V of 23S rRNA, namely A2476 and A2478, and residues around position 2660 in domain VI, the so-called sarcin-ricin loop. These footprints are generated by IF2 regardless of the presence of fMet-tRNA, GTP, mRNA, and IF1. IF2 causes no specific protection of 16S rRNA. We observe a decreased reactivity of residues A1418 and A1483, which is an indication that the initiation factor has a tightening effect on the association of ribosomal subunits. This result, confirmed by sucrose density gradient analysis, seems to be a universally conserved property of IF2. PMID:11497435

  12. Magnesium ions mediate contacts between phosphoryl oxygens at positions 2122 and 2176 of the 23S rRNA and ribosomal protein L1.

    PubMed Central

    Drygin, D; Zimmermann, R A

    2000-01-01

    The complex of ribosomal protein L1 with 23S rRNA from Escherichia coli is of great interest because of the unique structural and functional aspects of this ribonucleoprotein domain. We have minimized the binding site for protein L1 on the 23S rRNA to nt 2120-2129, 2159-2162, and 2167-2178. This RNA fragment consists of two helices as well as an interconnecting loop of unknown structure. RNA molecules corresponding to the minimized L1 binding site, in which G, A, U, or C were individually replaced by their deoxyribo- (dN) or alpha-thio- (rNaS) analogs have been synthesized by T7 transcription in vitro and analyzed for their ability to bind protein L1. It has been demonstrated that the substitution of rNaS at position 2122 or 2176 decreases the affinity of the RNA for the protein in the presence of magnesium five- to tenfold, whereas the same changes have little effect on binding in the presence of manganese. This suggests that Rp oxygens in the phosphates preceding positions 2122 and 2176 are coordinated with Mg2+ and may participate in L1-23S rRNA interaction via magnesium bridges. We have also shown that this interaction is impaired by the presence of dC at position 2122 coupled with the presence of deoxyribonucleotide(s) at other positions in the RNA. This study demonstrates that the ribose-phosphate backbone of the helix encompassing nt 2120-2124/2174-2178 is intimately involved in the interaction of protein L1 with the 23S rRNA. In particular, we suggest that this helix is positioned in the cleft between the two domains of protein L1. PMID:11142372

  13. Domain I of 23S rRNA competes with a paused transcription complex for ribosomal protein L4 of Escherichia coli.

    PubMed Central

    Zengel, J M; Lindahl, L

    1993-01-01

    Ribosomal protein L4 of Escherichia coli regulates expression of its own eleven gene S10 operon both by inhibiting translation and by stimulating premature termination of transcription. Both regulatory processes presumably involve L4 recognition of the S10 leader RNA. To help define L4's regulatory target, we have investigated the protein's cognate target on 23S rRNA. Binding of L4 to various fragments of the 23S rRNA was monitored by determining their ability to sequester L4 in an in vitro transcription system and thereby eliminate the protein's effect on transcription. Using this approach we identified a region of about 110 bases within domain I of 23S rRNA which binds L4. A two base deletion within this region, close to the base to which L4 has been cross-linked in intact 50S subunits, eliminates L4 binding. These results also confirm the prediction of the autogenous control model, that L4 bound to its target on rRNA is not active in regulating transcription of the S10 operon. Images PMID:7685080

  14. Lactobacillus species identification by amplified ribosomal 16S-23S rRNA restriction fragment length polymorphism analysis.

    PubMed

    Sandes, S H C; Alvin, L B; Silva, B C; Zanirati, D F; Jung, L R C; Nicoli, J R; Neumann, E; Nunes, A C

    2014-12-01

    Lactic acid bacteria strains are commonly used for animal and human consumption due to their probiotic properties. One of the major genera used is Lactobacillus, a highly diverse genus comprised of several closely related species. The selection of new strains for probiotic use, especially strains of Lactobacillus, is the focus of several research groups. Accurate identification to species level is fundamental for research on new strains, as well as for safety assessment and quality assurance. The 16S-23S internal transcribed spacer (ITS-1) is a deeply homologous region among prokaryotes that is commonly used for identification to the species level because it is able to acquire and accumulate mutations without compromising general bacterial metabolism. In the present study, 16S-23S ITS regions of 45 Lactobacillus species (48 strains) were amplified and subjected to independent enzymatic digestions, using 12 restriction enzymes that recognise six-base sequences. Twenty-nine species showed unique restriction patterns, and could therefore be precisely identified solely by this assay (64%). This approach proved to be reproducible, allowing us to establish simplified restriction patterns for each evaluated species. The restriction patterns of each species were similar among homologous strains, and to a large extent reflected phylogenetic relationships based on 16S rRNA sequences, demonstrating the promising nature of this region for evolutionary studies.

  15. SrmB, a DEAD-box helicase involved in Escherichia coli ribosome assembly, is specifically targeted to 23S rRNA in vivo

    PubMed Central

    Trubetskoy, Dmitrii; Proux, Florence; Allemand, Frédéric; Dreyfus, Marc; Iost, Isabelle

    2009-01-01

    DEAD-box proteins play specific roles in remodeling RNA or ribonucleoprotein complexes. Yet, in vitro, they generally behave as nonspecific RNA-dependent ATPases, raising the question of what determines their specificity in vivo. SrmB, one of the five Escherichia coli DEAD-box proteins, participates in the assembly of the large ribosomal subunit. Moreover, when overexpressed, it compensates for a mutation in L24, the ribosomal protein (r-protein) thought to initiate assembly. Here, using the tandem affinity purification (TAP) procedure, we show that SrmB forms a complex with r-proteins L4, L24 and a region near the 5′-end of 23S rRNA that binds these proteins. In vitro reconstitution experiments show that the stability of this complex reflects cooperative interactions of SrmB with L4, L24 and rRNA. These observations are consistent with an early role of SrmB in assembly and explain the genetic link between SrmB and L24. Besides its catalytic core, SrmB possesses a nonconserved C-terminal extension that, we show, is not essential for SrmB function and specificity. In this regard, SrmB differs from DbpA, another DEAD-box protein involved in ribosome assembly. PMID:19734346

  16. SrmB, a DEAD-box helicase involved in Escherichia coli ribosome assembly, is specifically targeted to 23S rRNA in vivo.

    PubMed

    Trubetskoy, Dmitrii; Proux, Florence; Allemand, Frédéric; Dreyfus, Marc; Iost, Isabelle

    2009-10-01

    DEAD-box proteins play specific roles in remodeling RNA or ribonucleoprotein complexes. Yet, in vitro, they generally behave as nonspecific RNA-dependent ATPases, raising the question of what determines their specificity in vivo. SrmB, one of the five Escherichia coli DEAD-box proteins, participates in the assembly of the large ribosomal subunit. Moreover, when overexpressed, it compensates for a mutation in L24, the ribosomal protein (r-protein) thought to initiate assembly. Here, using the tandem affinity purification (TAP) procedure, we show that SrmB forms a complex with r-proteins L4, L24 and a region near the 5'-end of 23S rRNA that binds these proteins. In vitro reconstitution experiments show that the stability of this complex reflects cooperative interactions of SrmB with L4, L24 and rRNA. These observations are consistent with an early role of SrmB in assembly and explain the genetic link between SrmB and L24. Besides its catalytic core, SrmB possesses a nonconserved C-terminal extension that, we show, is not essential for SrmB function and specificity. In this regard, SrmB differs from DbpA, another DEAD-box protein involved in ribosome assembly.

  17. The rluC gene of Escherichia coli codes for a pseudouridine synthase that is solely responsible for synthesis of pseudouridine at positions 955, 2504, and 2580 in 23 S ribosomal RNA.

    PubMed

    Conrad, J; Sun, D; Englund, N; Ofengand, J

    1998-07-17

    Escherichia coli ribosomal RNA contains 10 pseudouridines, one in the 16 S RNA and nine in the 23 S RNA. Previously, the gene for the synthase responsible for the 16 S RNA pseudouridine was identified and cloned, as was a gene for a synthase that makes a single pseudouridine in 23 S RNA. The yceC open reading frame of E. coli is one of a set of genes homologous to these previously identified ribosomal RNA pseudouridine synthases. In this work, the gene was cloned, overexpressed, and shown to code for a pseudouridine synthase able to react with in vitro transcripts of 23 S ribosomal RNA. Deletion of the gene and analysis of the 23 S RNA from the deletion strain for the presence of pseudouridine at its nine known sites revealed that this synthase is solely responsible in vivo for the synthesis of three of the nine pseudouridine residues, at positions 955, 2504, and 2580. Therefore, this gene has been renamed rluC. Despite the absence of one-third of the normal complement of pseudouridines, there was no change in the exponential growth rate in either LB or M-9 medium at temperatures ranging from 24 to 42 degrees C. From this work and our previous studies, we have now identified three synthases that account for 50% of the pseudouridines in the E. coli ribosome.

  18. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2011-07-01

    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 Å resolution.

  20. 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.

  1. A short fragment of 23S rRNA containing the binding sites for two ribosomal proteins, L24 and L4, is a key element for rRNA folding during early assembly.

    PubMed Central

    Stelzl, U; Nierhaus, K H

    2001-01-01

    Previously we described an in vitro selection variant abbreviated SERF (in vitro selection from random rRNA fragments) that identifies protein binding sites within large RNAs. With this method, a small rRNA fragment derived from the 23S rRNA was isolated that binds simultaneously and independently the ribosomal proteins L4 and L24 from Escherichia coli. Until now the rRNA structure within the ternary complex L24-rRNA-L4 could not be studied due to the lack of an appropriate experimental strategy. Here we tackle the issue by separating the various complexes via native gel-electrophoresis and analyzing the rRNA structure by in-gel iodine cleavage of phosphorothioated RNA. The results demonstrate that during the transition from either the L4 or L24 binary complex to the ternary complex the structure of the rRNA fragment changes significantly. The identified protein binding sites are in excellent agreement with the recently reported crystal structure of the 50S subunit. Because both proteins play a prominent role in early assembly of the large subunit, the results suggest that the identified rRNA fragment is a key element for the folding of the 23S RNA during early assembly. The introduced in-gel cleavage method should be useful when an RNA structure within mixed populations of different but related complexes should be studied. PMID:11345438

  2. A short fragment of 23S rRNA containing the binding sites for two ribosomal proteins, L24 and L4, is a key element for rRNA folding during early assembly.

    PubMed

    Stelzl, U; Nierhaus, K H

    2001-04-01

    Previously we described an in vitro selection variant abbreviated SERF (in vitro selection from random rRNA fragments) that identifies protein binding sites within large RNAs. With this method, a small rRNA fragment derived from the 23S rRNA was isolated that binds simultaneously and independently the ribosomal proteins L4 and L24 from Escherichia coli. Until now the rRNA structure within the ternary complex L24-rRNA-L4 could not be studied due to the lack of an appropriate experimental strategy. Here we tackle the issue by separating the various complexes via native gel-electrophoresis and analyzing the rRNA structure by in-gel iodine cleavage of phosphorothioated RNA. The results demonstrate that during the transition from either the L4 or L24 binary complex to the ternary complex the structure of the rRNA fragment changes significantly. The identified protein binding sites are in excellent agreement with the recently reported crystal structure of the 50S subunit. Because both proteins play a prominent role in early assembly of the large subunit, the results suggest that the identified rRNA fragment is a key element for the folding of the 23S RNA during early assembly. The introduced in-gel cleavage method should be useful when an RNA structure within mixed populations of different but related complexes should be studied.

  3. Localisation of a series of intra-RNA cross-links in the secondary and tertiary structure of 23S RNA, induced by ultraviolet irradiation of Escherichia coli 50S ribosomal subunits.

    PubMed Central

    Stiege, W; Glotz, C; Brimacombe, R

    1983-01-01

    Intra-RNA cross-links were introduced into E. coli 50S ribosomal subunits by mild ultraviolet irradiation. The subunits were partially digested with cobra venom nuclease, and the cross-linked RNA complexes were isolated by two-dimensional electrophoresis. Many of the complexes were submitted to a second partial digestion procedure. Oligonucleotide analysis of the RNA fragments obtained in this manner enabled cross-links between the following ribonuclease T1 oligonucleotides in the 23S RNA to be established: positions 292-296 and 339-350; 601-604 and 652-656; 1018-1022 and 1140-1149; 1433-1435 and 1556-1560; 1836-1839 and 1898-1903; 2832-2834 (tentative) and 2878-2885; 2849-2852 and 2865-2867 (tentative); 739-748 and 2609-2618; 571-577 and 2030-2032; 1777-1792 (tentative) and 2584-2588. The first seven of these cross-links lie within the secondary structure of the 23S RNA, whereas the last three are tertiary structural cross-links. The degree of precision of the individual determinations was variable, depending on the nucleotide sequence in the vicinity of the cross-link site concerned. Images PMID:6340065

  4. Analysis of 23S rRNA genes in metagenomes - a case study from the Global Ocean Sampling Expedition.

    PubMed

    Yilmaz, Pelin; Kottmann, Renzo; Pruesse, Elmar; Quast, Christian; Glöckner, Frank Oliver

    2011-09-01

    As an evolutionary marker, 23S ribosomal RNA (rRNA) offers more diagnostic sequence stretches and greater sequence variation than 16S rRNA. However, 23S rRNA is still not as widely used. Based on 80 metagenome samples from the Global Ocean Sampling (GOS) Expedition, the usefulness and taxonomic resolution of 23S rRNA were compared to those of 16S rRNA. Since 23S rRNA is approximately twice as large as 16S rRNA, twice as many 23S rRNA gene fragments were retrieved from the GOS reads than 16S rRNA gene fragments, with 23S rRNA gene fragments being generally about 100bp longer. Datasets for 16S and 23S rRNA sequences revealed similar relative abundances for major marine bacterial and archaeal taxa. However, 16S rRNA sequences had a better taxonomic resolution due to their significantly larger reference database. Reevaluation of the specificity of previously published PCR amplification primers and group specific fluorescence in situ hybridization probes on this metagenomic set of non-amplified 23S rRNA sequences revealed that out of 16 primers investigated, only two had more than 90% target group coverage. Evaluations of two probes, BET42a and GAM42a, were in accordance with previous evaluations, with a discrepancy in the target group coverage of the GAM42a probe when evaluated against the GOS metagenomic dataset.

  5. A second function for pseudouridine synthases: A point mutant of RluD unable to form pseudouridines 1911, 1915, and 1917 in Escherichia coli 23S ribosomal RNA restores normal growth to an RluD-minus strain.

    PubMed

    Gutgsell, N S; Del Campo, M; Raychaudhuri, S; Ofengand, J

    2001-07-01

    This laboratory previously showed that truncation of the gene for RluD, the Escherichia coli pseudouridine synthase responsible for synthesis of 23S rRNA pseudouridines 1911, 1915, and 1917, blocks pseudouridine formation and inhibits growth. We now show that RluD mutants at the essential aspartate 139 allow these two functions of RluD to be separated. In vitro, RluD with aspartate 139 replaced by threonine or asparagine is completely inactive. In vivo, the growth defect could be completely restored by transformation of an RluD-inactive strain with plasmids carrying genes for RluD with aspartate 139 replaced by threonine or asparagine. Pseudouridine sequencing of the 23S rRNA from these transformed strains demonstrated the lack of these pseudouridines. Pseudoreversion, which has previously been shown to restore growth without pseudouridine formation by mutation at a distant position on the chromosome, was not responsible because transformation with empty vector under identical conditions did not alter the growth rate.

  6. [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

  7. 23S rRNA nucleotides in the peptidyl transferase center are essential for tryptophanase operon induction.

    PubMed

    Yang, Rui; Cruz-Vera, Luis R; Yanofsky, Charles

    2009-06-01

    Distinct features of the ribosomal peptide exit tunnel are known to be essential for recognition of specific amino acids of a nascent peptidyl-tRNA. Thus, a tryptophan residue at position 12 of the peptidyl-tRNA TnaC-tRNA(Pro) leads to the creation of a free tryptophan binding site within the ribosome at which bound tryptophan inhibits normal ribosome functions. The ribosomal processes that are inhibited are hydrolysis of TnaC-tRNA(Pro) by release factor 2 and peptidyl transfer of TnaC of TnaC-tRNA(Pro) to puromycin. These events are normally performed in the ribosomal peptidyl transferase center. In the present study, changes of 23S rRNA nucleotides in the 2585 region of the peptidyl transferase center, G2583A and U2584C, were observed to reduce maximum induction of tna operon expression by tryptophan in vivo without affecting the concentration of tryptophan necessary to obtain 50% induction. The growth rate of strains with ribosomes with either of these changes was not altered appreciably. In vitro analyses with mutant ribosomes with these changes showed that tryptophan was not as efficient in protecting TnaC-tRNA(Pro) from puromycin action as wild-type ribosomes. However, added tryptophan did prevent sparsomycin action as it normally does with wild-type ribosomes. These findings suggest that these two mutational changes act by reducing the ability of ribosome-bound tryptophan to inhibit peptidyl transferase activity rather than by reducing the ability of the ribosome to bind tryptophan. Thus, the present study identifies specific nucleotides within the ribosomal peptidyl transferase center that appear to be essential for effective tryptophan induction of tna operon expression. PMID:19329641

  8. 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.

  9. 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...

  10. A DEAD box protein is required for formation of a hidden break in Arabidopsis chloroplast 23S rRNA.

    PubMed

    Nishimura, Kenji; Ashida, Hiroki; Ogawa, Taro; Yokota, Akiho

    2010-09-01

    In plant chloroplasts, the ribosomal RNA (rRNA) of the large subunit of the ribosome undergoes post-maturation fragmentation processing. This processing consists of site-specific cleavage that generates gapped, discontinuous rRNA molecules. However, the molecular mechanism underlying introduction of the gap structure (the 'hidden break') is poorly understood. Here, we found that the DEAD box protein RH39 plays a key role in introduction of the hidden break into the 23S rRNA in Arabidopsis chloroplasts. Genetic screening for an Arabidopsis plant with a drastically reduced level of ribulose-1,5-bisphosphate carboxylase/oxygenase identified an RH39 mutant. The levels of other chloroplast-encoded photosynthetic proteins were also severely reduced. The reductions were not due to a failure of transcription, but rather inefficiency in translation. RNA gel blotting revealed incomplete fragmentation of 23S rRNA in chloroplasts during maturation. In vitro analysis with recombinant RH39 suggested that the protein binds to the adjacent sequence upstream of the hidden break site to exert its function. We propose a molecular mechanism for the RH39-mediated fragmentation processing of 23S rRNA in chloroplasts.

  11. 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

  12. 5S rRNA and ribosome.

    PubMed

    Gongadze, G M

    2011-12-01

    5S rRNA is an integral component of the ribosome of all living organisms. It is known that the ribosome without 5S rRNA is functionally inactive. However, the question about the specific role of this RNA in functioning of the translation apparatus is still open. This review presents a brief history of the discovery of 5S rRNA and studies of its origin and localization in the ribosome. The previously expressed hypotheses about the role of this RNA in the functioning of the ribosome are discussed considering the unique location of 5S rRNA in the ribosome and its intermolecular contacts. Based on analysis of the current data on ribosome structure and its functional complexes, the role of 5S rRNA as an intermediary between ribosome functional domains is discussed.

  13. 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

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

    PubMed

    Jayasree Rajagopalan Nair, Parvathi; Singh, Sunita

    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

  15. The tmRNA ribosome rescue system

    PubMed Central

    Janssen, Brian D.; Hayes, Christopher S.

    2012-01-01

    The bacterial tmRNA quality control system monitors protein synthesis and recycles stalled translation complexes in a process termed “ribosome rescue”. During rescue, tmRNA acts first as a transfer RNA to bind stalled ribosomes, then as a messenger RNA to add the ssrA peptide tag to the C-terminus of the nascent polypeptide chain. The ssrA peptide targets tagged peptides for proteolysis, ensuring rapid degradation of potentially deleterious truncated polypeptides. Ribosome rescue also facilitates turnover of the damaged messages responsible for translational arrest. Thus, tmRNA increases the fidelity of gene expression by promoting the synthesis of full-length proteins. In addition to serving as a global quality control system, tmRNA also plays important roles in bacterial development, pathogenesis and environmental stress responses. This review focuses on the mechanism of tmRNA-mediated ribosome rescue and the role of tmRNA in bacterial physiology. PMID:22243584

  16. 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

  17. Identification of two Escherichia coli pseudouridine synthases that show multisite specificity for 23S RNA.

    PubMed

    Huang, L; Ku, J; Pookanjanatavip, M; Gu, X; Wang, D; Greene, P J; Santi, D V

    1998-11-10

    Several putative Escherichia coli pseudouridine (Psi) synthases have been identified by iterative searching of genomic databases for ORFs homologous to known Psi synthases [Gustafsson et al. (1996) Nucleic Acids Res. 24, 3756-3762]. Of these, yceC and yfiI were proposed to encode Psi synthases which modify 23S rRNA. In the present work, yceC and yfiI were cloned and overexpressed in E. coli, and the encoded enzymes, YceC and YfiI, were purified to homogeneity. Both proteins converted Urd residues of rRNA to Psi, thus confirming their identities as Psi synthases. However, in in vitro experiments both enzymes extensively modified Urd residues of both 23S rRNA and 16S rRNA. Gene-disruption of yceCresulted in the absence of Psi modification at positions U955, 2504, and 2580 of 23S RNA, thus identifying these sites as in vivo targets for YceC. Likewise, yfiI disruption resulted in the absence of Psi modification at positions U1911, 1917, and possibly 1915 of 23S RNA. Disruption of yceC did not affect the growth under the conditions tested, whereas yfiI-disrupted cells showed a dramatic decrease in growth rate. Since YceC and YfiI hypermodify RNA in vitro, factors in addition to ribonucleotide sequence must contribute to the in vivo specificity of these enzymes.

  18. 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

  19. Ribosomal RNA pseudouridines and pseudouridine synthases.

    PubMed

    Ofengand, James

    2002-03-01

    Pseudouridines are found in virtually all ribosomal RNAs but their function is unknown. There are four to eight times more pseudouridines in eukaryotes than in eubacteria. Mapping 19 Haloarcula marismortui pseudouridines on the three-dimensional 50S subunit does not show clustering. In bacteria, specific enzymes choose the site of pseudouridine formation. In eukaryotes, and probably also in archaea, selection and modification is done by a guide RNA-protein complex. No unique specific role for ribosomal pseudouridines has been identified. We propose that pseudouridine's function is as a molecular glue to stabilize required RNA conformations that would otherwise be too flexible.

  20. Rapid Diagnosis of Bacteremia by Universal Amplification of 23S Ribosomal DNA Followed by Hybridization to an Oligonucleotide Array

    PubMed Central

    Anthony, R. M.; Brown, T. J.; French, G. L.

    2000-01-01

    The rapid identification of bacteria in blood cultures and other clinical specimens is important for patient management and antimicrobial therapy. We describe a rapid (<4 h) detection and identification system that uses universal PCR primers to amplify a variable region of bacterial 23S ribosomal DNA, followed by reverse hybridization of the products to a panel of oligonucleotides. This procedure was successful in discriminating a range of bacteria in pure cultures. When this procedure was applied directly to 158 unselected positive blood culture broths on the day when growth was detected, 125 (79.7%) were correctly identified, including 4 with mixed cultures. Nine (7.2%) yielded bacteria for which no oligonucleotide targets were present in the oligonucleotide panel, and 16 culture-positive broths (10.3%) produced no PCR product. In seven of the remaining eight broths, streptococci were identified but not subsequently grown, and one isolate of Staphylococcus aureus was misidentified as a coagulase-negative staphylococcus. The accuracy, range, and discriminatory power of the assay can be continually extended by adding further oligonucleotides to the panel without significantly increasing complexity or cost. PMID:10655385

  1. 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. PMID:25739364

  2. 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.

  3. Patterns and regulation of ribosomal RNA transcription in Borrelia burgdorferi

    PubMed Central

    2011-01-01

    Background Borrelia burgdorferi contains one 16S and two tandem sets of 23S-5S ribosomal (r) RNA genes whose patterns of transcription and regulation are unknown but are likely to be critical for survival and persistence in its hosts. Results RT-PCR of B. burgdorferi N40 and B31 revealed three rRNA region transcripts: 16S rRNA-alanine transfer RNA (tRNAAla); tRNAIle; and both sets of 23S-5S rRNA. At 34°C, there were no differences in growth rate or in accumulation of total protein, DNA and RNA in B31 cultured in Barbour-Stoenner-Kelly (BSK)-H whether rabbit serum was present or not. At 23°C, B31 grew more slowly in serum-containing BSK-H than at 34°C. DNA per cell was higher in cells in exponential as compared to stationary phase at either temperature; protein per cell was similar at both temperatures in both phases. Similar amounts of rRNA were produced in exponential phase at both temperatures, and rRNA was down-regulated in stationary phase at either temperature. Interestingly, a relBbu deletion mutant unable to generate (p)ppGpp did not down-regulate rRNA at transition to stationary phase in serum-containing BSK-H at 34°C, similar to the relaxed phenotype of E. coli relA mutants. Conclusions We conclude that rRNA transcription in B. burgdorferi is complex and regulated both by growth phase and by the stringent response but not by temperature-modulated growth rate. PMID:21251259

  4. SOT1, a pentatricopeptide repeat protein with a small MutS-related domain, is required for correct processing of plastid 23S-4.5S rRNA precursors in Arabidopsis thaliana.

    PubMed

    Wu, Wenjuan; Liu, Sheng; Ruwe, Hannes; Zhang, Delin; Melonek, Joanna; Zhu, Yajuan; Hu, Xupeng; Gusewski, Sandra; Yin, Ping; Small, Ian D; Howell, Katharine A; Huang, Jirong

    2016-03-01

    Ribosomal RNA processing is essential for plastid ribosome biogenesis, but is still poorly understood in higher plants. Here, we show that SUPPRESSOR OF THYLAKOID FORMATION1 (SOT1), a plastid-localized pentatricopeptide repeat (PPR) protein with a small MutS-related domain, is required for maturation of the 23S-4.5S rRNA dicistron. Loss of SOT1 function leads to slower chloroplast development, suppression of leaf variegation, and abnormal 23S and 4.5S processing. Predictions based on the PPR motif sequences identified the 5' end of the 23S-4.5S rRNA dicistronic precursor as a putative SOT1 binding site. This was confirmed by electrophoretic mobility shift assay, and by loss of the abundant small RNA 'footprint' associated with this site in sot1 mutants. We found that more than half of the 23S-4.5S rRNA dicistrons in sot1 mutants contain eroded and/or unprocessed 5' and 3' ends, and that the endonucleolytic cleavage product normally released from the 5' end of the precursor is absent in a sot1 null mutant. We postulate that SOT1 binding protects the 5' extremity of the 23S-4.5S rRNA dicistron from exonucleolytic attack, and favours formation of the RNA structure that allows endonucleolytic processing of its 5' and 3' ends.

  5. A functional relationship between helix 1 and the 900 tetraloop of 16S ribosomal RNA within the bacterial ribosome.

    PubMed

    Bélanger, François; Théberge-Julien, Gabriel; Cunningham, Philip R; Brakier-Gingras, Léa

    2005-06-01

    The conserved 900 tetraloop that caps helix 27 of 16S ribosomal RNA (rRNA) interacts with helix 24 of 16S rRNA and also with helix 67 of 23S rRNA, forming the intersubunit bridge B2c, proximal to the decoding center. In previous studies, we investigated how the interaction between the 900 tetraloop and helix 24 participates in subunit association and translational fidelity. In the present study, we investigated whether the 900 tetraloop is involved in other undetected interactions with different regions of the Escherichia coli 16S rRNA. Using a genetic complementation approach, we selected mutations in 16S rRNA that compensate for a 900 tetraloop mutation, A900G, which severely impairs subunit association and translational fidelity. Mutations were randomly introduced in 16S rRNA, using either a mutagenic XL1-Red E. coli strain or an error-prone PCR strategy. Gain-offunction mutations were selected in vivo with a specialized ribosome system. Two mutations, the deletion of U12 and the U12C substitution, were thus independently selected in helix 1 of 16S rRNA. This helix is located in the vicinity of helix 27, but does not directly contact the 900 tetraloop in the crystal structures of the ribosome. Both mutations correct the subunit association and translational fidelity defects caused by the A900G mutation, revealing an unanticipated functional interaction between these two regions of 16S rRNA.

  6. 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

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

    PubMed

    Wang, Shuai; Bai, Ge; Wang, Shu; Yang, Leiyun; Yang, Fen; Wang, Yi; Zhu, Jian-Kang; Hua, Jian

    2016-05-01

    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

  8. Identification of Carnobacterium species by restriction fragment length polymorphism of the 16S-23S rRNA gene intergenic spacer region and species-specific PCR.

    PubMed

    Rachman, Cinta; Kabadjova, Petia; Valcheva, Rosica; Prévost, Hervé; Dousset, Xavier

    2004-08-01

    The genus Carnobacterium is currently divided into the following eight species: Carnobacterium piscicola, C. divergens, C. gallinarum, C. mobile, C. funditum, C. alterfunditum, C. inhibens, and C. viridans. An identification tool for the rapid differentiation of these eight Carnobacterium species was developed, based on the 16S-23S ribosomal DNA (rDNA) intergenic spacer region (ISR). PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of this 16S-23S rDNA ISR was performed in order to obtain restriction profiles for all of the species. Three PCR amplicons, which were designated small ISR (S-ISR), medium ISR (M-ISR), and large ISR (L-ISR), were obtained for all Carnobacterium species. The L-ISR sequence revealed the presence of two tRNA genes, tRNA(Ala) and tRNA(Ile), which were separated by a spacer region that varied from 24 to 38 bp long. This region was variable among the species, allowing the design of species-specific primers. These primers were tested and proved to be species specific. The identification method based on the 16S-23S rDNA ISR, using PCR-RFLP and specific primers, is very suitable for the rapid low-cost identification and discrimination of all of the Carnobacterium species from other phylogenetically related lactic acid bacteria.

  9. Evolutionary relationships amongst archaebacteria. A comparative study of 23 S ribosomal RNAs of a sulphur-dependent extreme thermophile, an extreme halophile and a thermophilic methanogen.

    PubMed

    Leffers, H; Kjems, J; Ostergaard, L; Larsen, N; Garrett, R A

    1987-05-01

    The 23 S RNA genes representative of each of the main archaebacterial subkingdoms, Desulfurococcus mobilis an extreme thermophile, Halococcus morrhuae an extreme halophile and Methanobacterium thermoautotrophicum a thermophilic methanogen, were cloned and sequenced. The inferred RNA sequences were aligned with all the available 23 S-like RNAs of other archaebacteria, eubacteria/chloroplasts and the cytoplasm of eukaryotes. Universal secondary structural models containing six major structural domains were refined, and extended, using the sequence comparison approach. Much of the present structure was confirmed but six new helices were added, including one that also exists in the eukaryotic 5.8 S RNA, and extensions were made to several existing helices. The data throw doubt on whether the 5' and 3' ends of the 23 S RNA interact, since no stable helix can form in either the extreme thermophile or the methanogen RNA. A few secondary structural features, specific to the archaebacterial RNAs were identified; two of these were supported by a comparison of the archaebacterial RNA sequences, and experimentally, using chemical and ribonuclease probes. Seven tertiary structural interactions, common to all 23 S-like RNAs, were predicted within unpaired regions of the secondary structural model on the basis of co-variation of nucleotide pairs; two lie in the region of the 23 S RNA corresponding to 5.8 S RNA but they are not conserved in the latter. The flanking sequences of each of the RNAs could base-pair to form long RNA processing stems. They were not conserved in sequence but each exhibited a secondary structural feature that is common to all the archaebacterial stems for both 16 S and 23 S RNAs and constitutes a processing site. Kingdom-specific nucleotides have been identified that are associated with antibiotic binding sites at functional centres in 23 S-like RNAs: in the peptidyl transferase centre (erythromycin-domain V) the archaebacterial RNAs classify with the

  10. 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

  11. Elucidation of pathways of ribosomal RNA degradation: an essential role for RNase E.

    PubMed

    Sulthana, Shaheen; Basturea, Georgeta N; Deutscher, Murray P

    2016-08-01

    Although normally stable in growing cells, ribosomal RNAs are degraded under conditions of stress, such as starvation, and in response to misassembled or otherwise defective ribosomes in a process termed RNA quality control. Previously, our laboratory found that large fragments of 16S and 23S rRNA accumulate in strains lacking the processive exoribonucleases RNase II, RNase R, and PNPase, implicating these enzymes in the later steps of rRNA breakdown. Here, we define the pathways of rRNA degradation in the quality control process and during starvation, and show that the essential endoribonuclease, RNase E, is required to make the initial cleavages in both degradative processes. We also present evidence that explains why the exoribonuclease, RNase PH, is required to initiate the degradation of rRNA during starvation. The data presented here provide the first detailed description of rRNA degradation in bacterial cells. PMID:27298395

  12. 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.

  13. Nature of polymorphisms in 16S-23S rRNA gene intergenic transcribed spacer fingerprinting of Bacillus and related genera.

    PubMed

    Daffonchio, Daniele; Cherif, Ameur; Brusetti, Lorenzo; Rizzi, Aurora; Mora, Diego; Boudabous, Abdellatif; Borin, Sara

    2003-09-01

    The intergenic transcribed spacers (ITS) between the 16S and 23S rRNA genetic loci are frequently used in PCR fingerprinting to discriminate bacterial strains at the species and intraspecies levels. We investigated the molecular nature of polymorphisms in ITS-PCR fingerprinting of low-G+C-content spore-forming bacteria belonging to the genera Bacillus, Brevibacillus, Geobacillus, and Paenibacillus: We found that besides the polymorphisms in the homoduplex fragments amplified by PCR, heteroduplex products formed during PCR between amplicons from different ribosomal operons, with or without tRNA genes in the ITS, contribute to the interstrain variability in ITS-PCR fingerprinting patterns obtained in polyacrylamide-based gel matrices. The heteroduplex nature of the discriminating bands was demonstrated by fragment separation in denaturing polyacrylamide gels, by capillary electrophoresis, and by cloning, sequencing, and recombination of purified short and tRNA gene-containing long ITS. We also found that heteroduplex product formation is enhanced by increasing the number of PCR cycles. Homoduplex-heteroduplex polymorphisms (HHP) in a conserved region, such as the 16S and 23S rRNA gene ITS, allowed discrimination of closely related strains and species undistinguishable by other methods, indicating that ITS-HHP analysis is an easy and reproducible additional tool for strain typing.

  14. Nature of polymorphisms in 16S-23S rRNA gene intergenic transcribed spacer fingerprinting of Bacillus and related genera.

    PubMed

    Daffonchio, Daniele; Cherif, Ameur; Brusetti, Lorenzo; Rizzi, Aurora; Mora, Diego; Boudabous, Abdellatif; Borin, Sara

    2003-09-01

    The intergenic transcribed spacers (ITS) between the 16S and 23S rRNA genetic loci are frequently used in PCR fingerprinting to discriminate bacterial strains at the species and intraspecies levels. We investigated the molecular nature of polymorphisms in ITS-PCR fingerprinting of low-G+C-content spore-forming bacteria belonging to the genera Bacillus, Brevibacillus, Geobacillus, and Paenibacillus: We found that besides the polymorphisms in the homoduplex fragments amplified by PCR, heteroduplex products formed during PCR between amplicons from different ribosomal operons, with or without tRNA genes in the ITS, contribute to the interstrain variability in ITS-PCR fingerprinting patterns obtained in polyacrylamide-based gel matrices. The heteroduplex nature of the discriminating bands was demonstrated by fragment separation in denaturing polyacrylamide gels, by capillary electrophoresis, and by cloning, sequencing, and recombination of purified short and tRNA gene-containing long ITS. We also found that heteroduplex product formation is enhanced by increasing the number of PCR cycles. Homoduplex-heteroduplex polymorphisms (HHP) in a conserved region, such as the 16S and 23S rRNA gene ITS, allowed discrimination of closely related strains and species undistinguishable by other methods, indicating that ITS-HHP analysis is an easy and reproducible additional tool for strain typing. PMID:12957895

  15. 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. PMID:11921103

  16. 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.

  17. Dissociability of free and peptidyl-tRNA bound ribosomes.

    PubMed

    Surguchov, A P; Fominykch, E S; Lyzlova, L V

    1978-06-16

    The influence of peptidyl-tRNA on the dissociation of yeast 80 S ribosomes into subunits was studied. For this purpose temperature-sensitive (ts) suppressor strain of yeast Saccharomyces cervisiae carrying a defect in peptide chain termination was used. It was found that peptidyl-tRNA did not influence the dissociation of ribosomes either at high salt concentration or in the presence of dissociation factor (DF) from yeast. After dissociation of yeast ribosomes in 0.5 M KCl, peptidyl-tRNA remains bound to the 60 S subunit. Some characteristics of the termination process and release of nascent polypeptides from yeast ribosomes are discussed. PMID:355860

  18. Metabolic Labeling in the Study of Mammalian Ribosomal RNA Synthesis.

    PubMed

    Stefanovsky, Victor Y; Moss, Tom

    2016-01-01

    RNA metabolic labeling is a method of choice in the study of dynamic changes in the rate of gene transcription and RNA processing. It is particularly applicable to transcription of the ribosomal RNA genes and their processing products due to the very high levels of ribosomal RNA synthesis. Metabolic labeling can detect changes in ribosomal RNA transcription that occur within a few minutes as opposed to the still widely used RT-PCR or Northern blot procedures that measure RNA pool sizes and at best are able to detect changes occurring over several hours or several days. Here, we describe a metabolic labeling technique applicable to the measurement of ribosomal RNA synthesis and processing rates, as well as to the determination of RNA Polymerase I transcription elongation rates. PMID:27576716

  19. 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...

  20. 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

  1. New Site of Modification of 23S rRNA Associated with Clarithromycin Resistance of Helicobacter pylori Clinical Isolates

    PubMed Central

    Fontana, Carla; Favaro, Marco; Minelli, Silvia; Criscuolo, Anna Angela; Pietroiusti, Antonio; Galante, Alberto; Favalli, Cartesio

    2002-01-01

    Resistance of Helicobacter pylori to clarithromycin occurs with a prevalence ranging from 0 to 15%. This has an important clinical impact on dual and triple therapies, in which clarithromycin seems to be the better choice to achieve H. pylori eradication. In order to evaluate the possibility of new mechanisms of clarithromycin resistance, a PCR assay that amplified a portion of 23S rRNA from H. pylori isolates was used. Gastric tissue biopsy specimens from 230 consecutive patients were cultured for H. pylori isolation. Eighty-six gastric biopsy specimens yielded H. pylori-positive results, and among these 12 isolates were clarithromycin resistant. The latter were studied to detect mutations in the 23S rRNA gene. Sequence analysis of the 1,143-bp PCR product (portion of the 23S rRNA gene) did not reveal mutation such as that described at position 2142 to 2143. On the contrary, our findings show, for seven isolates, a T-to-C transition at position 2717. This mutation conferred a low level of resistance, equivalent to the MIC for the isolates, selected using the E-test as well as using the agar dilution method: 1 μg/ml. Moreover, T2717C transition is located in a highly conserved region of the 23S RNA associated with functional sites: domain VI. This fact has a strong effect on the secondary structure of the 23S RNA and on its interaction with macrolide. Mutation at position 2717 also generated an HhaI restriction site; therefore, restriction analysis of the PCR product also permits a rapid detection of resistant isolates. PMID:12435674

  2. [Intracellular transport of nuclear ribosomal RNA in Acetabularia mediterranea].

    PubMed

    Naumova, L P; Pressman, E K; Sandakhchiev, L S

    1976-01-01

    The ribosomal RNA transport from a nucleus to a perinuclear cytoplasm and its following distribution in the cytoplasm of Acetabularia mediterranea cells were studied using transplantation of RNA-labeled rhizoid into unlabeled stalk. In addition rifamycin treatment was used for inhibition of cytoplasmic RNA synthesis. Acetabularia nuclei contain the stable RNA fractions similar to those present in some other eukaryotes. Nuclear 25S and 17S ribosomal RNA rapidly enter the rhizoid cytoplasm whereas the following trasfer of them to other regions of the cell is a very slow process. Within two days only an insignificant part of 25S and 17S ribosomal RNA is transferred from the rhizoid to the stalk and is distributed there over the base-apical gradient. No preferential transfer of the nuclear ribosomal RNA to the apical region was observed.

  3. Absolute Quantification of Enterococcal 23S rRNA Gene Using Digital PCR.

    PubMed

    Wang, Dan; Yamahara, Kevan M; Cao, Yiping; Boehm, Alexandria B

    2016-04-01

    We evaluated the ability of chip-based digital PCR (dPCR) to quantify enterococci, the fecal indicator recommended by the United States Environmental Protection Agency (USEPA) for water-quality monitoring. dPCR uses Poisson statistics to estimate the number of DNA fragments in a sample with a specific sequence. Underestimation may occur when a gene is redundantly encoded in the genome and multiple copies of that gene are on one DNA fragment. When genomic DNA (gDNA) was extracted using two commercial DNA extraction kits, we confirmed that dPCR could discern individual copies of the redundant 23s rRNA gene in the enterococcal genome. dPCR quantification was accurate when compared to the nominal concentration inferred from fluorometer measurements (linear regression slope = 0.98, intercept = 0.03, R(2) = 0.99, and p value <0.0001). dPCR quantification was also consistent with quantitative PCR (qPCR) measurements as well as cell counts for BioBall reference standard and 24 environmental water samples. qPCR and dPCR quantification of enterococci in the 24 environmental samples were significantly correlated (linear regression slope =1.08, R(2) of 0.96, and p value <0.0001); the group mean of the qPCR measurements was 0.19 log units higher than that of the dPCR measurements. At environmentally relevant concentrations, dPCR quantification was more precise (i.e., had narrower 95% confidence intervals than qPCR quantification). We observed that humic acid caused a similar level of inhibition in both dPCR and qPCR, but calcium inhibited dPCR to a lesser degree than qPCR. Inhibition of dPCR was partially relieved when the number of thermal cycles was increased. Based on these results, we conclude that dPCR is a viable option for enumerating enterococci in ambient water. PMID:26903207

  4. 16S-23S rRNA Gene Intergenic Spacer Region Variability Helps Resolve Closely Related Sphingomonads.

    PubMed

    Tokajian, Sima; Issa, Nahla; Salloum, Tamara; Ibrahim, Joe; Farah, Maya

    2016-01-01

    Sphingomonads comprise a physiologically versatile group many of which appear to be adapted to oligotrophic environments, but several also had features in their genomes indicative of host associations. In this study, the extent variability of the 16S-23S rDNA intergenic spacer (ITS) sequences of 14 ATCC reference sphingomonad strains and 23 isolates recovered from drinking water was investigated through PCR amplification and sequencing. Sequencing analysis of the 16S-23S rRNA gene ITS region revealed that the ITS sizes for all studied isolates varied between 415 and 849 bp, while their G+C content was 42.2-57.9 mol%. Five distinct ITS types were identified: ITS(none) (without tRNA genes), ITS(Ala(TGC)), ITS(Ala(TGC)+Ile(GAT)), ITS(Ile(GAT)+Ala(TGC)), and ITS (Ile(GAT)+Pseudo). All of the identified tRNA(Ala(TGC)) molecules consisted of 73 bases, and all of the tRNA(Ile(GAT)) molecules consisted of 74 bases. We also detected striking variability in the size of the ITS region among the various examined isolates. Highest variability was detected within the ITS-2. The importance of this study is that this is the first comparison of the 16S-23S rDNA ITS sequence similarities and tRNA genes from sphingomonads. Collectively the data obtained in this study revealed the heterogeneity and extent of variability within the ITS region compared to the 16S rRNA gene within closely related isolates. Sequence and length polymorphisms within the ITS region along with the ITS types (tRNA-containing or lacking and the type of tRNA) and ITS-2 size and sequence similarities allowed us to overcome the limitation we previously encountered in resolving closely related isolates based on the 16S rRNA gene sequence.

  5. 16S-23S rRNA Gene Intergenic Spacer Region Variability Helps Resolve Closely Related Sphingomonads.

    PubMed

    Tokajian, Sima; Issa, Nahla; Salloum, Tamara; Ibrahim, Joe; Farah, Maya

    2016-01-01

    Sphingomonads comprise a physiologically versatile group many of which appear to be adapted to oligotrophic environments, but several also had features in their genomes indicative of host associations. In this study, the extent variability of the 16S-23S rDNA intergenic spacer (ITS) sequences of 14 ATCC reference sphingomonad strains and 23 isolates recovered from drinking water was investigated through PCR amplification and sequencing. Sequencing analysis of the 16S-23S rRNA gene ITS region revealed that the ITS sizes for all studied isolates varied between 415 and 849 bp, while their G+C content was 42.2-57.9 mol%. Five distinct ITS types were identified: ITS(none) (without tRNA genes), ITS(Ala(TGC)), ITS(Ala(TGC)+Ile(GAT)), ITS(Ile(GAT)+Ala(TGC)), and ITS (Ile(GAT)+Pseudo). All of the identified tRNA(Ala(TGC)) molecules consisted of 73 bases, and all of the tRNA(Ile(GAT)) molecules consisted of 74 bases. We also detected striking variability in the size of the ITS region among the various examined isolates. Highest variability was detected within the ITS-2. The importance of this study is that this is the first comparison of the 16S-23S rDNA ITS sequence similarities and tRNA genes from sphingomonads. Collectively the data obtained in this study revealed the heterogeneity and extent of variability within the ITS region compared to the 16S rRNA gene within closely related isolates. Sequence and length polymorphisms within the ITS region along with the ITS types (tRNA-containing or lacking and the type of tRNA) and ITS-2 size and sequence similarities allowed us to overcome the limitation we previously encountered in resolving closely related isolates based on the 16S rRNA gene sequence. PMID:26904019

  6. 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.

  7. Adenosylcobalamin inhibits ribosome binding to btuB RNA.

    PubMed

    Nou, X; Kadner, R J

    2000-06-20

    Expression of the btuB gene encoding the outer membrane cobalamin transporter in Escherichia coli is strongly reduced on growth with cobalamins. Previous studies have shown that this regulation occurs in response to adenosylcobalamin (Ado-Cbl) and operates primarily at the translational level. Changes in the level and stability of btuB RNA are consequences of the modulated translation initiation. To examine how Ado-Cbl affects translation, the binding of E. coli 30S ribosomal subunits to btuB RNA was investigated by using a primer extension inhibition assay. Ribosome binding to btuB RNA was much less efficient than to other RNAs and was preferentially lost when the ribosomes were subjected to a high-salt wash. Ribosome binding to btuB RNA was inhibited by Ado-Cbl but not by cyanocobalamin, with half-maximal inhibition around 0.3 microM Ado-Cbl. Ribosome-binding activity was increased or decreased by mutations in the btuB leader region, which affected two predicted RNA hairpins and altered expression of btuB-lacZ reporters. Finally, the presence of Ado-Cbl elicited formation of a single primer extension-inhibition product with the same specificity and Cbl-concentration dependence as the inhibition of ribosome binding. These results indicate that btuB expression is controlled by the specific binding of Ado-Cbl to btuB RNA, which then affects access to its ribosome-binding sequence. PMID:10852957

  8. Adenosylcobalamin inhibits ribosome binding to btuB RNA

    PubMed Central

    Nou, Xiangwu; Kadner, Robert J.

    2000-01-01

    Expression of the btuB gene encoding the outer membrane cobalamin transporter in Escherichia coli is strongly reduced on growth with cobalamins. Previous studies have shown that this regulation occurs in response to adenosylcobalamin (Ado-Cbl) and operates primarily at the translational level. Changes in the level and stability of btuB RNA are consequences of the modulated translation initiation. To examine how Ado-Cbl affects translation, the binding of E. coli 30S ribosomal subunits to btuB RNA was investigated by using a primer extension inhibition assay. Ribosome binding to btuB RNA was much less efficient than to other RNAs and was preferentially lost when the ribosomes were subjected to a high-salt wash. Ribosome binding to btuB RNA was inhibited by Ado-Cbl but not by cyanocobalamin, with half-maximal inhibition around 0.3 μM Ado-Cbl. Ribosome-binding activity was increased or decreased by mutations in the btuB leader region, which affected two predicted RNA hairpins and altered expression of btuB-lacZ reporters. Finally, the presence of Ado-Cbl elicited formation of a single primer extension-inhibition product with the same specificity and Cbl-concentration dependence as the inhibition of ribosome binding. These results indicate that btuB expression is controlled by the specific binding of Ado-Cbl to btuB RNA, which then affects access to its ribosome-binding sequence. PMID:10852957

  9. 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

  10. Motion of individual ribosomes along mRNA

    NASA Astrophysics Data System (ADS)

    Visscher, Koen

    2004-11-01

    Ribosomes move along messenger RNA to translate a sequence of ribonucleotides into a corresponding sequence of amino acids that make up a protein. Efficient motion of ribosomes along the mRNA requires hydrolysis of GTP, converting chemical energy into mechanical work, like better known molecular motors such as kinesin. However, motion is just one of the many tasks of the ribosome, whereas for kinesin, motion itself is the main goal. In keeping with these functional differences, the ribosome is also much larger consisting of more than 50 proteins and with half of its mass made up of ribosomal RNA. Such structural complexity enables indirect ways of coupling GTP hydrolysis to directed motion. In order to elucidate the mechanochemical coupling in ribosomes we have developed a single-molecule assay based on using optical tweezers to record the motion of individual ribosomes along mRNA. Translation rates of 2-4 codons/s have been observed. However, when increasing the force opposing motion, we observe backward slippage of ribosomes along homopolymeric poly(U) messages. Currently, it is not clear if the motor operates in reverse or if backward motion has become completely uncoupled from GTP hydrolysis. Interestingly, force-induced backward motion is of biological relevance because of its possible role in -1 frameshifting, a mechanism used by viruses to regulate gene expression at the level of translation.

  11. 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.

  12. 16S–23S rRNA Gene Intergenic Spacer Region Variability Helps Resolve Closely Related Sphingomonads

    PubMed Central

    Tokajian, Sima; Issa, Nahla; Salloum, Tamara; Ibrahim, Joe; Farah, Maya

    2016-01-01

    Sphingomonads comprise a physiologically versatile group many of which appear to be adapted to oligotrophic environments, but several also had features in their genomes indicative of host associations. In this study, the extent variability of the 16S–23S rDNA intergenic spacer (ITS) sequences of 14 ATCC reference sphingomonad strains and 23 isolates recovered from drinking water was investigated through PCR amplification and sequencing. Sequencing analysis of the 16S–23S rRNA gene ITS region revealed that the ITS sizes for all studied isolates varied between 415 and 849 bp, while their G+C content was 42.2–57.9 mol%. Five distinct ITS types were identified: ITSnone (without tRNA genes), ITSAla(TGC), ITSAla(TGC)+Ile(GAT), ITSIle(GAT)+Ala(TGC), and ITS Ile(GAT)+Pseudo. All of the identified tRNAAla(TGC) molecules consisted of 73 bases, and all of the tRNAIle(GAT) molecules consisted of 74 bases. We also detected striking variability in the size of the ITS region among the various examined isolates. Highest variability was detected within the ITS-2. The importance of this study is that this is the first comparison of the 16S–23S rDNA ITS sequence similarities and tRNA genes from sphingomonads. Collectively the data obtained in this study revealed the heterogeneity and extent of variability within the ITS region compared to the 16S rRNA gene within closely related isolates. Sequence and length polymorphisms within the ITS region along with the ITS types (tRNA-containing or lacking and the type of tRNA) and ITS-2 size and sequence similarities allowed us to overcome the limitation we previously encountered in resolving closely related isolates based on the 16S rRNA gene sequence. PMID:26904019

  13. Two Cases of Mycoplasma pneumoniae Pneumonia with A2063G Mutation in the 23S rRNA Gene in Siblings

    PubMed Central

    Hong, Joo Hee; Chun, Jin Kyong; Oh, Ki Jin; Kim, Juwon; Yoon, Kap Jun

    2013-01-01

    We describe 2 cases of pneumonia caused by the same macrolide-resistant Mycoplasma pneumoniae in siblings. M. pneumoniae was identified using real-time PCR. Direct sequence analysis of the 23S rRNA gene revealed a point mutation in V domain (A2063G) of the 23S rRNA gene. PMID:23301225

  14. Phylogenetic relationships within the family Halomonadaceae based on comparative 23S and 16S rRNA gene sequence analysis.

    PubMed

    de la Haba, Rafael R; Arahal, David R; Márquez, M Carmen; Ventosa, Antonio

    2010-04-01

    A phylogenetic study of the family Halomonadaceae was carried out based on complete 16S rRNA and 23S rRNA gene sequences. Several 16S rRNA genes of type strains were resequenced, and 28 new sequences of the 23S rRNA gene were obtained. Currently, the family includes nine genera (Carnimonas, Chromohalobacter, Cobetia, Halomonas, Halotalea, Kushneria, Modicisalibacter, Salinicola and Zymobacter). These genera are phylogenetically coherent except Halomonas, which is polyphyletic. This genus comprises two clearly distinguished clusters: group 1 includes Halomonas elongata (the type species) and the species Halomonas eurihalina, H. caseinilytica, H. halmophila, H. sabkhae, H. almeriensis, H. halophila, H. salina, H. organivorans, H. koreensis, H. maura and H. nitroreducens. Group 2 comprises the species Halomonas aquamarina, H. meridiana, H. axialensis, H. magadiensis, H. hydrothermalis, H. alkaliphila, H. venusta, H. boliviensis, H. neptunia, H. variabilis, H. sulfidaeris, H. subterranea, H. janggokensis, H. gomseomensis, H. arcis and H. subglaciescola. Halomonas salaria forms a cluster with Chromohalobacter salarius and the recently described genus Salinicola, and their taxonomic affiliation requires further study. More than 20 Halomonas species are phylogenetically not within the core constituted by the Halomonas sensu stricto cluster (group 1) or group 2 and, since their positions on the different phylogenetic trees are not stable, they cannot be recognized as additional groups either. In general, there is excellent agreement between the phylogenies based on the two rRNA gene sequences, but the 23S rRNA gene showed higher resolution in the differentiation of species of the family Halomonadaceae.

  15. 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

  16. Crystal structure of the RNA binding ribosomal protein L1 from Thermus thermophilus.

    PubMed Central

    Nikonov, S; Nevskaya, N; Eliseikina, I; Fomenkova, N; Nikulin, A; Ossina, N; Garber, M; Jonsson, B H; Briand, C; Al-Karadaghi, S; Svensson, A; Aevarsson, A; Liljas, A

    1996-01-01

    L1 has a dual function as a ribosomal protein binding rRNA and as a translational repressor binding mRNA. The crystal structure of L1 from Thermus thermophilus has been determined at 1.85 angstroms resolution. The protein is composed of two domains with the N- and C-termini in domain I. The eight N-terminal residues are very flexible, as the quality of electron density map shows. Proteolysis experiments have shown that the N-terminal tail is accessible and important for 23S rRNA binding. Most of the conserved amino acids are situated at the interface between the two domains. They probably form the specific RNA binding site of L1. Limited non-covalent contacts between the domains indicate an unstable domain interaction in the present conformation. Domain flexibility and RNA binding by induced fit seems plausible. Images PMID:8635468

  17. 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

  18. Selecting rRNA binding sites for the ribosomal proteins L4 and L6 from randomly fragmented rRNA: application of a method called SERF.

    PubMed

    Stelzl, U; Spahn, C M; Nierhaus, K H

    2000-04-25

    Two-thirds of the 54 proteins of the Escherichia coli ribosome interact directly with the rRNAs, but the rRNA binding sites of only a very few proteins are known. We present a method (selection of random RNA fragments; SERF) that can identify the minimal binding region for proteins within ribonucleo-protein complexes such as the ribosome. The power of the method is exemplified with the ribosomal proteins L4 and L6. Binding sequences are identified for both proteins and characterized by phosphorothioate footprinting. Surprisingly, the binding region of L4, a 53-nt rRNA fragment of domain I of 23S rRNA, can simultaneously and independently bind L24, one of the two assembly initiator proteins of the large subunit.

  19. Case of Localized Recombination in 23S rRNA Genes from Divergent Bradyrhizobium Lineages Associated with Neotropical Legumes

    PubMed Central

    Parker, Matthew A.

    2001-01-01

    Enzyme electrophoresis and rRNA sequencing were used to analyze relationships of Bradyrhizobium sp. nodule bacteria from four papilionoid legumes (Clitoria javitensis, Erythrina costaricensis, Rhynchosia pyramidalis, and Desmodium axillare) growing on Barro Colorado Island (BCI), Panama. Bacteria with identical multilocus allele profiles were commonly found in association with two or more legume genera. Among the 16 multilocus genotypes (electrophoretic types [ETs]) detected, six ETs formed a closely related cluster that included isolates from all four legume taxa. Bacteria from two other BCI legumes (Platypodium and Machaerium) sampled in a previous study were also identical to certain ETs in this group. Isolates from different legume genera that had the same ET had identical nucleotide sequences for both a 5′ portion of the 23S rRNA and the nearly full-length 16S rRNA genes. These results suggest that Bradyrhizobium genotypes with low host specificity may be prevalent in this tropical forest. Parsimony analysis of 16S rRNA sequence variation indicated that most isolates were related to Bradyrhizobium japonicum USDA 110, although one ET sampled from C. javitensis had a 16S rRNA gene highly similar to that of Bradyrhizobium elkanii USDA 76. However, this isolate displayed a mosaic structure within the 5′ 23S rRNA region: one 84-bp segment was identical to that of BCI isolate Pe1-3 (a close relative of B. japonicum USDA 110, based on 16S rRNA data), while an adjacent 288-bp segment matched that of B. elkanii USDA 76. This mosaic structure is one of the first observations suggesting recombination in nature between Bradyrhizobium isolates related to B. japonicum versus B. elkanii. PMID:11319084

  20. Case of localized recombination in 23S rRNA genes from divergent bradyrhizobium lineages associated with neotropical legumes.

    PubMed

    Parker, M A

    2001-05-01

    Enzyme electrophoresis and rRNA sequencing were used to analyze relationships of Bradyrhizobium sp. nodule bacteria from four papilionoid legumes (Clitoria javitensis, Erythrina costaricensis, Rhynchosia pyramidalis, and Desmodium axillare) growing on Barro Colorado Island (BCI), Panama. Bacteria with identical multilocus allele profiles were commonly found in association with two or more legume genera. Among the 16 multilocus genotypes (electrophoretic types [ETs]) detected, six ETs formed a closely related cluster that included isolates from all four legume taxa. Bacteria from two other BCI legumes (Platypodium and Machaerium) sampled in a previous study were also identical to certain ETs in this group. Isolates from different legume genera that had the same ET had identical nucleotide sequences for both a 5' portion of the 23S rRNA and the nearly full-length 16S rRNA genes. These results suggest that Bradyrhizobium genotypes with low host specificity may be prevalent in this tropical forest. Parsimony analysis of 16S rRNA sequence variation indicated that most isolates were related to Bradyrhizobium japonicum USDA 110, although one ET sampled from C. javitensis had a 16S rRNA gene highly similar to that of Bradyrhizobium elkanii USDA 76. However, this isolate displayed a mosaic structure within the 5' 23S rRNA region: one 84-bp segment was identical to that of BCI isolate Pe1-3 (a close relative of B. japonicum USDA 110, based on 16S rRNA data), while an adjacent 288-bp segment matched that of B. elkanii USDA 76. This mosaic structure is one of the first observations suggesting recombination in nature between Bradyrhizobium isolates related to B. japonicum versus B. elkanii.

  1. Hierarchical RNA Processing Is Required for Mitochondrial Ribosome Assembly.

    PubMed

    Rackham, Oliver; Busch, Jakob D; Matic, Stanka; Siira, Stefan J; Kuznetsova, Irina; Atanassov, Ilian; Ermer, Judith A; Shearwood, Anne-Marie J; Richman, Tara R; Stewart, James B; Mourier, Arnaud; Milenkovic, Dusanka; Larsson, Nils-Göran; Filipovska, Aleksandra

    2016-08-16

    The regulation of mitochondrial RNA processing and its importance for ribosome biogenesis and energy metabolism are not clear. We generated conditional knockout mice of the endoribonuclease component of the RNase P complex, MRPP3, and report that it is essential for life and that heart and skeletal-muscle-specific knockout leads to severe cardiomyopathy, indicating that its activity is non-redundant. Transcriptome-wide parallel analyses of RNA ends (PARE) and RNA-seq enabled us to identify that in vivo 5' tRNA cleavage precedes 3' tRNA processing, and this is required for the correct biogenesis of the mitochondrial ribosomal subunits. We identify that mitoribosomal biogenesis proceeds co-transcriptionally because large mitoribosomal proteins can form a subcomplex on an unprocessed RNA containing the 16S rRNA. Taken together, our data show that RNA processing links transcription to translation via assembly of the mitoribosome. PMID:27498866

  2. In situ probing of gram-positive bacteria with high DNA G + C content using 23S rRNA-targeted oligonucleotides.

    PubMed

    Roller, C; Wagner, M; Amann, R; Ludwig, W; Schleifer, K H

    1994-10-01

    23S-rRNA-targeted oligonucleotide probes were designed for the phylogenetic group 'Gram-positive bacteria with high G + C content of DNA' (GPBHGC). A sequence idiosyncrasy in two adjacent base pairs in the stem of helix 69 in domain IV of the 23S rRNA is present in all hitherto analysed strains of GPBHGC. An oligonucleotide probe targeted to this region hybridized only with strains of GPBHGC and was successfully used for in situ monitoring of these cells in activated sludge. Another unique feature of the 23S rRNA molecules of GPBHGC is a large insertion in domain III. Fluorescent oligonucleotides targeted to the highly variable regions of the rRNA within the insertions of Corynebacterium glutamicum DSM 20300, Aureobacterium testaceum DSM 20166 and Brevibacterium sp. DSM 20165 hybridized specifically to their target strains, whereas probing with oligonucleotides complementary to the rRNA-coding strand of the 23S rDNA and to the spacer between 16S and 23S rRNA of C. glutamicum did not result in detectable fluorescence. This confirmed that the large 23S insertions are indeed present in 23S rRNAs of GPBHGC and provide potential target sites for highly specific nucleic acid probes.

  3. 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.

  4. 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.

  5. The binding sites for tRNA on eukaryotic ribosomes.

    PubMed

    Leader, D P; Machray, G C

    1975-07-01

    We have studied the non-enzymic binding of phe-tRNA to ribosomes from rat liver using deacylated tRNA to inhibit binding to the P-site and puromycin (5 x 10-minus3M) to inhibit binding to the A-site. We conclude that at a low concentration of magnesium ions (10mM) phe-tRNA is bound only at the A-site of 80S irbosomes, whereas at a high concentration of magnesium ions (40mM) phe-tRNA is also bound at the P-site. Studies with edeine indicate that, during non-enzymic binding of phe-tRNA, eukaryotic ribosomes (in contrast to prokarotic ribosomes) have the A-site of the 60S subunit and the initiation site of the 40S subunit juxtaposed. This may account for the differences observed, in formation of diphenylalanyl-tRNA and phenylalanyl-puromycin, between phe-tRNA bound non-enzymically to the P-sites of eukaryotic and prokaryotic ribosomes.

  6. The binding sites for tRNA on eukaryotic ribosomes.

    PubMed Central

    Leader, D P; Machray, G C

    1975-01-01

    We have studied the non-enzymic binding of phe-tRNA to ribosomes from rat liver using deacylated tRNA to inhibit binding to the P-site and puromycin (5 x 10-minus3M) to inhibit binding to the A-site. We conclude that at a low concentration of magnesium ions (10mM) phe-tRNA is bound only at the A-site of 80S irbosomes, whereas at a high concentration of magnesium ions (40mM) phe-tRNA is also bound at the P-site. Studies with edeine indicate that, during non-enzymic binding of phe-tRNA, eukaryotic ribosomes (in contrast to prokarotic ribosomes) have the A-site of the 60S subunit and the initiation site of the 40S subunit juxtaposed. This may account for the differences observed, in formation of diphenylalanyl-tRNA and phenylalanyl-puromycin, between phe-tRNA bound non-enzymically to the P-sites of eukaryotic and prokaryotic ribosomes. PMID:1098024

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

    PubMed Central

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

    1980-01-01

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

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

    PubMed

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

    1980-12-01

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

  9. Crystal structure of prokaryotic ribosomal protein L9: a bi-lobed RNA-binding protein.

    PubMed Central

    Hoffman, D W; Davies, C; Gerchman, S E; Kycia, J H; Porter, S J; White, S W; Ramakrishnan, V

    1994-01-01

    The crystal structure of protein L9 from the Bacillus stearothermophilus ribosome has been determined at 2.8 A resolution using X-ray diffraction methods. This primary RNA-binding protein has a highly elongated and unusual structure consisting of two separated domains joined by a long exposed alpha-helix. Conserved, positively charged and aromatic amino acids on the surfaces of both domains probably represent the sites of specific interactions with 23S rRNA. Comparisons with other prokaryotic L9 sequences show that while the length of the connecting alpha-helix is invariant, the sequence within the exposed central region is not conserved. This suggests that the alpha-helix has an architectural role and serves to fix the relative separation and orientation of the N- and C-terminal domains within the ribosome. The N-terminal domain has structural homology to the smaller ribosomal proteins L7/L12 and L30, and the eukaryotic RNA recognition motif (RRM). Images PMID:8306963

  10. Ribosomal RNA sequence suggests microsporidia are extremely ancient eukaryotes.

    PubMed

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

    The microsporidia are a group of unusual, obligately parasitic protists that infect a great variety of other eukaryotes, including vertebrates, arthropods, molluscs, annelids, nematodes, cnidaria and even various ciliates, myxosporidia and gregarines. They possess a number of unusual cytological and molecular characteristics. Their nuclear division is considered to be primitive, they have no mitochondria, their ribosomes and ribosomal RNAs are reported to be of prokaryotic size and their large ribosomal subunit contains no 5.8S rRNA. The uniqueness of the microsporidia may reflect their phylogenetic position, because comparative sequence analysis shows that the small subunit rRNA of the microsporidium Vairimorpha necatrix is more unlike those of other eukaryotes than any known eukaryote 18S rRNA sequence. We conclude that the lineage leading to microsporidia branched very early from that leading to other eukaryotes.

  11. Structural Insights into tRNA Dynamics on the Ribosome

    PubMed Central

    Agirrezabala, Xabier; Valle, Mikel

    2015-01-01

    High-resolution structures at different stages, as well as biochemical, single molecule and computational approaches have highlighted the elasticity of tRNA molecules when bound to the ribosome. It is well acknowledged that the inherent structural flexibility of the tRNA lies at the heart of the protein synthesis process. Here, we review the recent advances and describe considerations that the conformational changes of the tRNA molecules offer about the mechanisms grounded in translation. PMID:25941930

  12. The HIV Tat protein affects processing of ribosomal RNA precursor

    PubMed Central

    Ponti, Donatella; Troiano, Maria; Bellenchi, Gian Carlo; Battaglia, Piero A; Gigliani, Franca

    2008-01-01

    Background Inside the cell, the HIV Tat protein is mainly found in the nucleus and nucleolus. The nucleolus, the site of ribosome biogenesis, is a highly organized, non-membrane-bound sub-compartment where proteins with a high affinity for nucleolar components are found. While it is well known that Tat accumulates in the nucleolus via a specific nucleolar targeting sequence, its function in this compartment it still unknown. Results To clarify the significance of the Tat nucleolar localization, we induced the expression of the protein during oogenesis in Drosophila melanogaster strain transgenic for HIV-tat gene. Here we show that Tat localizes in the nucleoli of Drosophila oocyte nurse cells, where it specifically co-localizes with fibrillarin. Tat expression is accompanied by a significant decrease of cytoplasmic ribosomes, which is apparently related to an impairment of ribosomal rRNA precursor processing. Such an event is accounted for by the interaction of Tat with fibrillarin and U3 snoRNA, which are both required for pre-rRNA maturation. Conclusion Our data contribute to understanding the function of Tat in the nucleolus, where ribosomal RNA synthesis and cell cycle control take place. The impairment of nucleolar pre-rRNA maturation through the interaction of Tat with fibrillarin-U3snoRNA complex suggests a process by which the virus modulates host response, thus contributing to apoptosis and protein shut-off in HIV-uninfected cells. PMID:18559082

  13. Molecular architecture of the ribosome-bound Hepatitis C Virus internal ribosomal entry site RNA.

    PubMed

    Yamamoto, Hiroshi; Collier, Marianne; Loerke, Justus; Ismer, Jochen; Schmidt, Andrea; Hilal, Tarek; Sprink, Thiemo; Yamamoto, Kaori; Mielke, Thorsten; Bürger, Jörg; Shaikh, Tanvir R; Dabrowski, Marylena; Hildebrand, Peter W; Scheerer, Patrick; Spahn, Christian M T

    2015-12-14

    Internal ribosomal entry sites (IRESs) are structured cis-acting RNAs that drive an alternative, cap-independent translation initiation pathway. They are used by many viruses to hijack the translational machinery of the host cell. IRESs facilitate translation initiation by recruiting and actively manipulating the eukaryotic ribosome using only a subset of canonical initiation factor and IRES transacting factors. Here we present cryo-EM reconstructions of the ribosome 80S- and 40S-bound Hepatitis C Virus (HCV) IRES. The presence of four subpopulations for the 80S•HCV IRES complex reveals dynamic conformational modes of the complex. At a global resolution of 3.9 Å for the most stable complex, a derived atomic model reveals a complex fold of the IRES RNA and molecular details of its interaction with the ribosome. The comparison of obtained structures explains how a modular architecture facilitates mRNA loading and tRNA binding to the P-site. This information provides the structural foundation for understanding the mechanism of HCV IRES RNA-driven translation initiation. PMID:26604301

  14. Structure of psoralen-crosslinked ribosomal RNA from Drosophila melanogaster.

    PubMed Central

    Wollenzien, P L; Youvan, D C; Hearst, J E

    1978-01-01

    Ribosomal RNA from Drosophila melanogaster photoreacted with hydroxymethyltrioxsalen has been examined by electron microscopy. Reproducible patterns of hairpins were found in both the 26S and 18S RNA. The frequency of these hairpins and the amount of incorporated drug were dependent upon the conditions under which the crosslinking was performed. A prominent central hairpin occurs in the 26S RNA and the break that interrupts the continuity of the RNA chain is located within it. In addition to several small hairpins, the crosslinked 18S RNA contains a large open loop. Images PMID:417342

  15. Analysis of 16S-23S rRNA Intergenic Spacer Regions of Vibrio cholerae and Vibrio mimicus

    PubMed Central

    Chun, Jongsik; Huq, Anwarul; Colwell, Rita R.

    1999-01-01

    Vibrio cholerae identification based on molecular sequence data has been hampered by a lack of sequence variation from the closely related Vibrio mimicus. The two species share many genes coding for proteins, such as ctxAB, and show almost identical 16S DNA coding for rRNA (rDNA) sequences. Primers targeting conserved sequences flanking the 3′ end of the 16S and the 5′ end of the 23S rDNAs were used to amplify the 16S-23S rRNA intergenic spacer regions of V. cholerae and V. mimicus. Two major (ca. 580 and 500 bp) and one minor (ca. 750 bp) amplicons were consistently generated for both species, and their sequences were determined. The largest fragment contains three tRNA genes (tDNAs) coding for tRNAGlu, tRNALys, and tRNAVal, which has not previously been found in bacteria examined to date. The 580-bp amplicon contained tDNAIle and tDNAAla, whereas the 500-bp fragment had single tDNA coding either tRNAGlu or tRNAAla. Little variation, i.e., 0 to 0.4%, was found among V. cholerae O1 classical, O1 El Tor, and O139 epidemic strains. Slightly more variation was found against the non-O1/non-O139 serotypes (ca. 1% difference) and V. mimicus (2 to 3% difference). A pair of oligonucleotide primers were designed, based on the region differentiating all of V. cholerae strains from V. mimicus. The PCR system developed was subsequently evaluated by using representatives of V. cholerae from environmental and clinical sources, and of other taxa, including V. mimicus. This study provides the first molecular tool for identifying the species V. cholerae. PMID:10224020

  16. Discordant 16S and 23S rRNA gene phylogenies for the genus Helicobacter: implications for phylogenetic inference and systematics.

    PubMed

    Dewhirst, Floyd E; Shen, Zeli; Scimeca, Michael S; Stokes, Lauren N; Boumenna, Tahani; Chen, Tsute; Paster, Bruce J; Fox, James G

    2005-09-01

    Analysis of 16S rRNA gene sequences has become the primary method for determining prokaryotic phylogeny. Phylogeny is currently the basis for prokaryotic systematics. Therefore, the validity of 16S rRNA gene-based phylogenetic analyses is of fundamental importance for prokaryotic systematics. Discrepancies between 16S rRNA gene analyses and DNA-DNA hybridization and phenotypic analyses have been noted in the genus Helicobacter. To clarify these discrepancies, we sequenced the 23S rRNA genes for 55 helicobacter strains representing 41 taxa (>2,700 bases per sequence). Phylogenetic-tree construction using neighbor-joining, parsimony, and maximum likelihood methods for 23S rRNA gene sequence data yielded stable trees which were consistent with other phenotypic and genotypic methods. The 16S rRNA gene sequence-derived trees were discordant with the 23S rRNA gene trees and other data. Discrepant 16S rRNA gene sequence data for the helicobacters are consistent with the horizontal transfer of 16S rRNA gene fragments and the creation of mosaic molecules with loss of phylogenetic information. These results suggest that taxonomic decisions must be supported by other phylogenetically informative macromolecules, such as the 23S rRNA gene, when 16S rRNA gene-derived phylogeny is discordant with other credible phenotypic and genotypic methods. This study found Wolinella succinogenes to branch with the unsheathed-flagellum cluster of helicobacters by 23S rRNA gene analyses and whole-genome comparisons. This study also found intervening sequences (IVSs) in the 23S rRNA genes of strains of 12 Helicobacter species. IVSs were found in helices 10, 25, and 45, as well as between helices 31' and 27'. Simultaneous insertion of IVSs at three sites was found in H. mesocricetorum. PMID:16109952

  17. Genotypic Characterization of Bradyrhizobium Strains Nodulating Small Senegalese Legumes by 16S-23S rRNA Intergenic Gene Spacers and Amplified Fragment Length Polymorphism Fingerprint Analyses

    PubMed Central

    Doignon-Bourcier, Florence; Willems, Anne; Coopman, Renata; Laguerre, Gisele; Gillis, Monique; de Lajudie, Philippe

    2000-01-01

    We examined the genotypic diversity of 64 Bradyrhizobium strains isolated from nodules from 27 native leguminous plant species in Senegal (West Africa) belonging to the genera Abrus, Alysicarpus, Bryaspis, Chamaecrista, Cassia, Crotalaria, Desmodium, Eriosema, Indigofera, Moghania, Rhynchosia, Sesbania, Tephrosia, and Zornia, which play an ecological role and have agronomic potential in arid regions. The strains were characterized by intergenic spacer (between 16S and 23S rRNA genes) PCR and restriction fragment length polymorphism (IGS PCR-RFLP) and amplified fragment length polymorphism (AFLP) fingerprinting analyses. Fifty-three reference strains of the different Bradyrhizobium species and described groups were included for comparison. The strains were diverse and formed 27 groups by AFLP and 16 groups by IGS PCR-RFLP. The sizes of the IGS PCR products from the Bradyrhizobium strains that were studied varied from 780 to 1,038 bp and were correlated with the IGS PCR-RFLP results. The grouping of strains was consistent by the three methods AFLP, IGS PCR-RFLP, and previously reported 16S amplified ribosomal DNA restriction analysis. For investigating the whole genome, AFLP was the most discriminative technique, thus being of particular interest for future taxonomic studies in Bradyrhizobium, for which DNA is difficult to obtain in quantity and quality to perform extensive DNA:DNA hybridizations. PMID:10966419

  18. Genotypic characterization of Bradyrhizobium strains nodulating small Senegalese legumes by 16S-23S rRNA intergenic gene spacers and amplified fragment length polymorphism fingerprint analyses.

    PubMed

    Doignon-Bourcier, F; Willems, A; Coopman, R; Laguerre, G; Gillis, M; de Lajudie, P

    2000-09-01

    We examined the genotypic diversity of 64 Bradyrhizobium strains isolated from nodules from 27 native leguminous plant species in Senegal (West Africa) belonging to the genera Abrus, Alysicarpus, Bryaspis, Chamaecrista, Cassia, Crotalaria, Desmodium, Eriosema, Indigofera, Moghania, Rhynchosia, Sesbania, Tephrosia, and Zornia, which play an ecological role and have agronomic potential in arid regions. The strains were characterized by intergenic spacer (between 16S and 23S rRNA genes) PCR and restriction fragment length polymorphism (IGS PCR-RFLP) and amplified fragment length polymorphism (AFLP) fingerprinting analyses. Fifty-three reference strains of the different Bradyrhizobium species and described groups were included for comparison. The strains were diverse and formed 27 groups by AFLP and 16 groups by IGS PCR-RFLP. The sizes of the IGS PCR products from the Bradyrhizobium strains that were studied varied from 780 to 1,038 bp and were correlated with the IGS PCR-RFLP results. The grouping of strains was consistent by the three methods AFLP, IGS PCR-RFLP, and previously reported 16S amplified ribosomal DNA restriction analysis. For investigating the whole genome, AFLP was the most discriminative technique, thus being of particular interest for future taxonomic studies in Bradyrhizobium, for which DNA is difficult to obtain in quantity and quality to perform extensive DNA:DNA hybridizations.

  19. 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.

  20. Reverse Translocation of tRNA in the Ribosome

    PubMed Central

    Shoji, Shinichiro; Walker, Sarah E.; Fredrick, Kurt

    2009-01-01

    Summary A widely held view is that directional movement of tRNA in the ribosome is determined by an intrinsic mechanism and driven thermodynamically by transpeptidation. Here, we show that, in certain ribosomal complexes, the pretranslocation (PRE) state is thermodynamically favored over the posttranslocation (POST) state. Spontaneous and efficient conversion from the POST to PRE state is observed when EF-G is depleted from ribosomes in the POST state or when tRNA is added to the E site of ribosomes containing P-site tRNA. In the latter assay, the rate of tRNA movement is increased by streptomycin and neomycin, decreased by tetracycline, and not affected by the acylation state of the tRNA. In one case, we provide evidence that complex conversion occurs by reverse translocation (i.e., direct movement of the tRNAs from the E and P sites to the P and A sites, respectively). These findings have important implications for the energetics of translocation. PMID:17189194

  1. Ribosome heterogeneity in tumorigenesis: the rRNA point of view

    PubMed Central

    Marcel, Virginie; Catez, Frédéric; Diaz, Jean-Jacques

    2015-01-01

    The "specialized ribosome" concept proposes that ribosome variants are produced and differentially regulate translation. Examples supporting this notion demonstrated heterogeneity of ribosomal protein composition. However, ribosome translational activity is carried out by rRNA. We, and others, recently showed that rRNA heterogeneity regulates translation to generate distinct translatomes promoting tumorigenesis. PMID:27305893

  2. Study of the functional interaction of the 900 Tetraloop of 16S ribosomal RNA with helix 24 within the bacterial ribosome.

    PubMed

    Bélanger, François; Gagnon, Matthieu G; Steinberg, Sergey V; Cunningham, Philip R; Brakier-Gingras, Léa

    2004-05-01

    The 900 tetraloop that caps helix 27 of 16S ribosomal RNA (rRNA) is amongst the most conserved regions of rRNA. This tetraloop forms a GNRA motif that docks into the minor groove of three base-pairs at the bottom of helix 24 of 16S rRNA in the 30S subunit. Both the tetraloop and its receptor in helix 24 contact the 23S rRNA, forming the intersubunit bridge B2c. Here, we investigated the interaction between the 900 tetraloop and its receptor by genetic complementation. We used a specialized ribosome system in combination with an in vivo instant evolution approach to select mutations in helix 24 compensating for a mutation in the 900 tetraloop (A900G) that severely decreases ribosomal activity, impairing subunit association and translational fidelity. We selected two mutants where the G769-C810 base-pair of helix 24 was substituted with either U-A or C x A. When these mutations in helix 24 were investigated in the context of a wild-type 900 tetraloop, the C x A but not the U-A mutation severely impaired ribosome activity, interfering with subunit association and decreasing translational fidelity. In the presence of the A900G mutation, both mutations in helix 24 increased the ribosome activity to the same extent. Subunit association and translational fidelity were increased to the same level. Computer modeling was used to analyze the effect of the mutations in helix 24 on the interaction between the tetraloop and its receptor. This study demonstrates the functional importance of the interaction between the 900 tetraloop and helix 24.

  3. RNA structures regulating ribosomal protein biosynthesis in bacilli.

    PubMed

    Deiorio-Haggar, Kaila; Anthony, Jon; Meyer, Michelle M

    2013-07-01

    In Bacilli, there are three experimentally validated ribosomal-protein autogenous regulatory RNAs that are not shared with E. coli. Each of these RNAs forms a unique secondary structure that interacts with a ribosomal protein encoded by a downstream gene, namely S4, S15, and L20. Only one of these RNAs that interacts with L20 is currently found in the RNA Families Database. We created, or modified, existing structural alignments for these three RNAs and used them to perform homology searches. We have determined that each structure exhibits a narrow phylogenetic distribution, mostly relegated to the Firmicute class Bacilli. This work, in conjunction with other similar work, demonstrates that there are most likely many non-homologous RNA regulatory elements regulating ribosomal protein biosynthesis that still await discovery and characterization in other bacterial species. PMID:23611891

  4. RNA structures regulating ribosomal protein biosynthesis in bacilli

    PubMed Central

    Deiorio-Haggar, Kaila; Anthony, Jon; Meyer, Michelle M.

    2013-01-01

    In Bacilli, there are three experimentally validated ribosomal-protein autogenous regulatory RNAs that are not shared with E. coli. Each of these RNAs forms a unique secondary structure that interacts with a ribosomal protein encoded by a downstream gene, namely S4, S15, and L20. Only one of these RNAs that interacts with L20 is currently found in the RNA Families Database. We created, or modified, existing structural alignments for these three RNAs and used them to perform homology searches. We have determined that each structure exhibits a narrow phylogenetic distribution, mostly relegated to the Firmicute class Bacilli. This work, in conjunction with other similar work, demonstrates that there are most likely many non-homologous RNA regulatory elements regulating ribosomal protein biosynthesis that still await discovery and characterization in other bacterial species. PMID:23611891

  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. 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

  7. Ribosomal RNA metabolism in cucumber leaf mesophyll protoplasts.

    PubMed Central

    Coutts, R H; Barnett, A; Wood, K R

    1975-01-01

    Aspects of the metabolism of RNA have been studied in enzymatically isolated protoplasts from cotyledon and first leaf mesophyll tissue of two cultivars of cucumber. The first leaf mesophyll protoplasts incorporated (3H)-uridine into ribosomal RNA at a constant rate for up to 25 hr in a simple salts medium and for up to 45 hr in a growth medium. Pulse-chase labelling experiments on such preparations showed a rapid dilution of the intracellular (3H)-uridine pool(s) and a high metabolic rate in the cells in one cultivar but not in another. Gel electrophoretic analysis of the RNA from both cotyledon and first leaf protoplasts showed that both protoplast types incorporated either (14C)- or (3H)-uridine into ribosomal RNA species. Incorporation of (3H)-uridine into chloroplasts RNA was minimal in cotyledon protoplasts, but significant in leaf protoplasts. Greater incorporation into the chloroplast RNA species could be achieved by longer pulses. Synthesis of all of the ribosomal RNA species was sensitive to actinomycin D at 10 and 25 mug/ml concentrations in all protoplasts tested. PMID:1153330

  8. Molecular dynamics simulations suggest that RNA three-way junctions can act as flexible RNA structural elements in the ribosome

    PubMed Central

    Beššeová, Ivana; Réblová, Kamila; Leontis, Neocles B.; Šponer, Jiří

    2010-01-01

    We present extensive explicit solvent molecular dynamics analysis of three RNA three-way junctions (3WJs) from the large ribosomal subunit: the 3WJ formed by Helices 90–92 (H90–H92) of 23S rRNA; the 3WJ formed by H42–H44 organizing the GTPase associated center (GAC) of 23S rRNA; and the 3WJ of 5S rRNA. H92 near the peptidyl transferase center binds the 3′-CCA end of amino-acylated tRNA. The GAC binds protein factors and stimulates GTP hydrolysis driving protein synthesis. The 5S rRNA binds the central protuberance and A-site finger (ASF) involved in bridges with the 30S subunit. The simulations reveal that all three 3WJs possess significant anisotropic hinge-like flexibility between their stacked stems and dynamics within the compact regions of their adjacent stems. The A-site 3WJ dynamics may facilitate accommodation of tRNA, while the 5S 3WJ flexibility appears to be essential for coordinated movements of ASF and 5S rRNA. The GAC 3WJ may support large-scale dynamics of the L7/L12-stalk region. The simulations reveal that H42–H44 rRNA segments are not fully relaxed and in the X-ray structures they are bent towards the large subunit. The bending may be related to L10 binding and is distributed between the 3WJ and the H42–H97 contact. PMID:20507916

  9. Gram-positive bacteria with a high DNA G+C content are characterized by a common insertion within their 23S rRNA genes.

    PubMed

    Roller, C; Ludwig, W; Schleifer, K H

    1992-06-01

    An insertion of about 100 bases within the central part of the 23S rRNA genes was found to be a phylogenetic marker for the bacterial line of descent of Gram-positive bacteria with a high DNA G + C content. The insertion was present in 23S rRNA genes of 64 strains representing the major phylogenetic groups of Gram-positive bacteria with a high DNA G+C content, whereas it was not found in 23S rRNA genes of 55 (eu)bacteria representing Gram-positive bacteria with a low DNA G + C content and all other known (eu)bacterial phyla. The presence of the insertion could be easily demonstrated by comparative gel electrophoretic analysis of in vitro-amplified 23S rDNA fragments, which contained the insertion. The nucleotide sequences of the amplified fragments were determined and sequence similarities of at least 44% were found. The overall similarity values are lower than those of 16S and 23S rRNA sequences of the particular organism. Northern hybridization experiments indicated the presence of the insertion within the mature 23S rRNA of Corynebacterium glutamicum.

  10. 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

  11. 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

  12. Phylogeny of bradyrhizobia from Chinese cowpea miscellany inferred from 16S rRNA, atpD, glnII, and 16S-23S intergenic spacer sequences.

    PubMed

    Zhang, Sufang; Xie, Fuli; Yang, Jiangke; Li, Youguo

    2011-04-01

    The cowpea (Vigna unguiculata L.), peanut (Arachis hypogaea L.), and mung bean (Vigna radiata L.) belong to a group of plants known as the "cowpea miscellany" plants, which are widely cultivated throughout the tropic and subtropical zones of Africa and Asia. However, the phylogeny of the rhizobial strains that nodulate these plants is poorly understood. Previous studies have isolated a diversity of rhizobial strains from cowpea miscellany hosts and have suggested that, phylogenetically, they are from different species. In this work, the phylogeny of 42 slow-growing rhizobial strains, isolated from root nodules of cowpea, peanut, and mung bean from different geographical regions of China, was investigated using sequences from the 16S rRNA, atpD and glnII genes, and the 16S-23S rRNA intergenic spacer. The indigenous rhizobial strains from the cowpea miscellany could all be placed in the genus Bradyrhizobium , and Bradyrhizobium liaoningense and Bradyrhizobium yuanmingense were the main species. Phylogenies derived from housekeeping genes were consistent with phylogenies generated from the ribosomal gene. Mung bean rhizobia clustered only into B. liaoningense and B. yuanmingense and were phylogenetically less diverse than cowpea and peanut rhizobia. Geographical origin was significantly reflected in the phylogeny of mung bean rhizobia. Most cowpea rhizobia were more closely related to the 3 major groups B. liaoningense, B. yuanmingense, and Bradyrhizobium elkanii than to the minor groups Bradyrhizobium japonicum or Bradyrhizobium canariense . However, most peanut rhizobia were more closely related to the 2 major groups B. liaoningense and B. yuanmingense than to the minor group B. elkanii.

  13. 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.

  14. rrndb: the Ribosomal RNA Operon Copy Number Database.

    PubMed

    Klappenbach, J A; Saxman, P R; Cole, J R; Schmidt, T M

    2001-01-01

    The Ribosomal RNA Operon Copy Number Database (rrndb) is an Internet-accessible database containing annotated information on rRNA operon copy number among prokaryotes. Gene redundancy is uncommon in prokaryotic genomes, yet the rRNA genes can vary from one to as many as 15 copies. Despite the widespread use of 16S rRNA gene sequences for identification of prokaryotes, information on the number and sequence of individual rRNA genes in a genome is not readily accessible. In an attempt to understand the evolutionary implications of rRNA operon redundancy, we have created a phylogenetically arranged report on rRNA gene copy number for a diverse collection of prokaryotic microorganisms. Each entry (organism) in the rrndb contains detailed information linked directly to external websites including the Ribosomal Database Project, GenBank, PubMed and several culture collections. Data contained in the rrndb will be valuable to researchers investigating microbial ecology and evolution using 16S rRNA gene sequences. The rrndb web site is directly accessible on the WWW at http://rrndb.cme. msu.edu.

  15. Pyrosequencing of plastid 23S rRNA genes reveals diverse and dynamic cyanobacterial and algal populations in two eutrophic lakes.

    PubMed

    Steven, Blaire; McCann, Sage; Ward, Naomi L

    2012-12-01

    Pyrosequencing of plastid 23S rRNA genes was performed to determine the usefulness of this methodology for describing spatial and temporal patterns of algal diversity in two eutrophic lakes. The majority of the sequences were identified as known cyanobacteria or eukaryotic algae (> 70% of sequence reads), indicating this approach can specifically recover algal sequences from complex communities. Furthermore, estimated coverage of the data sets indicated that the majority of the 23S rRNA genetic diversity was recovered in these surveys. Communities from algal mats could be clearly distinguished from algae in the water column, and the communities could be readily differentiated between the two lakes, suggesting that the plastid 23S rRNA sequencing was able to distinguish niche and biogeographic partitioning of algal communities. Within the sequence data sets, the ratio of cyanobacteria to eukaryotic algae fluctuated over the course of sampling, with cyanobacteria 23S rRNA sequences being more abundant in later samples. In addition, the eukaryotic algae communities showed large shifts in composition over the course of sampling. Taken together, these data demonstrate the usefulness of targeted plastid 23S rRNA sequencing for describing the structure and dynamics of complex algal communities.

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

    PubMed

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

    2016-08-24

    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.

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

    PubMed

    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

  18. 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

  19. The unusually long small subunit ribosomal RNA of Phreatamoeba balamuthi.

    PubMed Central

    Hinkle, G; Leipe, D D; Nerad, T A; Sogin, M L

    1994-01-01

    The small subunit ribosomal RNA (rRNA) of the anaerobic amoeba Phreatamoeba balamuthi is the longest 16S-like rRNA sequenced to date. Secondary structure analysis suggests that the additional sequence is incorporated in canonical eukaryotic expansion regions and is not due to the presence of introns. Reverse transcriptase sequencing of total RNA extracts confirmed that two uncommonly long expansion regions are present in native P. balamuthi 16S-like rRNA. Primary sequence comparison and similar secondary structure indicate a 61 base stem and loop repeat within an expansion region; a mechanism whereby the repeat may have been incorporated is presented. P. balamuthi provides further evidence that 16S-like rRNA length does not correlate with phylogenetic position. PMID:8127686

  20. A model for the topology of active ribosomal RNA genes.

    PubMed

    Denissov, Serguei; Lessard, Frédéric; Mayer, Christine; Stefanovsky, Victor; van Driel, Marc; Grummt, Ingrid; Moss, Tom; Stunnenberg, Hendrik G

    2011-03-01

    The Christmas tree view of active ribosomal RNA (rRNA) genes suggests a gene topology in which a large number of nascent rRNA transcripts are prevented from intertwining. The way in which this is achieved has remained unclear. By using a combination of chromatin immunoprecipitation and chromosome conformation capture techniques, we show that the promoter, upstream region and terminator R3 of active rRNA genes are held together spatially throughout the cell cycle, forming a stable core around which the transcribed region is organized. We suggest a new core-helix model for the topology of rRNA genes, that provides a structural basis for the productive synthesis or rRNA.

  1. Depletion of Free 30S Ribosomal Subunits in Escherichia coli by Expression of RNA Containing Shine-Dalgarno-Like Sequences

    PubMed Central

    Mawn, Mary V.; Fournier, Maurille J.; Tirrell, David A.; Mason, Thomas L.

    2002-01-01

    We have constructed synthetic coding sequences for the expression of poly(α,l-glutamic acid) (PLGA) as fusion proteins with dihydrofolate reductase (DHFR) in Escherichia coli. These PLGA coding sequences use both GAA and GAG codons for glutamic acid and contain sequence elements (5′-GAGGAGG-3′) that resemble the consensus Shine-Dalgarno (SD) sequence found at translation initiation sites in bacterial mRNAs. An unusual feature of DHFR-PLGA expression is that accumulation of the protein is inversely related to the level of induction of its mRNA. Cellular protein synthesis was inhibited >95% by induction of constructs for either translatable or untranslatable PLGA RNAs. Induction of PLGA RNA resulted in the depletion of free 30S ribosomal subunits and the appearance of new complexes in the polyribosome region of the gradient. Unlike normal polyribosomes, these complexes were resistant to breakdown in the presence of puromycin. The novel complexes contained 16S rRNA, 23S rRNA, and PLGA RNA. We conclude that multiple noninitiator SD-like sequences in the PLGA RNA inhibit cellular protein synthesis by sequestering 30S small ribosomal subunits and 70S ribosomes in nonfunctional complexes on the PLGA mRNA. PMID:11751827

  2. 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

  3. Mechanisms for Ribotoxin-induced Ribosomal RNA Cleavage

    PubMed Central

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

    2012-01-01

    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 caspase 8, 9 and 3 concurrently with apoptosis further suggested rRNA cleavage occurred in parallel with both extrinsic and intrinsic pathways of programmed cell death. When specific inhibitors cathepsin 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. PMID:23022514

  4. 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.

  5. 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

  6. 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

  7. 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

  8. 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.

  9. Nucleotide sequence of the 16S - 23S spacer region in an rRNA gene cluster from tobacco chloroplast DNA.

    PubMed Central

    Takaiwa, F; Sugiura, M

    1982-01-01

    The nucleotide sequence of a spacer region between 16S and 23S rRNA genes from tobacco chloroplasts has been determined. The spacer region is 2080 bp long and encodes tRNAIle and tRNAAla genes which contain intervening sequences of 707 bp and 710 bp, respectively. Strong homology between the two intervening sequences is observed. These spacer tRNAs are synthesized as part of an 8.2 kb precursor molecule containing 16S and 23S rRNA sequences. Images PMID:6281739

  10. 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

  11. Interactions of the TnaC nascent peptide with rRNA in the exit tunnel enable the ribosome to respond to free tryptophan.

    PubMed

    Martínez, Allyson K; Gordon, Emily; Sengupta, Arnab; Shirole, Nitin; Klepacki, Dorota; Martinez-Garriga, Blanca; Brown, Lewis M; Benedik, Michael J; Yanofsky, Charles; Mankin, Alexander S; Vazquez-Laslop, Nora; Sachs, Matthew S; Cruz-Vera, Luis R

    2014-01-01

    A transcriptional attenuation mechanism regulates expression of the bacterial tnaCAB operon. This mechanism requires ribosomal arrest induced by the regulatory nascent TnaC peptide in response to free L-tryptophan (L-Trp). In this study we demonstrate, using genetic and biochemical analyses, that in Escherichia coli, TnaC residue I19 and 23S rRNA nucleotide A2058 are essential for the ribosome's ability to sense free L-Trp. We show that the mutational change A2058U in 23S rRNA reduces the concentration dependence of L-Trp-mediated tna operon induction, whereas the TnaC I19L change suppresses this phenotype, restoring the sensitivity of the translating A2058U mutant ribosome to free L-Trp. These findings suggest that interactions between TnaC residue I19 and 23S rRNA nucleotide A2058 contribute to the creation of a regulatory L-Trp binding site within the translating ribosome. PMID:24137004

  12. Phylogenetic relationships of Cryptosporidium determined by ribosomal RNA sequence comparison.

    PubMed

    Johnson, A M; Fielke, R; Lumb, R; Baverstock, P R

    1990-04-01

    Reverse transcription of total cellular RNA was used to obtain a partial sequence of the small subunit ribosomal RNA of Cryptosporidium, a protist currently placed in the phylum Apicomplexa. The semi-conserved regions were aligned with homologous sequences in a range of other eukaryotes, and the evolutionary relationships of Cryptosporidium were determined by two different methods of phylogenetic analysis. The prokaryotes Escherichia coli and Halobacterium cuti were included as outgroups. The results do not show an especially close relationship of Cryptosporidium to other members of the phylum Apicomplexa. PMID:2332273

  13. 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

  14. 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

  15. Studies on ribosomal RNA genes of mycobacteria including M. leprae.

    PubMed

    Katoch, V M; Shivannavar, C T; Datta, A K

    1989-01-01

    Information about specific genes specially of pathogenic mycobacteria could be used to unequivocally identify isolates of mycobacteria which are of clinical interest. Both eukaryotic and prokaryotic ribosomal RNA (rRNA) genes have been shown to comprise sequences which are conserved and others which are divergent. In the present study, rRNA genes from several cultivable mycobacteria including M. tuberculosis and armadillo derived M. leprae have been investigated. rRNA was isolated, made radioactive in vitro and then used to identify restriction fragments of DNA containing rRNA gene sequences. It was observed that restriction endonuclease patterns of rRNA genes are characteristic. By probing with homologous and heterologous rRNA probes, fragments hybridizing maximum with homologous probes could be identified and it appears that sequences flanking the rRNA genes are not identical. These fragments need to be further sequenced to identify the nucleotide sequences specific to rRNA gene cluster. It would also be necessary to analyse several isolates of each species including armadillo derived M. leprae before reaching any conclusions.

  16. Polysome analysis for determining mRNA and ribosome association in Saccharomyces cerevisiae.

    PubMed

    Hu, Wenqian; Coller, Jeff

    2013-01-01

    The control of mRNA translation is vital for gene expression and it is regulated under various physiological and pathological conditions (Groppo and Richter, 2009). For example, under some physiological conditions, translational initiation is impaired. Therefore, the association of mRNA with ribosomes and/or ribosomal subunits can provide a powerful means to dissect specific aspects of posttranscriptional mRNA regulation. This protocol describes a technique used to determine ribosome occupancy on mRNA.

  17. Rapid Detection of Mutations in the 23S rRNA Gene of Helicobacter pylori That Confers Resistance to Clarithromycin Treatment to the Bacterium

    PubMed Central

    Matsumura, Masayuki; Hikiba, Yoko; Ogura, Keiji; Togo, Goichi; Tsukuda, Izumi; Ushikawa, Kenji; Shiratori, Yasushi; Omata, Masao

    2001-01-01

    We developed a new method capable of detecting point mutations in the 23S rRNA gene of Helicobacter pylori using a LightCycler. Our method can detect a mutation in this gene in less than 1 h and can process many samples at once, thereby contributing to the selection of patients suitable for clarithromycin-based therapy. PMID:11158129

  18. Differentiation of non-pylori Helicobacter species based on PCR-restriction fragment length polymorphism of the 23S rRNA gene.

    PubMed

    Yadegar, Abbas; Alebouyeh, Masoud; Lawson, Andy J; Mirzaei, Tabassom; Nazemalhosseini Mojarad, Ehsan; Zali, Mohammad Reza

    2014-06-01

    Phenotypic identification of non-pylori Helicobacter species has always been problematic and time-consuming in comparison with many other bacteria. We developed a rapid two-step identification assay based on PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of the 23S rRNA gene for differentiating between non-pylori Helicobacter species. A new genus-specific primer pair based on all available complete and partial 23S rRNA sequences of Helicobacter species was designed. In silico restriction analysis of variable regions of the 23S rRNA gene suggested SmaI and HindIII endonucleases would provide a good level of differentiation. Analysis of the obtained 23S rRNA RFLP patterns divided all Helicobacter study strains into three species groups (groups A-C) and 12 unique restriction patterns. Wolinella succinogenes also gave a unique pattern. Our proposed PCR-RFLP method was found to be as a valuable tool for routine identification of non-pylori Helicobacter species from human or animal samples.

  19. Binding site for Xenopus ribosomal protein L5 and accompanying structural changes in 5S rRNA.

    PubMed

    Scripture, J Benjamin; Huber, Paul W

    2011-05-10

    The structure of the eukaryotic L5-5S rRNA complex was investigated in protection and interference experiments and is compared with the corresponding structure (L18-5S rRNA) in the Haloarcula marismortui 50S subunit. In close correspondence with the archaeal structure, the contact sites for the eukaryotic ribosomal protein are located primarily in helix III and loop C and secondarily in loop A and helix V. While the former is unique to L5, the latter is also a critical contact site for transcription factor IIIA (TFIIIA), accounting for the mutually exclusive binding of these two proteins to 5S RNA. The binding of L5 causes structural changes in loops B and C that expose nucleotides that contact the Xenopus L11 ortholog in H. marismortui. This induced change in the structure of the RNA reveals the origins of the cooperative binding to 5S rRNA that has been observed for the bacterial counterparts of these proteins. The native structure of helix IV and loop D antagonizes binding of L5, indicating that this region of the RNA is dynamic and also influenced by the protein. Examination of the crystal structures of Thermus thermophilus ribosomes in the pre- and post-translocation states identified changes in loop D and in the surrounding region of 23S rRNA that support the proposal that 5S rRNA acts to transmit information between different functional domains of the large subunit.

  20. The conserved endoribonuclease YbeY is required for chloroplast ribosomal RNA processing in Arabidopsis.

    PubMed

    Liu, Jinwen; Zhou, Wenbin; Liu, Guifeng; Yang, Chuanping; Sun, Yi; Wu, Wenjuan; Cao, Shenquan; Wang, Chong; Hai, Guanghui; Wang, Zhifeng; Bock, Ralph; Huang, Jirong; Cheng, Yuxiang

    2015-05-01

    Maturation of chloroplast ribosomal RNAs (rRNAs) comprises several endoribonucleolytic and exoribonucleolytic processing steps. However, little is known about the specific enzymes involved and the cleavage steps they catalyze. Here, we report the functional characterization of the single Arabidopsis (Arabidopsis thaliana) gene encoding a putative YbeY endoribonuclease. AtYbeY null mutants are seedling lethal, indicating that AtYbeY function is essential for plant growth. Knockdown plants display slow growth and show pale-green leaves. Physiological and ultrastructural analyses of atybeY mutants revealed impaired photosynthesis and defective chloroplast development. Fluorescent microcopy analysis showed that, when fused with the green fluorescence protein, AtYbeY is localized in chloroplasts. Immunoblot and RNA gel-blot assays revealed that the levels of chloroplast-encoded subunits of photosynthetic complexes are reduced in atybeY mutants, but the corresponding transcripts accumulate normally. In addition, atybeY mutants display defective maturation of both the 5' and 3' ends of 16S, 23S, and 4.5S rRNAs as well as decreased accumulation of mature transcripts from the transfer RNA genes contained in the chloroplast rRNA operon. Consequently, mutant plants show a severe deficiency in ribosome biogenesis, which, in turn, results in impaired plastid translational activity. Furthermore, biochemical assays show that recombinant AtYbeY is able to cleave chloroplast rRNAs as well as messenger RNAs and transfer RNAs in vitro. Taken together, our findings indicate that AtYbeY is a chloroplast-localized endoribonuclease that is required for chloroplast rRNA processing and thus for normal growth and development.

  1. The structure of the yeast ribosomal RNA genes. I. The complete nucleotide sequence of the 18S ribosomal RNA gene from Saccharomyces cerevisiae.

    PubMed

    Rubtsov, P M; Musakhanov, M M; Zakharyev, V M; Krayev, A S; Skryabin, K G; Bayev, A A

    1980-12-11

    The cloned 18 S ribosomal RNA gene from Saccharomyces cerevisiae have been sequenced, using the Maxam-Gilbert procedure. From this data the complete sequence of 1789 nucleotides of the 18 S RNA was deduced. Extensive homology with many eucaryotic as well as E. coli ribosomal small subunit rRNA (S-rRNA) has been observed in the 3'-end region of the rRNA molecule. Comparison of the yeast 18 S rRNA sequences with partial sequence data, available for rRNAs of the other eucaryotes provides strong evidence that a substantial portion of the 18 S RNA sequence has been conserved in evolution.

  2. 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.

  3. Detection of the new cosmopolitan genus Thermoleptolyngbya (Cyanobacteria, Leptolyngbyaceae) using the 16S rRNA gene and 16S-23S ITS region.

    PubMed

    Sciuto, Katia; Moro, Isabella

    2016-12-01

    Cyanobacteria are widespread prokaryotes that are able to live in extreme conditions such as thermal springs. Strains attributable to the genus Leptolyngbya are among the most common cyanobacteria sampled from thermal environments. Leptolyngbya is a character-poor taxon that was demonstrated to be polyphyletic based on molecular analyses. The recent joining of 16S rRNA gene phylogenies with 16S-23S ITS secondary structure analysis is a useful approach to detect new cryptic taxa and has led to the separation of new genera from Leptolyngbya and to the description of new species inside this genus and in other related groups. In this study, phylogenetic investigations based on both the 16S rRNA gene and the 16S-23S ITS region were performed alongside 16S rRNA and 16S-23S ITS secondary structure analyses on cyanobacteria of the family Leptolyngbyaceae. These analyses focused on filamentous strains sampled from thermal springs with a morphology ascribable to the genus Leptolyngbya. The phylogenetic reconstructions showed that the Leptolyngbya-like thermal strains grouped into a monophyletic lineage that was distinct from Leptolyngbya. The 16S-23S ITS secondary structure results supported the separation of this cluster. A new genus named Thermoleptolyngbya was erected to encompass these strains, and two species were described inside this new taxon: T. albertanoae and T. oregonensis. PMID:27546720

  4. Detection of the new cosmopolitan genus Thermoleptolyngbya (Cyanobacteria, Leptolyngbyaceae) using the 16S rRNA gene and 16S-23S ITS region.

    PubMed

    Sciuto, Katia; Moro, Isabella

    2016-12-01

    Cyanobacteria are widespread prokaryotes that are able to live in extreme conditions such as thermal springs. Strains attributable to the genus Leptolyngbya are among the most common cyanobacteria sampled from thermal environments. Leptolyngbya is a character-poor taxon that was demonstrated to be polyphyletic based on molecular analyses. The recent joining of 16S rRNA gene phylogenies with 16S-23S ITS secondary structure analysis is a useful approach to detect new cryptic taxa and has led to the separation of new genera from Leptolyngbya and to the description of new species inside this genus and in other related groups. In this study, phylogenetic investigations based on both the 16S rRNA gene and the 16S-23S ITS region were performed alongside 16S rRNA and 16S-23S ITS secondary structure analyses on cyanobacteria of the family Leptolyngbyaceae. These analyses focused on filamentous strains sampled from thermal springs with a morphology ascribable to the genus Leptolyngbya. The phylogenetic reconstructions showed that the Leptolyngbya-like thermal strains grouped into a monophyletic lineage that was distinct from Leptolyngbya. The 16S-23S ITS secondary structure results supported the separation of this cluster. A new genus named Thermoleptolyngbya was erected to encompass these strains, and two species were described inside this new taxon: T. albertanoae and T. oregonensis.

  5. 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

  6. 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

  7. 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

  8. Rapid ribosomal RNA sequencing and the phylogenetic analysis of protists.

    PubMed

    Johnson, A M; Baverstock, P R

    1989-04-01

    A newly described technique for rapidly obtaining the partial nucleotide sequence of ribosomal RNA is being applied to investigate phylogenetic relationships among living organisms. Alan Johnson and Peter Boverstock describe the importance of this method to parasitology in providing new information on the phylogenetic relationships of parasitic organisms previously placed in groups of convenience. The phylum Apicomplexo in particular, has been the object of much study using this technique, but the technology is likely to extend soon to the restructuring of the phylogenetic trees of many groups of parasites.

  9. Intragenomic heterogeneity of the 16S rRNA-23S rRNA internal transcribed spacer among Pseudomonas syringae and Pseudomonas fluorescens strains.

    PubMed

    Milyutina, Irina A; Bobrova, Vera K; Matveeva, Eugenia V; Schaad, Norman W; Troitsky, Alexey V

    2004-10-01

    The 16S-23S rRNA internal transcribed spacer regions (ITS1) from 14 strains of Pseudomonas syringae and P. fluorescens were sequenced. ITS1 exhibited significant sequence variability among different operons within a single genome. From 1 to 4 types of ITS1 were found in individual genomes of the P. syringae and P. fluorescens strains. A total of eight ITS1 types were identified among strains studied. The ITS1 nucleotide sequences consisted of conserved blocks including, among others, a stem-forming region of box B, tRNAIle and tRNAAla genes and several variable blocks. The differences in the variable regions were mostly due to insertions and/or deletions of nucleotide blocks. The intragenomic heterogeneity of ITS1 was brought about by different combinations of variable blocks, which possibly have resulted from recombination and horizontal transfer.

  10. 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.

  11. 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.

  12. 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.

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

    PubMed

    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

    2016-06-29

    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.

  14. 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.

  15. 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

  16. Efficient Detection of Pathogenic Leptospires Using 16S Ribosomal RNA

    PubMed Central

    Lindow, Janet; Wunder, Elsio A.; Reis, Mitermayer G.; Usmani-Brown, Sahar; Ledizet, Michel; Ko, Albert; Pal, Utpal

    2015-01-01

    Pathogenic Leptospira species cause a prevalent yet neglected zoonotic disease with mild to life-threatening complications in a variety of susceptible animals and humans. Diagnosis of leptospirosis, which primarily relies on antiquated serotyping methods, is particularly challenging due to presentation of non-specific symptoms shared by other febrile illnesses, often leading to misdiagnosis. Initiation of antimicrobial therapy during early infection to prevent more serious complications of disseminated infection is often not performed because of a lack of efficient diagnostic tests. Here we report that specific regions of leptospiral 16S ribosomal RNA molecules constitute a novel and efficient diagnostic target for PCR-based detection of pathogenic Leptospira serovars. Our diagnostic test using spiked human blood was at least 100-fold more sensitive than corresponding leptospiral DNA-based quantitative PCR assays, targeting the same 16S nucleotide sequence in the RNA and DNA molecules. The sensitivity and specificity of our RNA assay against laboratory-confirmed human leptospirosis clinical samples were 64% and 100%, respectively, which was superior then an established parallel DNA detection assay. Remarkably, we discovered that 16S transcripts remain appreciably stable ex vivo, including untreated and stored human blood samples, further highlighting their use for clinical detection of L. interrogans. Together, these studies underscore a novel utility of RNA targets, specifically 16S rRNA, for development of PCR-based modalities for diagnosis of human leptospirosis, and also may serve as paradigm for detection of additional bacterial pathogens for which early diagnosis is warranted. PMID:26091292

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

    PubMed Central

    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

    2016-01-01

    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. PMID:27354316

  18. Spontaneous reverse movement of mRNA-bound tRNA through the ribosome.

    PubMed

    Konevega, Andrey L; Fischer, Niels; Semenkov, Yuri P; Stark, Holger; Wintermeyer, Wolfgang; Rodnina, Marina V

    2007-04-01

    During the translocation step of protein synthesis, a complex of two transfer RNAs bound to messenger RNA (tRNA-mRNA) moves through the ribosome. The reaction is promoted by an elongation factor, called EF-G in bacteria, which, powered by GTP hydrolysis, induces an open, unlocked conformation of the ribosome that allows for spontaneous tRNA-mRNA movement. Here we show that, in the absence of EF-G, there is spontaneous backward movement, or retrotranslocation, of two tRNAs bound to mRNA. Retrotranslocation is driven by the gain in affinity when a cognate E-site tRNA moves into the P site, which compensates the affinity loss accompanying the movement of peptidyl-tRNA from the P to the A site. These results lend support to the diffusion model of tRNA movement during translocation. In the cell, tRNA movement is biased in the forward direction by EF-G, which acts as a Brownian ratchet and prevents backward movement.

  19. Rapid assay of A2058T-mutated 23S rRNA allelic profiles associated with high-level macrolide resistance in Moraxella catarrhalis.

    PubMed

    Saito, Ryoichi; Kasai, Ayako; Ogihara, Shinji; Yamada, Kageto; Tao, Kazuyuki

    2015-09-01

    We report on a restriction fragment-length polymorphism (HpyCH4III) assay for profile analysis of 23S rRNA gene A2058T-mutated alleles associated with high-level macrolide resistance in Moraxella catarrhalis. Our assay results were supported by DNA sequencing analysis, allowed for simultaneous testing of many strains, and produced results from pure-cultured colonies within 4 h.

  20. Circular non-coding RNA ANRIL modulates ribosomal RNA maturation and atherosclerosis in humans

    PubMed Central

    Holdt, Lesca M.; Stahringer, Anika; Sass, Kristina; Pichler, Garwin; Kulak, Nils A.; Wilfert, Wolfgang; Kohlmaier, Alexander; Herbst, Andreas; Northoff, Bernd H.; Nicolaou, Alexandros; Gäbel, Gabor; Beutner, Frank; Scholz, Markus; Thiery, Joachim; Musunuru, Kiran; Krohn, Knut; Mann, Matthias; Teupser, Daniel

    2016-01-01

    Circular RNAs (circRNAs) are broadly expressed in eukaryotic cells, but their molecular mechanism in human disease remains obscure. Here we show that circular antisense non-coding RNA in the INK4 locus (circANRIL), which is transcribed at a locus of atherosclerotic cardiovascular disease on chromosome 9p21, confers atheroprotection by controlling ribosomal RNA (rRNA) maturation and modulating pathways of atherogenesis. CircANRIL binds to pescadillo homologue 1 (PES1), an essential 60S-preribosomal assembly factor, thereby impairing exonuclease-mediated pre-rRNA processing and ribosome biogenesis in vascular smooth muscle cells and macrophages. As a consequence, circANRIL induces nucleolar stress and p53 activation, resulting in the induction of apoptosis and inhibition of proliferation, which are key cell functions in atherosclerosis. Collectively, these findings identify circANRIL as a prototype of a circRNA regulating ribosome biogenesis and conferring atheroprotection, thereby showing that circularization of long non-coding RNAs may alter RNA function and protect from human disease. PMID:27539542

  1. Ribosomal RNA methylation in Mycobacterium smegmatis SN2.

    PubMed

    Srivastava, R; Gopinathan, K P

    1987-12-01

    Ribosomal RNA (rRNA) from a fast growing nonpathogenic strain of mycobacteria, Mycobacterium smegmatis SN2, was analyzed for the presence of minor nucleotides. Of the sixteen modified nucleotides detected, the identity of twelve has been established and their molar ratios were determined. These nucleotides include m1A, m2A, m6A, m6(2)A, m7G, m5C, rT, CmpC, CmpG, GmpG, UmpG and UmpU. The distinct features of the mycobacterial rRNA modifications include: (i) relatively substantial level of methylation, a feature distinct from that of the tRNA species which are unique in being under methylated in these bacteria, (ii) N1 methyl adenine representing the bulk of the modified bases, (iii) the lack of ribose methylation on any two successive nucleotides, and (iv) the presence of N6,N6-dimethyl adenosines, which are the target sites of the antibiotic kasugamycin, although the bacterial growth is insensitive to the drug. PMID:3440025

  2. Evaluation of the 23S rRNA gene as target for qPCR based quantification of Frankia in soils.

    PubMed

    Samant, Suvidha; Amann, Rudolf I; Hahn, Dittmar

    2014-05-01

    The 23S rRNA gene was evaluated as target for the development of Sybr Green-based quantitative PCR (qPCR) for the analysis of nitrogen-fixing members of the genus Frankia or subgroups of these in soil. A qPCR with a primer combination targeting all nitrogen-fixing frankiae (clusters 1, 2 and 3) resulted in numbers similar to those obtained with a previously developed qPCR using nifH gene sequences, both with respect to introduced and indigenous Frankia populations. Primer combinations more specifically targeting three subgroups of the Alnus host infection group (cluster 1) or members of the Elaeagnus host infection group (cluster 3) were specific for introduced strains of the target group, with numbers corresponding to those obtained by quantification of nitrogen-fixing frankiae with both the 23S rRNA and nifH genes as target. Method verification on indigenous Frankia populations in soils, i.e. in depth profiles from four sites at an Alnus glutinosa stand, revealed declining numbers in the depth profiles, with similar abundance of all nitrogen-fixing frankiae independent of 23S rRNA or nifH gene targets, and corresponding numbers of one group of frankiae of the Alnus host infection only, with no detections of frankiae representing the Elaeagnus, Casuarina, or a second subgroup of the Alnus host infection groups. PMID:24315016

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

    PubMed Central

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

    2010-01-01

    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. PMID:20215440

  4. 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.

  5. 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

  6. 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…

  7. Specific contacts between protein S4 and ribosomal RNA are required at multiple stages of ribosome assembly.

    PubMed

    Mayerle, Megan; Woodson, Sarah A

    2013-04-01

    Assembly of bacterial 30S ribosomal subunits requires structural rearrangements to both its 16S rRNA and ribosomal protein components. Ribosomal protein S4 nucleates 30S assembly and associates rapidly with the 5' domain of the 16S rRNA. In vitro, transformation of initial S4-rRNA complexes to long-lived, mature complexes involves refolding of 16S helix 18, which forms part of the decoding center. Here we use targeted mutagenesis of Geobacillus stearothermophilus S4 to show that remodeling of S4-rRNA complexes is perturbed by ram alleles associated with reduced translational accuracy. Gel mobility shift assays, SHAPE chemical probing, and in vivo complementation show that the S4 N-terminal extension is required for RNA binding and viability. Alanine substitutions in Y47 and L51 that interact with 16S helix 18 decrease S4 affinity and destabilize the helix 18 pseudoknot. These changes to the protein-RNA interface correlate with no growth (L51A) or cold-sensitive growth, 30S assembly defects, and accumulation of 17S pre-rRNA (Y47A). A third mutation, R200A, over-stabilizes the helix 18 pseudoknot yet results in temperature-sensitive growth, indicating that complex stability is finely tuned by natural selection. Our results show that early S4-RNA interactions guide rRNA folding and impact late steps of 30S assembly.

  8. 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.

  9. Eukaryote-specific rRNA expansion segments function in ribosome biogenesis.

    PubMed

    Ramesh, Madhumitha; Woolford, John L

    2016-08-01

    The secondary structure of ribosomal RNA (rRNA) is largely conserved across all kingdoms of life. However, eukaryotes have evolved extra blocks of rRNA sequences, relative to those of prokaryotes, called expansion segments (ES). A thorough characterization of the potential roles of ES remains to be done, possibly because of limitations in the availability of robust systems to study rRNA mutants. We sought to systematically investigate the potential functions, if any, of the ES in 25S rRNA of Saccharomyces cerevisiae by deletion mutagenesis. We deleted 14 of the 16 different eukaryote-specific ES in yeast 25S rRNA individually and assayed their phenotypes. Our results show that all but two of the ES tested are necessary for optimal growth and are required for production of 25S rRNA, suggesting that ES play roles in ribosome biogenesis. Further, we classified expansion segments into groups that participate in early nucleolar, middle, and late nucleoplasmic steps of ribosome biogenesis, by assaying their pre-rRNA processing phenotypes. This study is the first of its kind to systematically identify the functions of eukaryote-specific expansion segments by showing that they play roles in specific steps of ribosome biogenesis. The catalog of phenotypes we identified, combined with previous investigations of the roles ribosomal proteins in large subunit biogenesis, leads us to infer that assembling ribosomes are composed of distinct RNA and protein structural neighborhood clusters that participate in specific steps of ribosome biogenesis. PMID:27317789

  10. Ribosome dynamics and tRNA movement by time-resolved electron cryomicroscopy.

    PubMed

    Fischer, Niels; Konevega, Andrey L; Wintermeyer, Wolfgang; Rodnina, Marina V; Stark, Holger

    2010-07-15

    The translocation step of protein synthesis entails large-scale rearrangements of the ribosome-transfer RNA (tRNA) complex. Here we have followed tRNA movement through the ribosome during translocation by time-resolved single-particle electron cryomicroscopy (cryo-EM). Unbiased computational sorting of cryo-EM images yielded 50 distinct three-dimensional reconstructions, showing the tRNAs in classical, hybrid and various novel intermediate states that provide trajectories and kinetic information about tRNA movement through the ribosome. The structures indicate how tRNA movement is coupled with global and local conformational changes of the ribosome, in particular of the head and body of the small ribosomal subunit, and show that dynamic interactions between tRNAs and ribosomal residues confine the path of the tRNAs through the ribosome. The temperature dependence of ribosome dynamics reveals a surprisingly flat energy landscape of conformational variations at physiological temperature. The ribosome functions as a Brownian machine that couples spontaneous conformational changes driven by thermal energy to directed movement.

  11. Crystal structure of the RluD pseudouridine synthase catalytic module, an enzyme that modifies 23S rRNA and is essential for normal cell growth of Escherichia coli.

    PubMed

    Sivaraman, J; Iannuzzi, Pietro; Cygler, Miroslaw; Matte, Allan

    2004-01-01

    Pseudouridine (5-beta-D-ribofuranosyluracil, Psi) is the most commonly found modified base in RNA. Conversion of uridine to Psi is performed enzymatically in both prokaryotes and eukaryotes by pseudouridine synthases (EC 4.2.1.70). The Escherichia coli Psi-synthase RluD modifies uridine to Psi at positions 1911, 1915 and 1917 within 23S rRNA. RluD also possesses a second function related to proper assembly of the 50S ribosomal subunit that is independent of Psi-synthesis. Here, we report the crystal structure of the catalytic module of RluD (residues 68-326; DeltaRluD) refined at 1.8A to a final R-factor of 21.8% (R(free)=24.3%). DeltaRluD is a monomeric enzyme having an overall mixed alpha/beta fold. The DeltaRluD molecule consists of two subdomains, a catalytic subdomain and C-terminal subdomain with the RNA-binding cleft formed by loops extending from the catalytic sub-domain. The catalytic sub-domain of DeltaRluD has a similar fold as in TruA, TruB and RsuA, with the location of the RNA-binding cleft, active-site and conserved, catalytic Asp residue superposing in all four structures. Superposition of the crystal structure of TruB bound to a T-stem loop with RluD reveals that similar RNA-protein interactions for the flipped-out uridine base would exist in both structures, implying that base-flipping is necessary for catalysis. This observation also implies that the specificity determinants for site-specific RNA-binding and recognition likely reside in parts of RluD beyond the active site.

  12. Stochastic theory of protein synthesis and polysome: ribosome profile on a single mRNA transcript.

    PubMed

    Sharma, Ajeet K; Chowdhury, Debashish

    2011-11-21

    The process of polymerizing a protein by a ribosome, using a messenger RNA (mRNA) as the corresponding template, is called translation. Ribosome may be regarded as a molecular motor for which the mRNA template serves also as the track. Often several ribosomes may translate the same (mRNA) simultaneously. The ribosomes bound simultaneously to a single mRNA transcript are the members of a polyribosome (or, simply, polysome). Experimentally measured polysome profile gives the distribution of polysome sizes. Recently a breakthrough in determining the instantaneous positions of the ribosomes on a given mRNA track has been achieved and the technique is called ribosome profiling (Ingolia et al., 2009; Guo et al., 2010). Motivated by the success of these techniques, we have studied the spatio-temporal organization of ribosomes by extending a theoretical model that we have reported elsewhere (Sharma and Chowdhury, 2011). This extended version of our model incorporates not only (i) mechano-chemical cycle of individual ribomes, and (ii) their steric interactions, but also (iii) the effects of (a) kinetic proofreading, (b) translational infidelity, (c) ribosome recycling, and (d) sequence inhomogeneities. The theoretical framework developed here will serve in guiding further experiments and in analyzing the data to gain deep insight into various kinetic processes involved in translation.

  13. tmRNA-SmpB: a journey to the centre of the bacterial ribosome.

    PubMed

    Weis, Félix; Bron, Patrick; Giudice, Emmanuel; Rolland, Jean-Paul; Thomas, Daniel; Felden, Brice; Gillet, Reynald

    2010-11-17

    Ribosomes mediate protein synthesis by decoding the information carried by messenger RNAs (mRNAs) and catalysing peptide bond formation between amino acids. When bacterial ribosomes stall on incomplete messages, the trans-translation quality control mechanism is activated by the transfer-messenger RNA bound to small protein B (tmRNA-SmpB ribonucleoprotein complex). Trans-translation liberates the stalled ribosomes and triggers degradation of the incomplete proteins. Here, we present the cryo-electron microscopy structures of tmRNA-SmpB accommodated or translocated into stalled ribosomes. Two atomic models for each state are proposed. This study reveals how tmRNA-SmpB crosses the ribosome and how, as the problematic mRNA is ejected, the tmRNA resume codon is placed onto the ribosomal decoding site by new contacts between SmpB and the nucleotides upstream of the tag-encoding sequence. This provides a structural basis for the transit of the large tmRNA-SmpB complex through the ribosome and for the means by which the tmRNA internal frame is set for translation to resume.

  14. 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.

  15. DNA homologies of ribosomal RNA genes of Neurospora species

    SciTech Connect

    Mukhopadhyay, D.K.; Mimiko, R.; Dutta, S.K.

    1980-01-01

    Ribosomal RNA genes (rDNAs) of Neurospora crassa contain DNA sequences which code for 17S, 5.8S, and 26S rRNAs, in addition to internal and external spacers. As has been reported for many eukaryotes, the DNA sequences which code for 17S, 5.8S, and 26S rRNAs in Neurospora species are probably conserved while the internal and external spacer regions are probably variable sequences. Extensive electron microscopic studies of 45S precursor rRNA of several cold and warm blooded animals confirm that spacer regions vary extensively from species to species. It was desirable to know whether such differences in rDNA sequences exist between Neurospora species. Any such difference should be detectable using standard procedures for DNA homology studies rDNA sequences were isolated from N. crassa mycelial cells using the procedure described previously. The purified rDNA was /sup 3/H-labeled (by nick translation) and reassociated with total DNA isolated from the heterothallic species N. crassa and from three homothalliospecies: N. dodgei, N. lineolata, and N. africana. In addition, /sup 32/P-labeled total DNA of N. crassa was reannealed with unlabeled bulk DNA from N. crassa, N. dodgei, and N. lineolata.

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

    PubMed Central

    Zuker, M; Jacobson, A B

    1995-01-01

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

  17. Low prevalence of clarithromycin-resistant Helicobacter pylori isolates with A2143G point mutation in the 23S rRNA gene in North India.

    PubMed

    Gehlot, Valentina; Mahant, Shweta; Mukhopadhyay, Asish Kumar; Das, Kunal; Alam, Jawed; Ghosh, Prachetash; Das, Rajashree

    2016-09-01

    Resistance of Helicobacter pylori to clarithromycin is associated with a single base substitution in the 23S rRNA gene. In this study, clarithromycin-resistant H. pylori isolates were analysed for the presence of 23S rRNA gene mutations. H. pylori were isolated from 68 patients suffering from various gastroduodenal diseases in North India. Minimum inhibitory concentrations (MICs) were determined by the agar dilution method, and point mutations in clarithromycin-resistant strains were identified by PCR-restriction fragment length polymorphism (PCR-RFLP) and DNA sequencing. Clarithromycin resistance was observed in 11.8% (8/68) of the H. pylori isolates in North India. The A2143G point mutation in the 23S rRNA gene was found in 87.5% (7/8) of the clarithromycin-resistant strains, and the A2142G mutation in association with the T2182C mutation was found in 12.5% (1/8). In conclusion, the continued high prevalence of clarithromycin-sensitive H. pylori strains (88.2%) observed in this study allows the use of the triple-therapy regimen for the treatment of H. pylori infection in this region. Surveillance studies need to be conducted at regular intervals for clarithromycin resistance in the population. To our knowledge, this is the first study in India to report that point mutations at position A2143G and at A2142G in association with T2182C are associated with clarithromycin resistance, confirming reports from other parts of the world. PMID:27530837

  18. Atypical processing in domain III of 23S rRNA of Rhizobium leguminosarum ATCC 10004(T) at a position homologous to an rRNA fragmentation site in protozoa.

    PubMed

    Klein, Franziska; Samorski, Regina; Klug, Gabriele; Evguenieva-Hackenberg, Elena

    2002-06-01

    For still unknown reasons, the 23S rRNA of many alpha-Proteobacteria shows a unique fragmentation pattern compared to other bacteria. The 23S rRNA processing involves RNase III and additional, yet unidentified enzymes. The alpha-proteobacterium Rhizobium leguminosarum ATCC 10004(T) possesses two fragmentation sites in its 23S rRNA. The first one harbors an intervening sequence in helix 9 which is cleaved by RNase III. We demonstrate that the mature 5' end of the resulting 2.6-kb rRNA fragment is generated by additional removal of helix 10. A fraction of the 2.6-kb rRNA is further processed in domain III, giving rise to two 1.3-kb rRNA fragments. We mapped the domain III fragmentation site and found it to be at a position which has only been reported for trypanosomatid protozoa. This fragmentation site is also unique in that it lacks an intervening sequence. We found that the simultaneous occurrence of 2.6-kb and 1.3-kb rRNA fragments is not due to interoperonal sequence differences but rather reflects slow processing. The different characteristics of the two fragmentation sites in the 23S rRNA suggest that they are processed by different mechanisms. Interestingly, the amount of 2.6-kb rRNA varies during culture growth. We observed a transient increase in the relative amount of 2.6-kb rRNA fragments during the first hours after inoculation, which points to changes in the ratio of rRNA synthesis rate to domain III processing rate during the growth of a culture.

  19. 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

  20. Discrimination of Bacillus anthracis and Closely Related Microorganisms by Analysis of 16S and 23S rRNA with Oligonucleotide Microarray

    PubMed Central

    Bavykin, Sergei G.; Mikhailovich, Vladimir M.; Zakharyev, Vladimir M.; Lysov, Yuri p.; Kelly, John J.; Alferov, Oleg S.; Gavin, Igor M.; Kukhtin, Alexander V.; Jackman, Joany; Stahl, David A.; Chandler, Darrell; Mirzabekov, Andrei D.

    2009-01-01

    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 minutes. 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

  1. High-level azithromycin resistance occurs in Neisseria gonorrhoeae as a result of a single point mutation in the 23S rRNA genes.

    PubMed

    Chisholm, Stephanie A; Dave, Jayshree; Ison, Catherine A

    2010-09-01

    High-level azithromycin resistance (AZM-HR), defined as a MIC of > or = 256 mg/liter, emerged in Neisseria gonorrhoeae in the United Kingdom in 2004. To determine the mechanism of this novel phenotype, isolates from the United Kingdom that were AZM-HR (n, 19), moderately AZM resistant (MICs, 2 to 8 mg/liter) (n, 26), or sensitive (MICs, 0.12 to 0.25 mg/liter) (n, 4) were screened for methylase (erm) genes and for mutations in the mtrR promoter region, associated with efflux pump upregulation. All AZM-resistant isolates and 12 sensitive isolates were screened for mutations in domain V of each 23S rRNA allele. All AZM-HR isolates contained the A2059G mutation (Escherichia coli numbering) in three (3 isolates) or four (16 isolates) 23S rRNA alleles. Most (22/26) moderately AZM resistant isolates contained the C2611T mutation in at least 3/4 alleles. The remainder contained four wild-type alleles, as did 8/12 sensitive isolates, while one allele was mutated in the remaining four sensitive isolates. Serial passage of AZM-sensitive colonies on an erythromycin-containing medium selected AZM-HR if the parent strain already contained mutation A2059G in one 23S rRNA allele. The resultant AZM-HR strains contained four mutated alleles. Eight isolates (five moderately AZM resistant and three AZM-HR) contained mutations in the mtrR promoter. No methylase genes were detected. This is the first evidence that AZM-HR in gonococci may result from a single point mutation (A2059G) in the peptidyltransferase loop in domain V of the 23S rRNA gene. Mutation of a single allele is insufficient to confer AZM-HR, but AZM-HR can develop under selection pressure. The description of a novel resistance mechanism will aid in screening for the AZM-HR phenotype. PMID:20585125

  2. 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

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

    PubMed

    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

    2015-01-30

    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.

  4. 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.

  5. The ribosome as a molecular machine: the mechanism of tRNA-mRNA movement in translocation.

    PubMed

    Rodnina, Marina V; Wintermeyer, Wolfgang

    2011-04-01

    Translocation of tRNA and mRNA through the ribosome is one of the most dynamic events during protein synthesis. In the cell, translocation is catalysed by EF-G (elongation factor G) and driven by GTP hydrolysis. Major unresolved questions are: how the movement is induced and what the moving parts of the ribosome are. Recent progress in time-resolved cryoelectron microscopy revealed trajectories of tRNA movement through the ribosome. Driven by thermal fluctuations, the ribosome spontaneously samples a large number of conformational states. The spontaneous movement of tRNAs through the ribosome is loosely coupled to the motions within the ribosome. EF-G stabilizes conformational states prone to translocation and promotes a conformational rearrangement of the ribosome (unlocking) that accelerates the rate-limiting step of translocation: the movement of the tRNA anticodons on the small ribosomal subunit. EF-G acts as a Brownian ratchet providing directional bias for movement at the cost of GTP hydrolysis.

  6. The human Shwachman-Diamond syndrome protein, SBDS, associates with ribosomal RNA.

    PubMed

    Ganapathi, Karthik A; Austin, Karyn M; Lee, Chung-Sheng; Dias, Anusha; Malsch, Maggie M; Reed, Robin; Shimamura, Akiko

    2007-09-01

    Shwachman-Diamond syndrome (SDS) is an autosomal recessive disorder characterized by bone marrow failure, exocrine pancreatic dysfunction, and leukemia predisposition. Mutations in the SBDS gene are identified in most patients with SDS. SBDS encodes a highly conserved protein of unknown function. Data from SBDS orthologs suggest that SBDS may play a role in ribosome biogenesis or RNA processing. Human SBDS is enriched in the nucleolus, the major cellular site of ribosome biogenesis. Here we report that SBDS nucleolar localization is dependent on active rRNA transcription. Cells from patients with SDS or Diamond-Blackfan anemia are hypersensitive to low doses of actinomycin D, an inhibitor of rRNA transcription. The addition of wild-type SBDS complements the actinomycin D hypersensitivity of SDS patient cells. SBDS migrates together with the 60S large ribosomal subunit in sucrose gradients and coprecipitates with 28S ribosomal RNA (rRNA). Loss of SBDS is not associated with a discrete block in rRNA maturation or with decreased levels of the 60S ribosomal subunit. SBDS forms a protein complex with nucleophosmin, a multifunctional protein implicated in ribosome biogenesis and leukemogenesis. Our studies support the addition of SDS to the growing list of human bone marrow failure syndromes involving the ribosome.

  7. The human Shwachman-Diamond syndrome protein, SBDS, associates with ribosomal RNA

    PubMed Central

    Ganapathi, Karthik A.; Austin, Karyn M.; Lee, Chung-Sheng; Dias, Anusha; Malsch, Maggie M.; Reed, Robin

    2007-01-01

    Shwachman-Diamond syndrome (SDS) is an autosomal recessive disorder characterized by bone marrow failure, exocrine pancreatic dysfunction, and leukemia predisposition. Mutations in the SBDS gene are identified in most patients with SDS. SBDS encodes a highly conserved protein of unknown function. Data from SBDS orthologs suggest that SBDS may play a role in ribosome biogenesis or RNA processing. Human SBDS is enriched in the nucleolus, the major cellular site of ribosome biogenesis. Here we report that SBDS nucleolar localization is dependent on active rRNA transcription. Cells from patients with SDS or Diamond-Blackfan anemia are hypersensitive to low doses of actinomycin D, an inhibitor of rRNA transcription. The addition of wild-type SBDS complements the actinomycin D hypersensitivity of SDS patient cells. SBDS migrates together with the 60S large ribosomal subunit in sucrose gradients and coprecipitates with 28S ribosomal RNA (rRNA). Loss of SBDS is not associated with a discrete block in rRNA maturation or with decreased levels of the 60S ribosomal subunit. SBDS forms a protein complex with nucleophosmin, a multifunctional protein implicated in ribosome biogenesis and leukemogenesis. Our studies support the addition of SDS to the growing list of human bone marrow failure syndromes involving the ribosome. PMID:17475909

  8. 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

  9. Variations in the 16S-23S rRNA internal transcribed spacer of fibrolytic Butyrivibrio isolates from the reindeer rumen.

    PubMed

    Præsteng, Kirsti E; Mackie, Roderick I; Cann, Isaac K O; Mathiesen, Svein D; Sundset, Monica A

    2011-07-01

    Strains of Butyrivibrio are principal cellulytic bacteria in the rumen of the High Arctic Svalbard reindeer ( Rangifer tarandus platyrhynchus ). According to phylogenetic analysis based on 16S rRNA gene sequencing, Butyrivibrio can be divided into three subgroups within the Clostridia class of the phylum Firmicutes, but the current phenotypic and genotypic differentiation within the family Lachnospiraceae is insufficient. This current study describes the sequence diversity of the 16S-23S rRNA intergenic transcribed spacer (ITS) region of Butyrivibrio isolates from reindeer. A total of 17 different ITS sequences with sizes between 449 and 784 nt were obtained. Genes encoding tRNA(Ile) and tRNA(Ala) were identified in four of the sequences. Phylogenetic neighbor-joining trees were constructed based on the ITS sequence and compared with a phylogenetic neighbor-joining tree based on 16S rRNA gene sequences previously obtained for the same isolates. These comparisons indicated a better differentiation between strains in the ITS sequence than the 16S rRNA gene based tree. Through this study, a better means for identifying and tracking fibrolytic and potentially probiotic Butyrivibrio strains in reindeer and other ruminants has been provided.

  10. 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.

  11. 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.

  12. The Mycoplasma gallisepticum 16S-23S rRNA intergenic spacer region sequence as a novel tool for epizootiological studies.

    PubMed

    Raviv, Ziv; Callison, S; Ferguson-Noel, N; Laibinis, V; Wooten, R; Kleven, S H

    2007-06-01

    Mycoplasma gallisepticum (MG) contains two sets of rRNA genes (5S, 16S and 23S) in its genome, but only one of the two is organized in an operon cluster and contains a unique 660-nucleotide intergenic spacer region (IGSR) between the 16S and the 23S rRNA genes. We designed a polymerase chain reaction (PCR) for the specific amplification of the complete MG IGSR segment. The MG IGSR PCR was tested on 18 avian mollicute species and was confirmed as MG specific. The reaction sensitivity was demonstrated by comparing it to the well-established MG mgc2 PCR. The MG IGSR sequence was found to be highly variable (discrimination [D] index of 0.950) among a variety of MG laboratory strains, vaccine strains, and field isolates. The sequencing of the MG IGSR appears to be a valuable single-locus sequence typing (SLST) tool for MG isolate differentiation in diagnostic cases and epizootiological studies. PMID:17626483

  13. 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. PMID:23269456

  14. Ribosome rescue: tmRNA tagging activity and capacity in Escherichia coli.

    PubMed

    Moore, Sean D; Sauer, Robert T

    2005-10-01

    When protein synthesis stalls in bacteria, tmRNA acts first as a surrogate tRNA and then as an mRNA in a series of reactions that append a peptide tag to the nascent polypeptide and 'rescue' the ribosome. The peptide tag encoded by wild-type tmRNA promotes rapid degradation of rescued proteins. Using a mutant tmRNA that encodes a tag that does not lead to degradation, we demonstrate that the synthesis of approximately 0.4% of all proteins terminates with tagging and ribosome rescue during normal exponential growth of Escherichia coli. The frequency of tagging was not significantly increased in cells expressing very high levels of tmRNA and its binding protein SmpB, suggesting that recognition of 'stalled' ribosomes does not involve competition between tmRNA and other translation factors for A-sites that are unoccupied transiently during protein synthesis. When the demand for ribosome rescue was increased artificially by overproduction of a non-stop mRNA, tmRNA levels did not increase but tmRNA-mediated tagging increased substantially. Thus, the ribosome-rescue system usually operates well below capacity.

  15. Identification of Lactobacillus Isolates from the Gastrointestinal Tract, Silage, and Yoghurt by 16S-23S rRNA Gene Intergenic Spacer Region Sequence Comparisons

    PubMed Central

    Tannock, G. W.; Tilsala-Timisjarvi, A.; Rodtong, S.; Ng, J.; Munro, K.; Alatossava, T.

    1999-01-01

    Lactobacillus isolates were identified by PCR amplification and sequencing of the region between the 16S and 23S rRNA genes (spacer region). The sequences obtained from the isolates were compared to those of reference strains held in GenBank. A similarity of 97.5% or greater was considered to provide identification. To check the reliability of the method, the V2-V3 region of the 16S rRNA gene was amplified and sequenced in the case of isolates whose spacer region sequences were less than 99% similar to that of a reference strain. Confirmation of identity was obtained in all instances. Spacer region sequencing provided rapid and accurate identification of Lactobacillus isolates obtained from gastrointestinal, yoghurt, and silage samples. It had an advantage over 16S V2-V3 sequence comparisons because it distinguished between isolates of Lactobacillus casei and Lactobacillus rhamnosus. PMID:10473450

  16. 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.

  17. 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.

  18. 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.

  19. Translation Initiation is Controlled by RNA Folding Kinetics via a Ribosome Drafting Mechanism.

    PubMed

    Espah Borujeni, Amin; Salis, Howard M

    2016-06-01

    RNA folding plays an important role in controlling protein synthesis as well as other cellular processes. Existing models have focused on how RNA folding energetics control translation initiation rate under equilibrium conditions but have largely ignored the effects of nonequilibrium RNA folding. We introduce a new mechanism, called "ribosome drafting", that explains how a mRNA's folding kinetics and the ribosome's binding rate collectively control its translation initiation rate. During cycles of translation, ribosome drafting emerges whenever successive ribosomes bind to a mRNA faster than the mRNA can refold, maintaining it in a nonequilibrium state with an acceleration of protein synthesis. Using computational design, time-correlated single photon counting, and expression measurements, we demonstrate that slow-folding and fast-folding RNA structures with equivalent folding energetics can vary protein synthesis rates by 1000-fold. We determine the necessary conditions for ribosome drafting by characterizing mRNAs with rationally designed ribosome binding rates, folding kinetics, and folding energetics, confirming the predictions of a nonequilibrium Markov model of translation. Our results have widespread implications, illustrating how competitive folding and assembly kinetics can shape the gene expression machinery's sequence-structure-function relationship inside cells. PMID:27199273

  20. 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.

  1. Functional analysis of the residues C770 and G771 of E. coli 16S rRNA implicated in forming the intersubunit bridge B2c of the ribosome.

    PubMed

    Kim, Hong-Man; Yeom, Ji-Hyun; Ha, Hye-Jung; Kim, Jong-Myung; Lee, Kangseok

    2007-07-01

    Structural analyses have shown that nucleotides at the positions 770 and 771 of Escherichia coli 16S rRNA are implicated in forming one of highly conserved intersubunit bridges of the ribosome, B2c. To examine a functional role of these residues, base substitutions were introduced at these positions and mutant ribosomes were analyzed for their protein synthesis ability using a specialized ribosome system. The results showed requirement of a pyrimidine at the position 770 for ribosome function regardless of the nucleotide identity at the position 771. Sucrose gradient profiles of ribosomes revealed that the loss of protein-synthesis ability of mutant ribosome bearing a base substitution from C to G at the position 770 stems from its inability to form 70S ribosomes. These findings indicate involvement of nucleotide at the position 770, not 771, in ribosomal subunit association and provide a useful rRNA mutation that can be used as a target to investigate the physical interaction between 16S and 23S rRNA.

  2. Characterization of the Lancefield group C streptococcus 16S-23S RNA gene intergenic spacer and its potential for identification and sub-specific typing.

    PubMed Central

    Chanter, N.; Collin, N.; Holmes, N.; Binns, M.; Mumford, J.

    1997-01-01

    The 16S-23S RNA gene intergenic spacers of isolates of Streptococcus equi (n = 5), S. zooepidemicus (n = 5), S. equisimilis (n = 3) and S. dysgalactiae (n = 2) were sequenced and compared. There were distinct regions within the spacer, arranged in the order 1-9 for all S. equi and one S. zooepidemicus isolate and 1,2 and 4-9 for the remaining isolates. Region 4 was identical to the tRNA(ala) gene found in the 16S-23S intergenic spacers of other streptococci. Regions 1, 5, 6 and 7 had distinct variations, each conserved in different isolates. However, amongst the intergenic spacers there were different combinations of variant regions, suggesting a role for DNA recombination in their evolution. The intergenic spacer of all isolates of S. equi and one S. zooepidemicus isolate were almost identical. Primers derived from the variant sequences of regions 1 and 5 to 6 were used to group all S. zooepidemicus (n = 17) and S. equi (n = 5) into 1 of 8 types by polymerase chain reaction; three S. zooepidemicus isolates typed the same as S. equi. S. equi and S. zooepidemicus were clearly distinguishable from S. equisimilis and S. dysgalactiae which had shorter regions 5 and 6 and no region 7. Most homology for the group C sequences was found in previously published sequences for the 16S-23S intergenic spacers of S. anginosis, S. constellatus, S. intermedius, S. salivarius and S. agalactiae. A 75-90 nucleotide length shared with S. anginosus and S. intermedius in opposite orientations in the two main variants of region 6 supported the role for DNA recombination in the evolution of the spacer. The 16S-23S intergenic spacers indicate that S. zooepidemicus was the archetypal species for S. equi and that both are genetically more distant from S. equisimilis and S. dysgalactiae. The intergenic spacer can be used to identify specifically the group C streptococci and as an epidemiological marker for S. zooepidemicus. PMID:9129589

  3. 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.

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

    PubMed

    Xie, Ping

    2014-07-01

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

  5. What do we know about ribosomal RNA methylation in Escherichia coli?

    PubMed

    Sergeeva, O V; Bogdanov, A A; Sergiev, P V

    2015-10-01

    A ribosome is a ribonucleoprotein that performs the synthesis of proteins. Ribosomal RNA of all organisms includes a number of modified nucleotides, such as base or ribose methylated and pseudouridines. Methylated nucleotides are highly conserved in bacteria and some even universally. In this review we discuss available data on a set of modification sites in the most studied bacteria, Escherichia coli. While most rRNA modification enzymes are known for this organism, the function of the modified nucleotides is rarely identified.

  6. Comparison of Ribosomal RNA Removal Methods for Transcriptome Sequencing Workflows in Teleost Fish.

    PubMed

    Abernathy, Jason; Overturf, Ken

    2016-01-01

    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. These methods have been well documented in mammals but typically require some optimization for lower vertebrates. Three commercial kits, including Dynabeads mRNA Purification Kit, RiboMinus Eukaryote System v2, and Ribo-Zero Gold rRNA Removal Kit were examined for the ability to remove rRNAs from rainbow trout (Oncorhynchus mykiss) RNA isolations. Total RNA was isolated from liver and muscle tissue samples (n = 24) and rRNAs removed using one of the three kits. Samples were analyzed visually on the Agilent Bioanalyzer and by Illumina RNA-seq, screening for Oncorhynchus rRNAs. There were significant differences between the kits in regards to their ability to remove rRNA, ranging from 2.74% - 10.94% rRNA sequences left behind per kit on average. Using the Bioanalyzer to evaluate ribosomal contamination in rRNA-depleted samples for RNA-Seq was good for detecting samples with higher concentrations of rRNA (>5%), but not very accurate at lower levels. Although all three kits were able to remove a substantial portion of the rRNA from different fish tissues, the Ribo-Zero Gold rRNA Removal Kit eliminated significantly more contaminating ribosomal RNAs than the others.

  7. The size and conformation of Artemia (brine-shrimp) ribosomal RNA free in solution.

    PubMed Central

    Donceel, K; Nieuwenhuysen, P; Clauwaert, J

    1982-01-01

    The RNA was isolated from the large ribosomal subunits of the brine shrimp Artemia, and its conformation free in solution was studied by determining its sedimentation and diffusion coefficients. A comparison was made of the hydrodynamic radius of the ribosomal subunit and its isolated RNA in various buffers. The conformation of the rRNA free in solution is more extended than when it is incorporated in the ribosome. This is not only the case when the rRNA solution lacks bivalent and polyvalent cations, but even in the presence of Mg2+ and spermidine, which cause a tightening of RNA. Thus the ribosomal proteins should induce a further tightening of the rRNA during the assembly of the ribosome. In the discussion, the reported data on Escherichia coli rRNA species are presented in such a way that large discrepancies between various studied are revealed, and that they can be compared with the data reported here on the larger rRNA of an eukaryote. PMID:7150228

  8. Eukaryotic ribosomal RNA determinants of aminoglycoside resistance and their role in translational fidelity.

    PubMed

    Fan-Minogue, Hua; Bedwell, David M

    2008-01-01

    Recent studies of prokaryotic ribosomes have dramatically increased our knowledge of ribosomal RNA (rRNA) structure, functional centers, and their interactions with antibiotics. However, much less is known about how rRNA function differs between prokaryotic and eukaryotic ribosomes. The core decoding sites are identical in yeast and human 18S rRNAs, suggesting that insights obtained in studies with yeast rRNA mutants can provide information about ribosome function in both species. In this study, we examined the importance of key nucleotides of the 18S rRNA decoding site on ribosome function and aminoglycoside susceptibility in Saccharomyces cerevisiae cells expressing homogeneous populations of mutant ribosomes. We found that residues G577, A1755, and A1756 (corresponding to Escherichia coli residues G530, A1492, and A1493, respectively) are essential for cell viability. We also found that residue G1645 (A1408 in E. coli) and A1754 (G1491 in E. coli) both make significant and distinct contributions to aminoglycoside resistance. Furthermore, we found that mutations at these residues do not alter the basal level of translational accuracy, but influence both paromomycin-induced misreading of sense codons and readthrough of stop codons. This study represents the most comprehensive mutational analysis of the eukaryotic decoding site to date, and suggests that many fundamental features of decoding site function are conserved between prokaryotes and eukaryotes.

  9. Nucleotide sequence of a crustacean 18S ribosomal RNA gene and secondary structure of eukaryotic small subunit ribosomal RNAs.

    PubMed

    Nelles, L; Fang, B L; Volckaert, G; Vandenberghe, A; De Wachter, R

    1984-12-11

    The primary structure of the gene for 18 S rRNA of the crustacean Artemia salina was determined. The sequence has been aligned with 13 other small ribosomal subunit RNA sequences of eukaryotic, archaebacterial, eubacterial, chloroplastic and plant mitochondrial origin. Secondary structure models for these RNAs were derived on the basis of previously proposed models and additional comparative evidence found in the alignment. Although there is a general similarity in the secondary structure models for eukaryotes and prokaryotes, the evidence seems to indicate a different topology in a central area of the structures.

  10. Accumulation of a mRNA decay intermediate by ribosomal pausing at a stop codon.

    PubMed Central

    Björnsson, A; Isaksson, L A

    1996-01-01

    A RNA fragment which is protected from degradation by ribosome pausing at a stop codon has been identified in growing Escherichia coli. The fragment is 261 nt long and corresponds to the 3'-end of the mRNA expressed from a semi-synthetic model gene. The 5'-end of the RNA fragment, denoted rpRNA (ribosomal pause RNA), is located 13 bases upstream of the stop codon. In vivo decay of the complete mRNA and accumulation of rpRNA are dependent on the nature of the stop codon and its codon context. The data indicate that the rpRNA fragment arises from interrupted decay of the S3A'mRNA in the 5'-->m3'direction, in connection with a ribosomal pause at the stop codon. RF-2 decoding of UGA is less efficient than RF-1 decoding of UAG in identical codon contexts, as judged from rpRNA steady-state levels. The half-life of UGA-containing rpRNAs is at least 5 min, indicating that ribosomal pausing can be a major factor in stabilising downstream regions of messenger RNAs. PMID:8649996

  11. 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.

  12. Ribosomal protein S7 from Escherichia coli uses the same determinants to bind 16S ribosomal RNA and its messenger RNA

    PubMed Central

    Robert, Francis; Brakier-Gingras, Léa

    2001-01-01

    Ribosomal protein S7 from Escherichia coli binds to the lower half of the 3′ major domain of 16S rRNA and initiates its folding. It also binds to its own mRNA, the str mRNA, and represses its translation. Using filter binding assays, we show in this study that the same mutations that interfere with S7 binding to 16S rRNA also weaken its affinity for its mRNA. This suggests that the same protein regions are responsible for mRNA and rRNA binding affinities, and that S7 recognizes identical sequence elements within the two RNA targets, although they have dissimilar secondary structures. Overexpression of S7 is known to inhibit bacterial growth. This phenotypic growth defect was relieved in cells overexpressing S7 mutants that bind poorly the str mRNA, confirming that growth impairment is controlled by the binding of S7 to its mRNA. Interestingly, a mutant with a short deletion at the C-terminus of S7 was more detrimental to cell growth than wild-type S7. This suggests that the C-terminal portion of S7 plays an important role in ribosome function, which is perturbed by the deletion. PMID:11160889

  13. Ribosomal protein S7 from Escherichia coli uses the same determinants to bind 16S ribosomal RNA and its messenger RNA.

    PubMed

    Robert, F; Brakier-Gingras, L

    2001-02-01

    Ribosomal protein S7 from Escherichia coli binds to the lower half of the 3' major domain of 16S rRNA and initiates its folding. It also binds to its own mRNA, the str mRNA, and represses its translation. Using filter binding assays, we show in this study that the same mutations that interfere with S7 binding to 16S rRNA also weaken its affinity for its mRNA. This suggests that the same protein regions are responsible for mRNA and rRNA binding affinities, and that S7 recognizes identical sequence elements within the two RNA targets, although they have dissimilar secondary structures. Overexpression of S7 is known to inhibit bacterial growth. This phenotypic growth defect was relieved in cells overexpressing S7 mutants that bind poorly the str mRNA, confirming that growth impairment is controlled by the binding of S7 to its mRNA. Interestingly, a mutant with a short deletion at the C-terminus of S7 was more detrimental to cell growth than wild-type S7. This suggests that the C-terminal portion of S7 plays an important role in ribosome function, which is perturbed by the deletion.

  14. 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.

  15. 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

  16. Cap-dependent translation is mediated by 'RNA looping' rather than 'ribosome scanning'.

    PubMed

    Jang, Sung Key; Paek, Ki Young

    2016-01-01

    The 40S ribosomal subunit cannot directly recognize the start codon of eukaryotic mRNAs. Instead, it recognizes the start codon after its association with the 5'-cap structure via translation initiation factors. Base-by-base inspection of the 5'UTR by a scanning ribosome is the generally accepted hypothesis of start codon selection. As part of an effort to confirm the underlying mechanism of start codon selection by the 40S ribosome, we investigated the role of eIF4G, which participates in the recruitment of 40S ribosomes to various translation enhancers, such as 5'-cap structure, poly(A) tail, and several internal ribosome entry sites. We found that an artificial translation factor composed of recombinant eIF4G fused with MS2 greatly enhanced translation of an upstream reporter gene when it was tethered to the 3'UTR. These data suggest that the 40S ribosome recruited to a translation enhancer can find the start codon by looping of the intervening RNA segment. The 'RNA-looping' hypothesis of translation start codon recognition was further supported by an analysis of the effect of 5'UTR length on translation efficiency and the mathematically predicted probability of RNA-loop-mediated interactions between the start codon and the 40S ribosome associated at the 5'-end. PMID:26515582

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

    NASA Astrophysics Data System (ADS)

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

    2008-12-01

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

  18. 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

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

    PubMed

    Sunita, S; Zhenxing, H; Swaathi, J; Cygler, Miroslaw; Matte, Allan; Sivaraman, J

    2006-06-16

    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 2.6A 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.

  20. Chloroplast RH3 DEAD box RNA helicases in maize and Arabidopsis function in splicing of specific group II introns and affect chloroplast ribosome biogenesis.

    PubMed

    Asakura, Yukari; Galarneau, Erin; Watkins, Kenneth P; Barkan, Alice; van Wijk, Klaas J

    2012-07-01

    Chloroplasts in angiosperms contain at least seven nucleus-encoded members of the DEAD box RNA helicase family. Phylogenetic analysis shows that five of these plastid members (RH22, -39, -47, -50, and -58) form a single clade and that RH3 forms a clade with two mitochondrial RH proteins (PMH1 and -2) functioning in intron splicing. The function of chloroplast RH3 in maize (Zea mays; ZmRH3) and Arabidopsis (Arabidopsis thaliana; AtRH3) was determined. ZmRH3 and AtRH3 are both under strong developmental control, and ZmRH3 abundance sharply peaked in the sink-source transition zone of developing maize leaves, coincident with the plastid biogenesis machinery. ZmRH3 coimmunoprecipitated with a specific set of plastid RNAs, including several group II introns, as well as pre23S and 23S ribosomal RNA (rRNA), but not 16S rRNA. Furthermore, ZmRH3 associated with 50S preribosome particles as well as nucleoids. AtRH3 null mutants are embryo lethal, whereas a weak allele (rh3-4) results in pale-green seedlings with defects in splicing of several group II introns and rRNA maturation as well as reduced levels of assembled ribosomes. These results provide strong evidence that RH3 functions in the splicing of group II introns and possibly also contributes to the assembly of the 50S ribosomal particle. Previously, we observed 5- to 10-fold up-regulation of AtRH3 in plastid Caseinolytic protease mutants. The results shown here indicate that AtRH3 up-regulation was not a direct consequence of reduced proteolysis but constituted a compensatory response at both RH3 transcript and protein levels to impaired chloroplast biogenesis; this response demonstrates that cross talk between the chloroplast and the nucleus is used to regulate RH3 levels.

  1. Binding of 16S rRNA to chloroplast 30S ribosomal proteins blotted on nitrocellulose.

    PubMed

    Rozier, C; Mache, R

    1984-10-11

    Protein-RNA associations were studied by a method using proteins blotted on a nitrocellulose sheet. This method was assayed with Escherichia Coli 30S ribosomal components. In stringent conditions (300 mM NaCl or 20 degrees C) only 9 E. coli ribosomal proteins strongly bound to the 16S rRNA: S4, S5, S7, S9, S12, S13, S14, S19, S20. 8 of these proteins have been previously found to bind independently to the 16S rRNA. The same method was applied to determine protein-RNA interactions in spinach chloroplast 30S ribosomal subunits. A set of only 7 proteins was bound to chloroplast rRNA in stringent conditions: chloroplast S6, S10, S11, S14, S15, S17 and S22. They also bound to E. coli 16S rRNA. This set includes 4 chloroplast-synthesized proteins: S6, S11, S15 and S22. The core particles obtained after treatment by LiCl of chloroplast 30S ribosomal subunit contained 3 proteins (S6, S10 and S14) which are included in the set of 7 binding proteins. This set of proteins probably play a part in the early steps of the assembly of the chloroplast 30S ribosomal subunit.

  2. DExD-box RNA-helicases in Listeria monocytogenes are important for growth, ribosomal maturation, rRNA processing and virulence factor expression

    PubMed Central

    Bäreclev, Caroline; Vaitkevicius, Karolis; Netterling, Sakura; Johansson, Jörgen

    2014-01-01

    RNA-helicases are proteins required for the unwinding of occluding secondary RNA structures, especially at low temperatures. In this work, we have deleted all 4 DExD-box RNA helicases in various combinations in the Gram-positive pathogen Listeria monocytogenes. Our results show that 3 out of 4 RNA-helicases were important for growth at low temperatures, whereas the effect was less prominent at 37°C. Over-expression of one RNA-helicase, Lmo1450, was able to overcome the reduced growth of the quadruple mutant strain at temperatures above 26°C, but not at lower temperatures. The maturation of ribosomes was affected in different degrees in the various strains at 20°C, whereas the effect was marginal at 37°C. This was accompanied by an increased level of immature 23S rRNA precursors in some of the RNA-helicase mutants at low temperatures. Although the expression of the PrfA regulated virulence factors ActA and LLO decreased in the quadruple mutant strain, this strain showed a slightly increased infection ability. Interestingly, even though the level of the virulence factor LLO was decreased in the quadruple mutant strain as compared with the wild-type strain, the hly-transcript (encoding LLO) was increased. Hence, our results could suggest a role for the RNA-helicases during translation. In this work, we show that DExD-box RNA-helicases are involved in bacterial virulence gene-expression and infection of eukaryotic cells. PMID:25590644

  3. Depletion of ribosomal protein S19 causes a reduction of rRNA synthesis

    PubMed Central

    Juli, Giada; Gismondi, Angelo; Monteleone, Valentina; Caldarola, Sara; Iadevaia, Valentina; Aspesi, Anna; Dianzani, Irma; Proud, Christopher G.; Loreni, Fabrizio

    2016-01-01

    Ribosome biogenesis plays key roles in cell growth by providing increased capacity for protein synthesis. It requires coordinated production of ribosomal proteins (RP) and ribosomal RNA (rRNA), including the processing of the latter. Here, we show that, the depletion of RPS19 causes a reduction of rRNA synthesis in cell lines of both erythroid and non-erythroid origin. A similar effect is observed upon depletion of RPS6 or RPL11. The deficiency of RPS19 does not alter the stability of rRNA, but instead leads to an inhibition of RNA Polymerase I (Pol I) activity. In fact, results of nuclear run-on assays and ChIP experiments show that association of Pol I with the rRNA gene is reduced in RPS19-depleted cells. The phosphorylation of three known regulators of Pol I, CDK2, AKT and AMPK, is altered during ribosomal stress and could be involved in the observed downregulation. Finally, RNA from patients with Diamond Blackfan Anemia (DBA), shows, on average, a lower level of 47S precursor. This indicates that inhibition of rRNA synthesis could be one of the molecular alterations at the basis of DBA. PMID:27734913

  4. 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.

  5. 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

  6. The development of peptide ligands that target helix 69 rRNA of bacterial ribosomes.

    PubMed

    Dremann, Danielle N; Chow, Christine S

    2016-09-15

    Antibiotic resistance prevents successful treatment of common bacterial infections, making it clear that new target locations and drugs are required to resolve this ongoing challenge. The bacterial ribosome is a common target for antibacterials due to its essential contribution to cell viability. The focus of this work is a region of the ribosome called helix 69 (H69), which was recently identified as a secondary target site for aminoglycoside antibiotics. H69 has key roles in essential ribosomal processes such as subunit association, ribosome recycling, and tRNA selection. Conserved across phylogeny, bacterial H69 also contains two pseudouridines and one 3-methylpseudouridine. Phage display revealed a heptameric peptide sequence that targeted H69. Using solid-phase synthesis, peptide variants with higher affinity and improved selectivity to modified H69 were generated. Electrospray ionization mass spectrometry was used to determine relative apparent dissociation constants of the RNA-peptide complexes. PMID:27492196

  7. 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

    2016-01-01

    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

  8. 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

  9. Murine Leukemia Virus Nucleocapsid Mutant Particles Lacking Viral RNA Encapsidate Ribosomes

    PubMed Central

    Muriaux, Delphine; Mirro, Jane; Nagashima, Kunio; Harvin, Demetria; Rein, Alan

    2002-01-01

    A single retroviral protein, termed Gag, is sufficient for assembly of retrovirus-like particles in mammalian cells. Gag normally selects the genomic RNA of the virus with high specificity; the nucleocapsid (NC) domain of Gag plays a crucial role in this selection process. However, encapsidation of the viral RNA is completely unnecessary for particle assembly. We previously showed that mutant murine leukemia virus (MuLV) particles that lack viral RNA because of a deletion in the cis-acting packaging signal (“Ψ”) in the genomic RNA compensate for the loss of the viral RNA by incorporating cellular mRNA. The RNA in wild-type and Ψ− particles was also found to be necessary for virion core structure. In the present work, we explored the role of RNA in MuLV particles that lack genomic RNA because of mutations in the NC domain of Gag. Using a fluorescent dye assay, we observed that NC mutant particles contain the same amount of RNA that wild-type virions do. Surprisingly enough, these particles contained large amounts of rRNAs. Furthermore, ribosomal proteins were detected by immunoblotting, and ribosomes were observed inside the particles by electron microscopy. The biological significance of the presence of ribosomes in NC mutant particles lacking genomic RNA is discussed. PMID:12388701

  10. Both endonucleolytic and exonucleolytic cleavage mediate ITS1 removal during human ribosomal RNA processing.

    PubMed

    Sloan, Katherine E; Mattijssen, Sandy; Lebaron, Simon; Tollervey, David; Pruijn, Ger J M; Watkins, Nicholas J

    2013-03-01

    Human ribosome production is up-regulated during tumorogenesis and is defective in many genetic diseases (ribosomopathies). We have undertaken a detailed analysis of human precursor ribosomal RNA (pre-rRNA) processing because surprisingly little is known about this important pathway. Processing in internal transcribed spacer 1 (ITS1) is a key step that separates the rRNA components of the large and small ribosomal subunits. We report that this was initiated by endonuclease cleavage, which required large subunit biogenesis factors. This was followed by 3' to 5' exonucleolytic processing by RRP6 and the exosome, an enzyme complex not previously linked to ITS1 removal. In contrast, RNA interference-mediated knockdown of the endoribonuclease MRP did not result in a clear defect in ITS1 processing. Despite the apparently high evolutionary conservation of the pre-rRNA processing pathway and ribosome synthesis factors, each of these features of human ITS1 processing is distinct from those in budding yeast. These results also provide significant insight into the links between ribosomopathies and ribosome production in human cells. PMID:23439679

  11. 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.

  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. Protein-RNA Dynamics in the Central Junction Control 30S Ribosome Assembly.

    PubMed

    Baker, Kris Ann; Lamichhane, Rajan; Lamichhane, Tek; Rueda, David; Cunningham, Philip R

    2016-09-11

    Interactions between ribosomal proteins (rproteins) and ribosomal RNA (rRNA) facilitate the formation of functional ribosomes. S15 is a central domain primary binding protein that has been shown to trigger a cascade of conformational changes in 16S rRNA, forming the functional structure of the central domain. Previous biochemical and structural studies in vitro have revealed that S15 binds a three-way junction of helices 20, 21, and 22, including nucleotides 652-654 and 752-754. All junction nucleotides except 653 are highly conserved among the Bacteria. To identify functionally important motifs within the junction, we subjected nucleotides 652-654 and 752-754 to saturation mutagenesis and selected and analyzed functional mutants. Only 64 mutants with greater than 10% ribosome function in vivo were isolated. S15 overexpression complemented mutations in the junction loop in each of the partially active mutants, although mutations that produced inactive ribosomes were not complemented by overexpression of S15. Single-molecule Förster or fluorescence resonance energy transfer (smFRET) was used to study the Mg(2+)- and S15-induced conformational dynamics of selected junction mutants. Comparison of the structural dynamics of these mutants with the wild type in the presence and absence of S15 revealed specific sequence and structural motifs in the central junction that are important in ribosome function. PMID:27192112

  14. 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

  15. Beyond ribosome rescue: tmRNA and co-translational processes

    PubMed Central

    Hayes, Christopher S.; Keiler, Kenneth C.

    2009-01-01

    tmRNA is a unique bi-functional RNA that acts as both a tRNA and an mRNA to enter stalled ribosomes and direct the addition of a peptide tag to the C terminus of nascent polypeptides. Despite a reasonably clear understanding of tmRNA activity, the reason for its absolute conservation throughout the eubacteria is unknown. Although tmRNA plays many physiological roles in different bacterial systems, recent studies suggest a general role for trans-translation in monitoring protein folding and perhaps other co-translational processes. This review will focus on these new hypotheses and the data that support them. PMID:19914241

  16. Ribosomal RNA and protein transcripts persist in the cysts of Entamoeba invadens.

    PubMed

    Ojha, Sandeep; Ahamad, Jamaluddin; Bhattacharya, Alok; Bhattacharya, Sudha

    2014-06-01

    In most organisms rDNA transcription ceases under conditions of growth stress. However, we have earlier shown that pre-rRNA accumulates during encystation in Entamoeba invadens. We labeled newly-synthesized rRNA during encystation, with [methyl-(3)H] methionine in the presence of chitinase to enable uptake of isotope. Incorporation rate reduced after 24h, and then increased to reach levels comparable with normal cells. The label was rapidly chased to the ribosomal pellet in dividing cells, while at late stages of encystation the ratio of counts going to the pellet dropped 3-fold. The transcript levels of selected ribosomal protein genes also went down initially but went up again at later stages of encystation. This suggested that rRNA and ribosomal protein transcription may be coordinately regulated. Our data shows that encysting E. invadens cells accumulate transcripts of both the RNA and protein components of the ribosome, which may ensure rapid synthesis of new ribosomes when growth resumes.

  17. 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.

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

    PubMed Central

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

    2014-01-01

    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. PMID:24569352

  19. [Affinity modification of Escherichia coli ribosomes with photoactivated analogs of mRNA].

    PubMed

    Gimautdinova, O I; Zenkova, M A; Karpova, G G; Podust, L M

    1984-01-01

    Oligoribonucleotide derivatives containing the photoactivated arylazidogroup at 5'-end of the oligonucleotide fragment [2-(N-2,4-dinitro-5-azidophenyl) aminoethyl] phosphamides of the oligoribonucleotides, azido-NH (CH2)2NHpN (pN) n-1, were prepared. It was demonstrated that azido-NH(CH2)2NHpA(pA)4 and azido-NH (CH2)2NHpU (pU)3 stimulate the binding of the codonspecific aminoacyl-tRNA with ribosome. After irradiation of the ternary complex ribosome-azido-NH (CH2)2NHpU (pU) n-1 X tRNA with UV-light (lambda greater than 350 nm) covalent binding of the reagent to ribosome occurs. Up to 10% of the reagent, bound in the ternary complex with ribosome, is cross-linked with the ribosomal proteins of 30S and 50S subunits. The ribosomal RNA are not modified by azido-NH (CH2)2NHpU (pU) n-1. The proteins of 30S and 50S subunits, modified with azido-NH (CH2)2NHpU (pU) n-1 with n = 4,7 and 8, were identified. It is shown that proteins of 30S subunits S3, S4, S9, S11, S12, S14, S17, S19, S20 undergo modification. The proteins of 50S subunits L2, L13, L16, L27, L32, L33 are modified. The set of the modified proteins essentially depends on the length of the oligonucleotide part of the reagent and on occupancy of ribosome A-site by a molecule of tRNA.

  20. 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

  1. Proceedings of the international workshop on Ribosomal RNA technology, April 7-9, 2008, Bremen, Germany.

    PubMed

    Amaral-Zettler, Linda; Peplies, Jörg; Ramette, Alban; Fuchs, Bernhard; Ludwig, Wolfgang; Glöckner, Frank Oliver

    2008-09-01

    Thirty years have passed since Carl Woese proposed three primary domains of life based on the phylogenetic analysis of ribosomal RNA (rRNA) genes. Adopted by researchers worldwide, rRNA has become the "gold-standard" for molecular taxonomy, biodiversity analysis and the identification of microorganisms. The more than 700,000 rRNA sequences in public databases constitute an unprecedented hallmark of the richness of microbial biodiversity on earth. The International Workshop on Ribosomal RNA Technology convened on April 7-9, 2008 in Bremen, Germany (http://www.arb-silva.de/rrna-workshop) to summarize the current status of the field and strategize on the best ways of proceeding on both biological and technological fronts. In five sessions, 26 leading international speakers and approximately 120 participants representing diverse disciplines discussed new technological approaches to address three basic ecological questions: "Who is out there?" "How many are there?" and "What are they doing?".

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

    PubMed

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

    2016-07-14

    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. PMID:27421425

  3. Development of a 16S-23S rRNA intergenic spacer-based quantitative PCR assay for improved detection and enumeration of Lactococcus garvieae.

    PubMed

    Thanh, Hien Dang; Park, Hee Kuk; Kim, Wonyong; Shin, Hyoung-Shik

    2013-02-01

    Lactococcus garvieae is an important foodborne pathogen causing lactococcosis associated with hemorrhagic septicemia in fish worldwide. A real-time quantitative polymerase chain reaction (qPCR) protocol targeting the 16S-23S rRNA intergenic spacer (ITS) region was developed for the detection and enum-eration of L. garvieae. The specificity was evaluated using genomic DNAs extracted from 66 cocci strains. Fourteen L. garvieae strains tested were positive, whereas 52 other strains including Lactococcus lactis ssp. lactis, Lactococcus lactis ssp. hordniae and Lactococcus lactis ssp. cremoris did not show a specific signal. The minimal limit of detection was 2.63 fg of purified genomic DNA, equivalent to 1 genome of L. garvieae. The optimized protocol was applied for the survey of L. garvieae in naturally contaminated fish samples. Our results suggest that the qPCR protocol using ITS is a sensitive and efficient tool for the rapid detection and enumeration of L. garvieae in fish and fish-containing foods.

  4. Distribution of rRNA introns in the three-dimensional structure of the ribosome.

    PubMed

    Jackson, Scott; Cannone, Jamie; Lee, Jung; Gutell, Robin; Woodson, Sarah

    2002-10-11

    More than 1200 introns have been documented at over 150 unique sites in the small and large subunit ribosomal RNA genes (as of February 2002). Nearly all of these introns are assigned to one of four main types: group I, group II, archaeal and spliceosomal. This sequence information has been organized into a relational database that is accessible through the Comparative RNA Web Site (http://www.rna.icmb.utexas.edu/) While the rRNA introns are distributed across the entire tree of life, the majority of introns occur within a few phylogenetic groups. We analyzed the distributions of rRNA introns within the three-dimensional structures of the 30S and 50S ribosomes. Most sites in rRNA genes that contain introns contain only one type of intron. While the intron insertion sites occur at many different coordinates, the majority are clustered near conserved residues that form tRNA binding sites and the subunit interface. Contrary to our expectations, many of these positions are not accessible to solvent in the mature ribosome. The correlation between the frequency of intron insertions and proximity of the insertion site to functionally important residues suggests an association between intron evolution and rRNA function.

  5. Correlation between mechanical strength of messenger RNA pseudoknots and ribosomal frameshifting

    PubMed Central

    Hansen, Thomas M.; Reihani, S. Nader S.; Oddershede, Lene B.; Sørensen, Michael A.

    2007-01-01

    Programmed ribosomal frameshifting is often used by viral pathogens including HIV. Slippery sequences present in some mRNAs cause the ribosome to shift reading frame. The resulting protein is thus encoded by one reading frame upstream from the slippery sequence and by another reading frame downstream from the slippery sequence. Although the mechanism is not well understood, frameshifting is known to be stimulated by an mRNA structure such as a pseudoknot. Here, we show that the efficiency of frameshifting relates to the mechanical strength of the pseudoknot. Two pseudoknots derived from the Infectious Bronchitis Virus were used, differing by one base pair in the first stem. In Escherichia coli, these two pseudoknots caused frameshifting frequencies that differed by a factor of two. We used optical tweezers to unfold the pseudoknots. The pseudoknot giving rise to the highest degree of frameshifting required a nearly 2-fold larger unfolding force than the other. The observed energy difference cannot be accounted for by any existing model. We propose that the degree of ribosomal frameshifting is related to the mechanical strength of RNA pseudoknots. Our observations support the “9 Å model” that predicts some physical barrier is needed to force the ribosome into the −1 frame. Also, our findings support the recent observation made by cryoelectron microscopy that mechanical interaction between a ribosome and a pseudoknot causes a deformation of the A-site tRNA. The result has implications for the understanding of genetic regulation, reading frame maintenance, tRNA movement, and unwinding of mRNA secondary structures by ribosomes. PMID:17389398

  6. 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

  7. Early life stress inhibits expression of ribosomal RNA in the developing hippocampus.

    PubMed

    Wei, Lan; Hao, Jin; Kaffman, Arie

    2014-01-01

    Children that are exposed to abuse or neglect show abnormal hippocampal function. However, the developmental mechanisms by which early life stress (ELS) impairs normal hippocampal development have not been elucidated. Here we propose that exposure to ELS blunts normal hippocampal growth by inhibiting the availability of ribosomal RNA (rRNA). In support of this hypothesis, we show that the normal mouse hippocampus undergoes a growth-spurt during the second week of life, followed by a gradual decrease in DNA and RNA content that persists into adulthood. This developmental pattern is associated with accelerated ribosomal RNA (rRNA) synthesis during the second week of life, followed by a gradual decline in rRNA levels that continue into adulthood. Levels of DNA methylation at the ribosomal DNA (rDNA) promoter are lower during the second week of life compared to earlier development or adulthood. Exposure to brief daily separation (BDS), a mouse model of early life stress, increased DNA methylation at the ribosomal DNA promoter, decreased rRNA levels, and blunted hippocampal growth during the second week of life. Exposure to acute (3 hrs) maternal separation decreased rRNA and increased DNA methylation at the rDNA proximal promoter, suggesting that exposure to stress early in life can rapidly regulate the availability of rRNA levels in the developing hippocampus. Given the critical role that rRNA plays in supporting normal growth and development, these findings suggest a novel molecular mechanism to explain how stress early in life impairs hippocampus development in the mouse. PMID:25517398

  8. Molecular contacts of ribose-phosphate backbone of mRNA with human ribosome.

    PubMed

    Sharifulin, Dmitri E; Grosheva, Anastasia S; Bartuli, Yulia S; Malygin, Alexey A; Meschaninova, Maria I; Ven'yaminova, Aliya G; Stahl, Joachim; Graifer, Dmitri M; Karpova, Galina G

    2015-08-01

    In this work, intimate contacts of riboses of mRNA stretch from nucleotides in positions +3 to +12 with respect to the first nucleotide of the P site codon were studied using cross-linking of short mRNA analogs with oxidized 3'-terminal riboses bound to human ribosomes in the complexes stabilized by codon-anticodon interactions and in the binary complexes. It was shown that in all types of complexes cross-links of the mRNA analogs to ribosomal protein (rp) uS3 occur and the yield of these cross-links does not depend on the presence of tRNA and on sequences of the mRNA analogs. Site of the mRNA analogs cross-linking in rp uS3 was mapped to the peptide in positions 55-64 that is located away from the mRNA binding site. Additionally, in complexes with P site-bound tRNA, riboses of mRNA nucleotides in positions +4 to +7 cross-linked to the C-terminal tail of rp uS19 displaying a contact specific to the decoding site of the mammalian ribosome, and tRNA bound at the A site completely blocked this cross-linking. Remarkably, rps uS3 and uS19 were also able to cross-link to the fragment of HCV IRES containing unstructured 3'-terminal part restricted by the AUGC tetraplet with oxidized 3'-terminal ribose. However, no cross-linking to rp uS3 was observed in the 48S preinitiation complex assembled in reticulocyte lysate with this HCV IRES derivative. The results obtained show an ability of rp uS3 to interact with single-stranded RNAs. Possible roles of rp uS3 region 55-64 in the functioning of ribosomes are discussed.

  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. Elongation factor 4 remodels the A-site tRNA on the ribosome

    PubMed Central

    Gagnon, Matthieu G.; Lin, Jinzhong; Steitz, Thomas A.

    2016-01-01

    During translation, a plethora of protein factors bind to the ribosome and regulate protein synthesis. Many of those factors are guanosine triphosphatases (GTPases), proteins that catalyze the hydrolysis of guanosine 5′-triphosphate (GTP) to promote conformational changes. Despite numerous studies, the function of elongation factor 4 (EF-4/LepA), a highly conserved translational GTPase, has remained elusive. Here, we present the crystal structure at 2.6-Å resolution of the Thermus thermophilus 70S ribosome bound to EF-4 with a nonhydrolyzable GTP analog and A-, P-, and E-site tRNAs. The structure reveals the interactions of EF-4 with the A-site tRNA, including contacts between the C-terminal domain (CTD) of EF-4 and the acceptor helical stem of the tRNA. Remarkably, EF-4 induces a distortion of the A-site tRNA, allowing it to interact simultaneously with EF-4 and the decoding center of the ribosome. The structure provides insights into the tRNA-remodeling function of EF-4 on the ribosome and suggests that the displacement of the CCA-end of the A-site tRNA away from the peptidyl transferase center (PTC) is functionally significant. PMID:27092003

  11. 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

  12. Translating the genome in time and space: specialized ribosomes, RNA regulons, and RNA-binding proteins.

    PubMed

    Shi, Zhen; Barna, Maria

    2015-01-01

    A central question in cell and developmental biology is how the information encoded in the genome is differentially interpreted to generate a diverse array of cell types. A growing body of research on posttranscriptional gene regulation is revealing that both global protein synthesis rates and the translation of specific mRNAs are highly specialized in different cell types. How this exquisite translational regulation is achieved is the focus of this review. Two levels of regulation are discussed: the translation machinery and cis-acting elements within mRNAs. Recent evidence shows that the ribosome itself directs how the genome is translated in time and space and reveals surprising functional specificity in individual components of the core translation machinery. We are also just beginning to appreciate the rich regulatory information embedded in the untranslated regions of mRNAs, which direct the selective translation of transcripts. These hidden RNA regulons may interface with a myriad of RNA-binding proteins and specialized translation machinery to provide an additional layer of regulation to how transcripts are spatiotemporally expressed. Understanding this largely unexplored world of translational codes hardwired in the core translation machinery is an exciting new research frontier fundamental to our understanding of gene regulation, organismal development, and evolution.

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

    PubMed

    Irigoyen, Nerea; Firth, Andrew E; Jones, Joshua D; Chung, Betty Y-W; Siddell, Stuart G; Brierley, Ian

    2016-02-01

    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

  14. 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

  15. A Nonradioactive Assay to Measure Production and Processing of Ribosomal RNA by 4sU-Tagging.

    PubMed

    Burger, Kaspar; Eick, Dirk

    2016-01-01

    In vivo metabolic pulse labeling is a classical approach to assess production and processing of ribosomal RNA (rRNA). However, conventional labeling techniques can be indirect and require work with radioactivity. Here, we describe in detail a protocol for in vivo metabolic labeling, purification, and readout of nascent rRNA by 4-thiouridine (4sU). We propose 4sU labeling as standard nonradioactive technique for the analysis of rRNA metabolism during ribosome biogenesis. PMID:27576715

  16. Analysis of Pteridium ribosomal RNA sequences by rapid direct sequencing.

    PubMed

    Tan, M K

    1991-08-01

    A total of 864 bases from 5 regions interspersed in the 18S and 26S rRNA molecules from various clones of Pteridium covering the general geographical distribution of the genus was analysed using a rapid rRNA sequencing technique. No base difference has been detected amongst the three major lineages, two of which apparently separated before the breakup of the ancient supercontinent, Pangaea. These regions of the rRNA sequences have thus been conserved for at least 160 million years and are here compared with other eukaryotic, especially plant rRNAs.

  17. Ribosomal protein-dependent orientation of the 16 S rRNA environment of S15.

    PubMed

    Jagannathan, Indu; Culver, Gloria M

    2004-01-30

    Ribosomal protein S15 binds specifically to the central domain of 16 S ribosomal RNA (16 S rRNA) and directs the assembly of four additional proteins to this domain. The central domain of 16 S rRNA along with these five proteins form the platform of the 30 S subunit. Previously, directed hydroxyl radical probing from Fe(II)-S15 in small ribonucleoprotein complexes was used to study assembly of the central domain of 16 S rRNA. Here, this same approach was used to understand the 16 S rRNA environment of Fe(II)-S15 in 30 S subunits and to determine the ribosomal proteins that are involved in forming the mature S15-16 S rRNA environment. We have identified additional sites of Fe(II)-S15-directed cleavage in 30S subunits compared to the binary complex of Fe(II)-S15/16 S rRNA. Along with novel targets in the central domain, sites within the 5' and 3' minor domains are also cleaved. This suggests that during the course of 30S subunit assembly these elements are positioned in the vicinity of S15. Besides the previously determined role for S8, roles for S5, S6+S18, and S16 in altering the 16 S rRNA environment of S15 were established. These studies reveal that ribosomal proteins can alter the assembly of regions of the 30 S subunit from a considerable distance and influence the overall conformation of this ribonucleoprotein particle.

  18. 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.

  19. Protein Folding Activity of Ribosomal RNA Is a Selective Target of Two Unrelated Antiprion Drugs

    PubMed Central

    Tribouillard-Tanvier, Déborah; Dos Reis, Suzana; Gug, Fabienne; Voisset, Cécile; Béringue, Vincent; Sabate, Raimon; Kikovska, Ema; Talarek, Nicolas; Bach, Stéphane; Huang, Chenhui; Desban, Nathalie; Saupe, Sven J.; Supattapone, Surachai; Thuret, Jean-Yves; Chédin, Stéphane; Vilette, Didier; Galons, Hervé; Sanyal, Suparna; Blondel, Marc

    2008-01-01

    Background 6-Aminophenanthridine (6AP) and Guanabenz (GA, a drug currently in use for the treatment of hypertension) were isolated as antiprion drugs using a yeast-based assay. These structurally unrelated molecules are also active against mammalian prion in several cell-based assays and in vivo in a mouse model for prion-based diseases. Methodology/Principal Findings Here we report the identification of cellular targets of these drugs. Using affinity chromatography matrices for both drugs, we demonstrate an RNA-dependent interaction of 6AP and GA with the ribosome. These specific interactions have no effect on the peptidyl transferase activity of the ribosome or on global translation. In contrast, 6AP and GA specifically inhibit the ribosomal RNA-mediated protein folding activity of the ribosome. Conclusion/Significance 6AP and GA are therefore the first compounds to selectively inhibit the protein folding activity of the ribosome. They thus constitute precious tools to study the yet largely unexplored biological role of this protein folding activity. PMID:18478094

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

    PubMed

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

    2015-11-19

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

  1. 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.

  2. PHF6 regulates cell cycle progression by suppressing ribosomal RNA synthesis.

    PubMed

    Wang, Jiadong; Leung, Justin Wai-chung; Gong, Zihua; Feng, Lin; Shi, Xiaobing; Chen, Junjie

    2013-02-01

    Mutation of PHF6, which results in the X-linked mental retardation disorder Börjeson-Forssman-Lehmann syndrome, is also present in about 38% of adult T-cell acute lymphoblastic leukemias and 3% of adult acute myeloid leukemias. However, it remains to be determined exactly how PHF6 acts in vivo and what functions of PHF6 may be associated with its putative tumor suppressor function. Here, we demonstrate that PHF6 is a nucleolus, ribosomal RNA promoter-associated protein. PHF6 directly interacts with upstream binding factor (UBF) through its PHD1 domain and suppresses ribosomal RNA (rRNA) transcription by affecting the protein level of UBF. Knockdown of PHF6 impairs cell proliferation and arrests cells at G(2)/M phase, which is accompanied by an increased level of phosphorylated H2AX, indicating that PHF6 deficiency leads to the accumulation of DNA damage in the cell. We found that increased DNA damage occurs at the ribosomal DNA (rDNA) locus in PHF6-deficient cells. This effect could be reversed by knocking down UBF or overexpressing RNASE1, which removes RNA-DNA hybrids, suggesting that there is a functional link between rRNA synthesis and genomic stability at the rDNA locus. Together, these results reveal that the key function of PHF6 is involved in regulating rRNA synthesis, which may contribute to its roles in cell cycle control, genomic maintenance, and tumor suppression.

  3. Affinity chromatography of Drosophila melanogaster ribosomal proteins to 5S rRNA.

    PubMed

    Stark, B C; Chooi, W Y

    1985-02-20

    The binding of Drosophila melanogaster ribosomal proteins to D. melanogaster 5S rRNA was studied using affinity chromatography of total ribosomal proteins (TP80) on 5S rRNA linked via adipic acid dihydrazide to Sepharose 4B. Ribosomal proteins which bound 5S rRNA at 0.3 M potassium chloride and were eluted at 1 M potassium chloride were identified as proteins 1, L4, 2/3, L14/L16, and S1, S2, S3, S4, S5, by two-dimensional polyacrylamide gel electrophoresis. Using poly A-Sepharose 4B columns as a model of non-specific binding, we found that a subset of TP80 proteins is also bound. This subset, while containing some of the proteins bound by 5S rRNA columns, was distinctly different from the latter subset, indicating that the binding to 5S rRNA was specific for that RNA species. PMID:3923010

  4. 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.

  5. 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.

  6. Complementarity between the mRNA 5' untranslated region and 18S ribosomal RNA can inhibit translation.

    PubMed

    Verrier, S B; Jean-Jean, O

    2000-04-01

    In eubacteria, base pairing between the 3' end of 16S rRNA and the ribosome-binding site of mRNA is required for efficient initiation of translation. An interaction between the 18S rRNA and the mRNA was also proposed for translation initiation in eukaryotes. Here, we used an antisense RNA approach in vivo to identify the regions of 18S rRNA that might interact with the mRNA 5' untranslated region (5' UTR). Various fragments covering the entire mouse 18S rRNA gene were cloned 5' of a cat reporter gene in a eukaryotic vector, and translation products were analyzed after transient expression in human cells. For the largest part of 18S rRNA, we show that the insertion of complementary fragments in the mRNA 5' UTR do not impair translation of the downstream open reading frame (ORF). When translation inhibition is observed, reduction of the size of the complementary sequence to less than 200 nt alleviates the inhibitory effect. A single fragment complementary to the 18S rRNA 3' domain retains its inhibitory potential when reduced to 100 nt. Deletion analyses show that two distinct sequences of approximately 25 nt separated by a spacer sequence of 50 nt are required for the inhibitory effect. Sucrose gradient fractionation of polysomes reveals that mRNAs containing the inhibitory sequences accumulate in the fractions with 40S ribosomal subunits, suggesting that translation is blocked due to stalling of initiation complexes. Our results support an mRNA-rRNA base pairing to explain the translation inhibition observed and suggest that this region of 18S rRNA is properly located for interacting with mRNA.

  7. 5S ribosomal RNA is an essential component of a nascent ribosomal precursor complex that regulates the Hdm2-p53 checkpoint.

    PubMed

    Donati, Giulio; Peddigari, Suresh; Mercer, Carol A; Thomas, George

    2013-07-11

    Recently, we demonstrated that RPL5 and RPL11 act in a mutually dependent manner to inhibit Hdm2 and stabilize p53 following impaired ribosome biogenesis. Given that RPL5 and RPL11 form a preribosomal complex with noncoding 5S ribosomal RNA (rRNA) and the three have been implicated in the p53 response, we reasoned they may be part of an Hdm2-inhibitory complex. Here, we show that small interfering RNAs directed against 5S rRNA have no effect on total or nascent levels of the noncoding rRNA, though they prevent the reported Hdm4 inhibition of p53. To achieve efficient inhibition of 5S rRNA synthesis, we targeted TFIIIA, a specific RNA polymerase III cofactor, which, like depletion of either RPL5 or RPL11, did not induce p53. Instead, 5S rRNA acts in a dependent manner with RPL5 and RPL11 to inhibit Hdm2 and stabilize p53. Moreover, depletion of any one of the three components abolished the binding of the other two to Hdm2, explaining their common dependence. Finally, we demonstrate that the RPL5/RPL11/5S rRNA preribosomal complex is redirected from assembly into nascent 60S ribosomes to Hdm2 inhibition as a consequence of impaired ribosome biogenesis. Thus, the activation of the Hdm2-inhibitory complex is not a passive but a regulated event, whose potential role in tumor suppression has been recently noted.

  8. Structural constraints identified with covariation analysis in ribosomal RNA.

    PubMed

    Shang, Lei; Xu, Weijia; Ozer, Stuart; Gutell, Robin R

    2012-01-01

    Covariation analysis is used to identify those positions with similar patterns of sequence variation in an alignment of RNA sequences. These constraints on the evolution of two positions are usually associated with a base pair in a helix. While mutual information (MI) has been used to accurately predict an RNA secondary structure and a few of its tertiary interactions, early studies revealed that phylogenetic event counting methods are more sensitive and provide extra confidence in the prediction of base pairs. We developed a novel and powerful phylogenetic events counting method (PEC) for quantifying positional covariation with the Gutell lab's new RNA Comparative Analysis Database (rCAD). The PEC and MI-based methods each identify unique base pairs, and jointly identify many other base pairs. In total, both methods in combination with an N-best and helix-extension strategy identify the maximal number of base pairs. While covariation methods have effectively and accurately predicted RNAs secondary structure, only a few tertiary structure base pairs have been identified. Analysis presented herein and at the Gutell lab's Comparative RNA Web (CRW) Site reveal that the majority of these latter base pairs do not covary with one another. However, covariation analysis does reveal a weaker although significant covariation between sets of nucleotides that are in proximity in the three-dimensional RNA structure. This reveals that covariation analysis identifies other types of structural constraints beyond the two nucleotides that form a base pair.

  9. CED-4 is an mRNA-binding protein that delivers ced-3 mRNA to ribosomes.

    PubMed

    Wang, Miao-xing; Itoh, Masanori; Li, Shimo; Hida, Yoko; Ohta, Kazunori; Hayakawa, Miki; Nishida, Emika; Ueda, Masashi; Islam, Saiful; Tana; Nakagawa, Toshiyuki

    2016-01-29

    Cell death abnormal (ced)-3 and ced-4 genes regulate apoptosis to maintain tissue homeostasis in Caenorhabditis elegans. Apoptosome formation and CED-4 translocation drive CED-3 activation. However, the precise role of CED-4 translocation is not yet fully understood. In this study, using a combination of immunoprecipitation and reverse transcription-polymerase chain reaction methods in cells and a glutathione-S-transferase pull down assay in a cell-free system, we show that CED-4 binds ced-3 mRNA. In the presence of ced-3 mRNA, CED-4 protein is enriched in the microsomal fraction and interacts with ribosomal protein L10a in mammalian cells, increasing the levels of CED-3. These results suggest that CED-4 forms a complex with ced-3 mRNA and delivers it to ribosomes for translation.

  10. Steric interactions lead to collective tilting motion in the ribosome during mRNA-tRNA translocation.

    PubMed

    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

  11. Steric interactions lead to collective tilting motion in the ribosome during mRNA-tRNA translocation

    NASA Astrophysics Data System (ADS)

    Nguyen, Kien; Whitford, Paul C.

    2016-02-01

    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.

  12. Primary sequence of the 16S ribosomal RNA of Escherichia coli.

    PubMed Central

    Ehresmann, C; Stiegler, P; Mackie, G A; Zimmermann, R A; Ebel, J P; Fellner, P

    1975-01-01

    Recent progress in the nucleotide sequence analysis of the 16S ribosomal RNA from E. coli is described. The sequence which has been partially or completely determined so far encompasses 1520 nucleotides, i.e. about 95% of the molecule. Possible features of the secondary structure are suggested on the basis of the nucleotide sequence and data on sequence heterogeneities, repetitions and the location of modified nucleotides are presented. In the accompanying paper, the use of the nucleotide sequence data in studies of the ribosomal protein binding sites is described. PMID:1091918

  13. 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.

  14. Human mitochondrial ribosomes can switch their structural RNA composition

    PubMed Central

    Rorbach, Joanna; Gao, Fei; Powell, Christopher A.; D’Souza, Aaron; Lightowlers, Robert N.; Minczuk, Michal; Chrzanowska-Lightowlers, Zofia M.

    2016-01-01

    The recent developments in cryo-EM have revolutionized our access to previously refractory structures. In particular, such studies of mammalian mitoribosomes have confirmed the absence of any 5S rRNA species and revealed the unexpected presence of a mitochondrially encoded tRNA (mt-tRNA) that usurps this position. Although the cryo-EM structures resolved the conundrum of whether mammalian mitoribosomes contain a 5S rRNA, they introduced a new dilemma: Why do human and porcine mitoribosomes integrate contrasting mt-tRNAs? Human mitoribosomes have been shown to integrate mt-tRNAVal compared with the porcine use of mt-tRNAPhe. We have explored this observation further. Our studies examine whether a range of mt-tRNAs are used by different mammals, or whether the mt-tRNA selection is strictly limited to only these two species of the 22 tRNAs encoded by the mitochondrial genome (mtDNA); whether there is tissue-specific variation within a single organism; and what happens to the human mitoribosome when levels of the mt-tRNAVal are depleted. Our data demonstrate that only mt-tRNAVal or mt-tRNAPhe are found in the mitoribosomes of five different mammals, each mammal favors the same mt-tRNA in all tissue types, and strikingly, when steady-state levels of mt-tRNAVal are reduced, human mitoribosome biogenesis displays an adaptive response by switching to the incorporation of mt-tRNAPhe to generate translationally competent machinery. PMID:27729525

  15. Intervening sequences in ribosomal RNA genes and bobbed phenotype in Drosophila hydei.

    PubMed

    Franz, G; Kunz, W

    1981-08-13

    The "bobbed' (bb) mutation in Drosophila is represented phenotypically by shortened and abnormally thin scutellar bristles and by delayed development. There is a direct correlation between bristle size and ribosomal RNA (rRNA) synthesis, and the bb mutation was at first explained as a deficiency of rRNA genes (rDNA). However, the bb phenotype can occur in Drosophila melanogaster and Drosophila hydei with high rDNA content, while phenotypically wild-type flies are known with few rRNA genes, suggesting that what matters is not the number of rRNA genes but their transcriptional activity. In D. melanogaster, it has recently emerged that rRNA genes interrupted by an intervening sequence are not transcribed. We now report that in D. hydei, the length of the scutellar bristle is directly proportional to the number of rRNA genes without this intervening sequence.

  16. Biological significance of 5S rRNA import into human mitochondria: role of ribosomal protein MRP-L18.

    PubMed

    Smirnov, Alexandre; Entelis, Nina; Martin, Robert P; Tarassov, Ivan

    2011-06-15

    5S rRNA is an essential component of ribosomes of all living organisms, the only known exceptions being mitochondrial ribosomes of fungi, animals, and some protists. An intriguing situation distinguishes mammalian cells: Although the mitochondrial genome contains no 5S rRNA genes, abundant import of the nuclear DNA-encoded 5S rRNA into mitochondria was reported. Neither the detailed mechanism of this pathway nor its rationale was clarified to date. In this study, we describe an elegant molecular conveyor composed of a previously identified human 5S rRNA import factor, rhodanese, and mitochondrial ribosomal protein L18, thanks to which 5S rRNA molecules can be specifically withdrawn from the cytosolic pool and redirected to mitochondria, bypassing the classic nucleolar reimport pathway. Inside mitochondria, the cytosolic 5S rRNA is shown to be associated with mitochondrial ribosomes.

  17. Chemical accessibility of the 4.5S RNA in spinach chloroplast ribosomes.

    PubMed Central

    Kumagai, I; Bartsch, M; Subramanian, A R; Erdmann, V A

    1983-01-01

    We have examined the accessibility to diethylpyrocarbonate of spinach chloroplast 4.5S ribosomal RNA when free and when it is part of the ribosomal structure. The modifications in free 4.5S RNA were found mostly in single-stranded regions of the secondary structure model proposed in our previous paper (Kumagai, I. et al. (1982) J.B.C. 257, 12924-28): adenines at positions 17, 19, 33, 36, 54, 55, 60, 64, 68, 72, 77, 86 and 87 were identified as the reactive residues. On the other hand, in 4.5S RNA in 70S ribosomes or 50S subunits, adenine 33 was exclusively modified, and its reactivity was much higher than in free 4.5S RNA. This highly accessible A33 of spinach 4.5S RNA is located within a characteristic seven nucleotide sequence, which is found in the 4.5S rRNAs from spinach, tobacco and a fern but deleted in 4.5S RNAs from maize and wheat. Images PMID:6828382

  18. Head swivel on the ribosome facilitates translocation via intra-subunit tRNA hybrid sites

    PubMed Central

    Ratje, Andreas H.; Loerke, Justus; Mikolajka, Aleksandra; Brünner, Matthias; Hildebrand, Peter W.; Starosta, Agata L.; Dönhöfer, Alexandra; Connell, Sean R.; Fucini, Paola; Mielke, Thorsten; Whitford, Paul C.; Onuchic, Jose’ N; Yu, Yanan; Sanbonmatsu, Karissa Y.; Hartmann, Roland K.; Penczek, Pawel A.; Wilson, Daniel N.; Spahn, Christian M.T.

    2011-01-01

    The elongation cycle of protein synthesis involves the delivery of aminoacyl-tRNAs to the A-site of the ribosome, followed by peptide-bond formation and translocation of the tRNAs through the ribosome to reopen the A-site1,2. The translocation reaction is catalyzed by elongation factor G (EF-G) in a GTP-dependent fashion3. Despite the availability of structures of various EF-G-ribosome complexes, the precise mechanism by which tRNAs move through the ribosome still remains unclear. Here we use multiparticle cryo-EM analysis to resolve two previously unseen subpopulations within EF-G-ribosome complexes at sub-nanometer resolution, one of them with a partially translocated tRNA. Comparison of these sub-states reveals that translocation of tRNA on the 30S subunit parallels the swiveling of the 30S-head and is coupled to un-ratcheting of the 30S-body. Since the tRNA maintains contact with the P-site on the 30S-head and simultaneously establishes interaction with the E-site on the 30S-platform, a novel intra-subunit pe/E hybrid state is formed. This state is stabilized by domain IV of EF-G, which interacts with the swiveled 30S-head conformation. These findings provide direct structural and mechanistic insight into the “missing link” in terms of tRNA intermediates involved in the universally conserved translocation process. PMID:21124459

  19. Comparative survey of the relative impact of mRNA features on local ribosome profiling read density

    PubMed Central

    O'Connor, Patrick B. F.; Andreev, Dmitry E.; Baranov, Pavel V.

    2016-01-01

    Ribosome profiling (Ribo-seq), a promising technology for exploring ribosome decoding rates, is characterized by the presence of infrequent high peaks in ribosome footprint density and by long alignment gaps. Here, to reduce the impact of data heterogeneity we introduce a simple normalization method, Ribo-seq Unit Step Transformation (RUST). RUST is robust and outperforms other normalization techniques in the presence of heterogeneous noise. We illustrate how RUST can be used for identifying mRNA sequence features that affect ribosome footprint densities globally. We show that a few parameters extracted with RUST are sufficient for predicting experimental densities with high accuracy. Importantly the application of RUST to 30 publicly available Ribo-seq data sets revealed a substantial variation in sequence determinants of ribosome footprint frequencies, questioning the reliability of Ribo-seq as an accurate representation of local ribosome densities without prior quality control. This emphasizes our incomplete understanding of how protocol parameters affect ribosome footprint densities. PMID:27698342

  20. 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

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

    PubMed

    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

  2. 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.

  3. 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

  4. Phylogenetic analysis of ruminant Theileria spp. from China based on 28S ribosomal RNA gene.

    PubMed

    Gou, Huitian; Guan, Guiquan; Ma, Miling; Liu, Aihong; Liu, Zhijie; Xu, Zongke; Ren, Qiaoyun; Li, Youquan; Yang, Jifei; Chen, Ze; Yin, Hong; Luo, Jianxun

    2013-10-01

    Species identification using DNA sequences is the basis for DNA taxonomy. In this study, we sequenced the ribosomal large-subunit RNA gene sequences (3,037-3,061 bp) in length of 13 Chinese Theileria stocks that were infective to cattle and sheep. The complete 28S rRNA gene is relatively difficult to amplify and its conserved region is not important for phylogenetic study. Therefore, we selected the D2-D3 region from the complete 28S rRNA sequences for phylogenetic analysis. Our analyses of 28S rRNA gene sequences showed that the 28S rRNA was useful as a phylogenetic marker for analyzing the relationships among Theileria spp. in ruminants. In addition, the D2-D3 region was a short segment that could be used instead of the whole 28S rRNA sequence during the phylogenetic analysis of Theileria, and it may be an ideal DNA barcode.

  5. 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.

  6. DksA Guards Elongating RNA Polymerase Against Ribosome-Stalling-Induced Arrest

    PubMed Central

    Zhang, Yan; Mooney, Rachel A.; Grass, Jeffrey A.; Sivaramakrishnan, Priya; Herman, Christophe; Landick, Robert; Wang, Jue D.

    2014-01-01

    Summary In bacteria, translation-transcription coupling inhibits RNA polymerase (RNAP) stalling. We present evidence suggesting that, upon amino acid starvation, inactive ribosomes promote rather than inhibit RNAP stalling. We developed an algorithm to evaluate genome-wide polymerase progression independently of local noise, and used it to reveal that the transcription factor DksA inhibits promoter-proximal pausing and increases RNAP elongation when uncoupled from translation by depletion of charged tRNAs. DksA has minimal effect on RNAP elongation in vitro and on untranslated RNAs in vivo. In these cases, transcripts can form RNA structures that prevent backtracking. Thus, the effect of DksA on transcript elongation may occur primarily upon ribosome slowing/stalling or at promoter-proximal locations that limit the potential for RNA structure. We propose that inactive ribosomes prevent formation of backtrack-blocking mRNA structures and that, in this circumstance, DksA acts as a transcription elongation factor in vivo. PMID:24606919

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

    PubMed

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

    2004-06-01

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

  8. A cluster of methylations in the domain IV of 25S rRNA is required for ribosome stability

    PubMed Central

    Gigova, Andriana; Duggimpudi, Sujitha; Pollex, Tim; Schaefer, Matthias

    2014-01-01

    In all three domains of life ribosomal RNAs are extensively modified at functionally important sites of the ribosome. These modifications are believed to fine-tune the ribosome structure for optimal translation. However, the precise mechanistic effect of modifications on ribosome function remains largely unknown. Here we show that a cluster of methylated nucleotides in domain IV of 25S rRNA is critical for integrity of the large ribosomal subunit. We identified the elusive cytosine-5 methyltransferase for C2278 in yeast as Rcm1 and found that a combined loss of cytosine-5 methylation at C2278 and ribose methylation at G2288 caused dramatic ribosome instability, resulting in loss of 60S ribosomal subunits. Structural and biochemical analyses revealed that this instability was caused by changes in the structure of 25S rRNA and a consequent loss of multiple ribosomal proteins from the large ribosomal subunit. Our data demonstrate that individual RNA modifications can strongly affect structure of large ribonucleoprotein complexes. PMID:25125595

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

    PubMed Central

    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. PMID:26872259

  10. A pathogenic non-coding RNA induces changes in dynamic DNA methylation of ribosomal RNA genes in host plants

    PubMed Central

    Martinez, German; Castellano, Mayte; Tortosa, Maria; Pallas, Vicente; Gomez, Gustavo

    2014-01-01

    Viroids are plant-pathogenic non-coding RNAs able to interfere with as yet poorly known host-regulatory pathways and to cause alterations recognized as diseases. The way in which these RNAs coerce the host to express symptoms remains to be totally deciphered. In recent years, diverse studies have proposed a close interplay between viroid-induced pathogenesis and RNA silencing, supporting the belief that viroid-derived small RNAs mediate the post-transcriptional cleavage of endogenous mRNAs by acting as elicitors of symptoms expression. Although the evidence supporting the role of viroid-derived small RNAs in pathogenesis is robust, the possibility that this phenomenon can be a more complex process, also involving viroid-induced alterations in plant gene expression at transcriptional levels, has been considered. Here we show that plants infected with the ‘Hop stunt viroid’ accumulate high levels of sRNAs derived from ribosomal transcripts. This effect was correlated with an increase in the transcription of ribosomal RNA (rRNA) precursors during infection. We observed that the transcriptional reactivation of rRNA genes correlates with a modification of DNA methylation in their promoter region and revealed that some rRNA genes are demethylated and transcriptionally reactivated during infection. This study reports a previously unknown mechanism associated with viroid (or any other pathogenic RNA) infection in plants providing new insights into aspects of host alterations induced by the viroid infectious cycle. PMID:24178032

  11. Quantitative studies of mRNA recruitment to the eukaryotic ribosome.

    PubMed

    Fraser, Christopher S

    2015-07-01

    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.

  12. 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

  13. [Eukaryogenesis: a model derivated from ribosomal RNA molecular phylogenise].

    PubMed

    Perasso, R; Baroin-Tourancheau, A

    1992-01-01

    We have undertaken the construction of a broad molecular phylogeny of protists through the comparison of 28S rRNA molecules. The sequences from several major protistan phyla were aligned and combined with a broad database of metazoans, metaphytes, fungi and bacteria and we have derived dendrograms from both distance matrix and parsimony methods. In agreement with classical systematics, a number of monophyletic groups separated by large evolutionary distances were observed (those of the ciliates, the chlorophytes, etc.). From this analysis, several inferences on the eukaryogenesis can be made among which the ancient origin of the cytoskeleton, the late occurrence of the chloroplastic endosymbiosis and the simultaneous emergence of the triploblastic and diploblastic metazoan patterns. PMID:1339595

  14. [Eukaryogenesis: a model derivated from ribosomal RNA molecular phylogenise].

    PubMed

    Perasso, R; Baroin-Tourancheau, A

    1992-01-01

    We have undertaken the construction of a broad molecular phylogeny of protists through the comparison of 28S rRNA molecules. The sequences from several major protistan phyla were aligned and combined with a broad database of metazoans, metaphytes, fungi and bacteria and we have derived dendrograms from both distance matrix and parsimony methods. In agreement with classical systematics, a number of monophyletic groups separated by large evolutionary distances were observed (those of the ciliates, the chlorophytes, etc.). From this analysis, several inferences on the eukaryogenesis can be made among which the ancient origin of the cytoskeleton, the late occurrence of the chloroplastic endosymbiosis and the simultaneous emergence of the triploblastic and diploblastic metazoan patterns.

  15. 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.

  16. The evolution of the Vahlkampfiidae as deduced from 16S-like ribosomal RNA analysis.

    PubMed

    Hinkle, G; Sogin, M L

    1993-01-01

    The amoebae, a phenotypically diverse, paraphyletic group of protists, have been largely neglected by molecular phylogeneticists. To better understand the evolution of amoebae, we sequenced and analyzed the 16S-like ribosomal RNA genes of three vahlkampfiid amoebae: Paratetramitus jugosus, Tetramitus rostratus and Vahlkampfia lobospinosa. The Vahlkampfiidae lineage is monophyletic, branches early along the eukaryotic line of descent, and is not a close relative of the multicellular amoebae that also reversibly transform from amoebae to flagellates.

  17. RPFdb: a database for genome wide information of translated mRNA generated from ribosome profiling

    PubMed Central

    Xie, Shang-Qian; Nie, Peng; Wang, Yan; Wang, Hongwei; Li, Hongyu; Yang, Zhilong; Liu, Yizhi; Ren, Jian; Xie, Zhi

    2016-01-01

    Translational control is crucial in the regulation of gene expression and deregulation of translation is associated with a wide range of cancers and human diseases. Ribosome profiling is a technique that provides genome wide information of mRNA in translation based on deep sequencing of ribosome protected mRNA fragments (RPF). RPFdb is a comprehensive resource for hosting, analyzing and visualizing RPF data, available at www.rpfdb.org or http://sysbio.sysu.edu.cn/rpfdb/index.html. The current version of database contains 777 samples from 82 studies in 8 species, processed and reanalyzed by a unified pipeline. There are two ways to query the database: by keywords of studies or by genes. The outputs are presented in three levels. (i) Study level: including meta information of studies and reprocessed data for gene expression of translated mRNAs; (ii) Sample level: including global perspective of translated mRNA and a list of the most translated mRNA of each sample from a study; (iii) Gene level: including normalized sequence counts of translated mRNA on different genomic location of a gene from multiple samples and studies. To explore rich information provided by RPF, RPFdb also provides a genome browser to query and visualize context-specific translated mRNA. Overall our database provides a simple way to search, analyze, compare, visualize and download RPF data sets. PMID:26433228

  18. Expedited quantification of mutant ribosomal RNA by binary deoxyribozyme (BiDz) sensors.

    PubMed

    Gerasimova, Yulia V; Yakovchuk, Petro; Dedkova, Larisa M; Hecht, Sidney M; Kolpashchikov, Dmitry M

    2015-10-01

    Mutations in ribosomal RNA (rRNA) have traditionally been detected by the primer extension assay, which is a tedious and multistage procedure. Here, we describe a simple and straightforward fluorescence assay based on binary deoxyribozyme (BiDz) sensors. The assay uses two short DNA oligonucleotides that hybridize specifically to adjacent fragments of rRNA, one of which contains a mutation site. This hybridization results in the formation of a deoxyribozyme catalytic core that produces the fluorescent signal and amplifies it due to multiple rounds of catalytic action. This assay enables us to expedite semi-quantification of mutant rRNA content in cell cultures starting from whole cells, which provides information useful for optimization of culture preparation prior to ribosome isolation. The method requires less than a microliter of a standard Escherichia coli cell culture and decreases analysis time from several days (for primer extension assay) to 1.5 h with hands-on time of ∼10 min. It is sensitive to single-nucleotide mutations. The new assay simplifies the preliminary analysis of RNA samples and cells in molecular biology and cloning experiments and is promising in other applications where fast detection/quantification of specific RNA is required.

  19. 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.

  20. 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.

  1. 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.

  2. 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

  3. Highly conserved base A55 of 16S ribosomal RNA is important for the elongation cycle of protein synthesis.

    PubMed

    Sahu, Bhubanananda; Khade, Prashant K; Joseph, Simpson

    2013-09-24

    Accurate decoding of mRNA requires the precise interaction of protein factors and tRNAs with the ribosome. X-ray crystallography and cryo-electron microscopy have provided detailed structural information about the 70S ribosome with protein factors and tRNAs trapped during translation. Crystal structures showed that one of the universally conserved 16S rRNA bases, A55, in the shoulder domain of the 30S subunit interacts with elongation factors Tu and G (EF-Tu and EF-G, respectively). The exact functional role of A55 in protein synthesis is not clear. We changed A55 to U and analyzed the effect of the mutation on the elongation cycle of protein synthesis using functional assays. Expression of 16S rRNA with the A55U mutation in cells confers a dominant lethal phenotype. Additionally, ribosomes with the A55U mutation in 16S rRNA show substantially reduced in vitro protein synthesis activity. Equilibrium binding studies showed that the A55U mutation considerably inhibited the binding of the EF-Tu·GTP·tRNA ternary complex to the ribosome. Furthermore, the A55U mutation slightly inhibited the peptidyl transferase reaction, the binding of EF-G·GTP to the ribosome, and mRNA-tRNA translocation. These results indicate that A55 is important for fine-tuning the activity of the ribosome during the elongation cycle of protein synthesis.

  4. A resource of ribosomal RNA-depleted RNA-Seq data from different normal adult and fetal human tissues.

    PubMed

    Choy, Jocelyn Y H; Boon, Priscilla L S; Bertin, Nicolas; Fullwood, Melissa J

    2015-01-01

    Gene expression is the most fundamental level at which the genotype leads to the phenotype of the organism. Enabled by ultra-high-throughput next-generation DNA sequencing, RNA-Seq involves shotgun sequencing of fragmented RNA transcripts by next-generation sequencing followed by in silico assembly, and is rapidly becoming the most popular method for gene expression analysis. Poly[A]+ RNA-Seq analyses of normal human adult tissue samples such as Illumina's Human BodyMap 2.0 Project and the RNA-Seq atlas have provided a useful global resource and framework for comparisons with diseased tissues such as cancer. However, these analyses have failed to provide information on poly[A]-RNA, which is abundant in our cells. The most recent advances in RNA-Seq analyses use ribosomal RNA-depletion to provide information on both poly[A]+ and poly[A]-RNA. In this paper, we describe the use of Illumina's HiSeq 2000 to generate high quality rRNA-depleted RNA-Seq datasets from human fetal and adult tissues. The datasets reported here will be useful in understanding the different expression profiles in different tissues.

  5. Slow growth and unstable ribosomal RNA lacking pseudouridine in mouse embryonic fibroblast cells expressing catalytically inactive dyskerin

    PubMed Central

    Gu, Bai-Wei; Ge, Jingping; Fan, Jian-Meng; Bessler, Monica; Mason, Philip J.

    2013-01-01

    Pseudouridine is the most abundant modified nucleotide in ribosomal RNA throughout eukaryotes and archaea but its role is not known. Here we produced mouse embryonic fibroblast cells expressing only catalytically inactive dyskerin, the pseudouridine synthase that converts uridine to pseudouridine in ribosomal RNA. The mutant dyskerin protein, D125A, was extremely unstable but cells were able to divide and grow very slowly. Abnormalities in ribosome RNA synthesis were apparent but mature cytoplasmic RNAs lacking pseudouridine were produced and were very unstable. We conclude that pseudouridine is required to stabilize the secondary structure of ribosomal RNA that is essential for its function. Structured summary of protein interactions∷ fibrillarin and Dkc1 colocalize by fluorescence microscopy (View interaction) PMID:23726835

  6. 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.

  7. Insights into RNA binding by the anticancer drug cisplatin from the crystal structure of cisplatin-modified ribosome

    PubMed Central

    Melnikov, Sergey V.; Söll, Dieter; Steitz, Thomas A.; Polikanov, Yury S.

    2016-01-01

    Cisplatin is a widely prescribed anticancer drug, which triggers cell death by covalent binding to a broad range of biological molecules. Among cisplatin targets, cellular RNAs remain the most poorly characterized molecules. Although cisplatin was shown to inactivate essential RNAs, including ribosomal, spliceosomal and telomeric RNAs, cisplatin binding sites in most RNA molecules are unknown, and therefore it remains challenging to study how modifications of RNA by cisplatin contributes to its toxicity. Here we report a 2.6Å-resolution X-ray structure of cisplatin-modified 70S ribosome, which describes cisplatin binding to the ribosome and provides the first nearly atomic model of cisplatin–RNA complex. We observe nine cisplatin molecules bound to the ribosome and reveal consensus structural features of the cisplatin-binding sites. Two of the cisplatin molecules modify conserved functional centers of the ribosome—the mRNA-channel and the GTPase center. In the mRNA-channel, cisplatin intercalates between the ribosome and the messenger RNA, suggesting that the observed inhibition of protein synthesis by cisplatin is caused by impaired mRNA-translocation. Our structure provides an insight into RNA targeting and inhibition by cisplatin, which can help predict cisplatin-binding sites in other cellular RNAs and design studies to elucidate a link between RNA modifications by cisplatin and cisplatin toxicity. PMID:27079977

  8. Identification of bacterial sRNA regulatory targets using ribosome profiling

    PubMed Central

    Wang, Jing; Rennie, William; Liu, Chaochun; Carmack, Charles S.; Prévost, Karine; Caron, Marie-Pier; Massé, Eric; Ding, Ye; Wade, Joseph T.

    2015-01-01

    Bacteria express large numbers of non-coding, regulatory RNAs known as ‘small RNAs’ (sRNAs). sRNAs typically regulate expression of multiple target messenger RNAs (mRNAs) through base-pairing interactions. sRNA:mRNA base-pairing often results in altered mRNA stability and/or altered translation initiation. Computational identification of sRNA targets is challenging due to the requirement for only short regions of base-pairing that can accommodate mismatches. Experimental approaches have been applied to identify sRNA targets on a genomic scale, but these focus only on those targets regulated at the level of mRNA stability. Here, we utilize ribosome profiling (Ribo-seq) to experimentally identify regulatory targets of the Escherichia coli sRNA RyhB. We not only validate a majority of known RyhB targets using the Ribo-seq approach, but also discover many novel ones. We further confirm regulation of a selection of known and novel targets using targeted reporter assays. By mutating nucleotides in the mRNA of a newly discovered target, we demonstrate direct regulation of this target by RyhB. Moreover, we show that Ribo-seq distinguishes between mRNAs regulated at the level of RNA stability and those regulated at the level of translation. Thus, Ribo-seq represents a powerful approach for genome-scale identification of sRNA targets. PMID:26546513

  9. Structure of the small ribosomal subunit RNA of the pulmonate snail, Limicolaria kambeul, and phylogenetic analysis of the Metazoa.

    PubMed

    Winnepennickx, B; Backeljau, T; van de Peer, Y; De Wachter, R

    1992-09-01

    The complete nucleotide sequence of the small ribosomal subunit RNA of the gastropod, Limicolaria kambeul, was determined and used to infer a secondary structure model. In order to clarify the phylogenetic position of the Mollusca among the Metazoa, an evolutionary tree was constructed by neighbor-joining, starting from an alignment of small ribosomal subunit RNA sequences. The Mollusca appear to be a monophyletic group, related to Arthropoda and Chordata in an unresolved trichotomy. PMID:1505675

  10. 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.

  11. 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

  12. An intercistronic region and ribosome-binding site in bacterial messenger RNA.

    PubMed Central

    Platt, T; Yanofsky, C

    1975-01-01

    A messenger RNA fragment about 220 nucleotides long has been isolated from 32-P-labeled tryptophan operon mRNA of Escherichia coli. When point mutations at the end of trpB and the beginning of trpA were introduced, the resulting nucleotide changes were found; hence the mRNA fragment must include the trpB-trpA intercistronic region. Most of the nucleotide sequences can be assigned to specific locations in the structural genes, based on the amino-acid sequences of the trpB and trpA proteins. In vitro, ribosomes bind to this piece of mRNA and protect from nuclease attack a region about 40 nucleotides long, containing a central AUG codon. The triplet codons to the 3' side of this AUG correspond to the first seven amino acids of the trpA protein; the codons to the 5' side correspond to the last six amino acids of the trpB protein. Translation of trpB is terminated by single UGA codon, which overlaps the trpA AUG initiation codon: UGAUG. Thus the untranslated "intercistronic" region consists of only two nucleotides. The RNA sequence spanning this region undoubtedly fulfills two functions, specifying ribosome recognition signals as well as encoding amino-acid sequences. Images PMID:1094468

  13. A unique combination of rare mitochondrial ribosomal RNA variants affects the kinetics of complex I assembly.

    PubMed

    Porcelli, Anna Maria; Calvaruso, Maria Antonietta; Iommarini, Luisa; Kurelac, Ivana; Zuntini, Roberta; Ferrari, Simona; Gasparre, Giuseppe

    2016-06-01

    Mitochondrial DNA (mtDNA) mutations in respiratory complexes subunits contribute to a large spectrum of human diseases. Nonetheless, ribosomal RNA variants remain largely under-investigated from a functional point of view. We here report a unique combination of two rare mitochondrial rRNA variants detected by serendipity in a subject with chronic granulomatous disease and never reported to co-occur within the same mitochondrial haplotype. In silico prediction of the mitochondrial ribosomal structure showed a dramatic rearrangement of the rRNA secondary structure. Functional investigation of cybrids carrying this unique haplotype demonstrated that the co-occurrence of the two rRNA variants determines a slow-down of the mitochondrial protein synthesis, especially in cells with an elevated metabolic rate, which impairs the assembly kinetics of Complex I, induces a bioenergetic defect and stimulates reactive oxygen species production. In conclusion, our results point to a sub-pathogenic role for these two rare mitochondrial rRNA variants, when found in the unique combination here reported in a single individual. PMID:27102412

  14. RNA mimicry by the fap7 adenylate kinase in ribosome biogenesis.

    PubMed

    Loc'h, Jérôme; Blaud, Magali; Réty, Stéphane; Lebaron, Simon; Deschamps, Patrick; Bareille, Joseph; Jombart, Julie; Robert-Paganin, Julien; Delbos, Lila; Chardon, Florian; Zhang, Elodie; Charenton, Clément; Tollervey, David; Leulliot, Nicolas

    2014-05-01

    During biogenesis of the 40S and 60S ribosomal subunits, the pre-40S particles are exported to the cytoplasm prior to final cleavage of the 20S pre-rRNA to mature 18S rRNA. Amongst the factors involved in this maturation step, Fap7 is unusual, as it both interacts with ribosomal protein Rps14 and harbors adenylate kinase activity, a function not usually associated with ribonucleoprotein assembly. Human hFap7 also regulates Cajal body assembly and cell cycle progression via the p53-MDM2 pathway. This work presents the functional and structural characterization of the Fap7-Rps14 complex. We report that Fap7 association blocks the RNA binding surface of Rps14 and, conversely, Rps14 binding inhibits adenylate kinase activity of Fap7. In addition, the affinity of Fap7 for Rps14 is higher with bound ADP, whereas ATP hydrolysis dissociates the complex. These results suggest that Fap7 chaperones Rps14 assembly into pre-40S particles via RNA mimicry in an ATP-dependent manner. Incorporation of Rps14 by Fap7 leads to a structural rearrangement of the platform domain necessary for the pre-rRNA to acquire a cleavage competent conformation.

  15. Specific interactions of the L10(L12)4 ribosomal protein complex with mRNA, rRNA, and L11.

    PubMed

    Iben, James R; Draper, David E

    2008-03-01

    Large ribosomal subunit proteins L10 and L12 form a pentameric protein complex, L10(L12) 4, that is intimately involved in the ribosome elongation cycle. Its contacts with rRNA or other ribosomal proteins have been only partially resolved by crystallography. In Escherichia coli, L10 and L12 are encoded from a single operon for which L10(L12) 4 is a translational repressor that recognizes a secondary structure in the mRNA leader. In this study, L10(L12) 4 was expressed from the moderate thermophile Bacillus stearothermophilus to quantitatively compare strategies for binding of the complex to mRNA and ribosome targets. The minimal mRNA recognition structure is widely distributed among bacteria and has the potential to form a kink-turn structure similar to one identified in the rRNA as part of the L10(L12) 4 binding site. Mutations in equivalent positions between the two sequences have similar effects on L10(L12) 4-RNA binding affinity and identify the kink-turn motif and a loop AA sequence as important recognition elements. In contrast to the larger rRNA structure, the mRNA apparently positions the kink-turn motif and loop for protein recognition without the benefit of Mg (2+)-dependent tertiary structure. The mRNA and rRNA fragments bind L10(L12) 4 with similar affinity ( approximately 10 (8) M (-1)), but fluorescence binding studies show that a nearby protein in the ribosome, L11, enhances L10(L12) 4 binding approximately 100-fold. Thus, mRNA and ribosome targets use similar RNA features, held in different structural contexts, to recognize L10(L12) 4, and the ribosome ensures the saturation of its L10(L12) 4 binding site by means of an additional protein-protein interaction. PMID:18247578

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

    PubMed Central

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

    2009-01-01

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

  17. 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

  18. The Comparative RNA Web (CRW) Site: an online database of comparative sequence and structure information for ribosomal, intron, and other RNAs

    PubMed Central

    2002-01-01

    Background Comparative analysis of RNA sequences is the basis for the detailed and accurate predictions of RNA structure and the determination of phylogenetic relationships for organisms that span the entire phylogenetic tree. Underlying these accomplishments are very large, well-organized, and processed collections of RNA sequences. This data, starting with the sequences organized into a database management system and aligned to reveal their higher-order structure, and patterns of conservation and variation for organisms that span the phylogenetic tree, has been collected and analyzed. This type of information can be fundamental for and have an influence on the study of phylogenetic relationships, RNA structure, and the melding of these two fields. Results We have prepared a large web site that disseminates our comparative sequence and structure models and data. The four major types of comparative information and systems available for the three ribosomal RNAs (5S, 16S, and 23S rRNA), transfer RNA (tRNA), and two of the catalytic intron RNAs (group I and group II) are: (1) Current Comparative Structure Models; (2) Nucleotide Frequency and Conservation Information; (3) Sequence and Structure Data; and (4) Data Access Systems. Conclusions This online RNA sequence and structure information, the result of extensive analysis, interpretation, data collection, and computer program and web development, is accessible at our Comparative RNA Web (CRW) Site http://www.rna.icmb.utexas.edu. In the future, more data and information will be added to these existing categories, new categories will be developed, and additional RNAs will be studied and presented at the CRW Site. PMID:11869452

  19. 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.

  20. Unusual transcription termination of the ribosomal RNA genes in Ascaris lumbricoides.

    PubMed Central

    Müller, E; Neuhaus, H; Tobler, H; Müller, F

    1990-01-01

    We studied termination of transcription of the ribosomal RNA genes in Ascaris lumbricoides, the first representative in the phylum of nemathelminthes analysed so far. RNase protection experiments in vivo reveal that the 3' end of the precursor rRNA coincides with the end of mature 26S rRNA. Promoter-containing miniplasmids are able to direct unique 3' end formation in vitro at a site identical to that observed in vivo, whereas deletion of these sequences abolishes 3' end formation throughout the entire spacer. A nuclear run-on experiment in vitro confirms the drop of polymerase I concentration down-stream of this site. The termination site for polymerase I transcription of the rDNA operon in A. lumbricoides is therefore unique, and located at the very end of the 26S rRNA gene. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. PMID:2390973

  1. 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.

  2. 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.

  3. 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.

  4. Oligonucleotide probes for Bordetella bronchiseptica based on 16S ribosomal RNA sequences.

    PubMed

    Taneda, A; Futo, S; Mitsuse, S; Seto, Y; Okada, M; Sakano, T

    1994-12-01

    Bordetella bronchiseptica 16S ribosomal RNA (rRNA) gene was cloned and identified. On the basis of information from computer-assisted sequence comparison of the B. bronchiseptica 16S RRNA sequences with that of other bacterial species, we constructed B. bronchiseptica-specific oligonucleotide probes complementary to variable regions in the 16S rRNA molecule. Specificity of these 32P-labeled oligo-nucleotide probes was tested in a RNA/DNA hybridization with B. bronchiseptica strains and other bacterial strains. Probe BB4 was more specific than three other oligonucleotide probes. This probe BB4 was sensitive enough to be able to detect 10(4) bacterial cells. PMID:9133055

  5. Ribosome biogenesis requires a highly diverged XRN family 5'->3' exoribonuclease for rRNA processing in Trypanosoma brucei.

    PubMed

    Sakyiama, Joseph; Zimmer, Sara L; Ciganda, Martin; Williams, Noreen; Read, Laurie K

    2013-10-01

    Although biogenesis of ribosomes is a crucial process in all organisms and is thus well conserved, Trypanosoma brucei ribosome biogenesis, of which maturation of rRNAs is an early step, has multiple points of divergence. Our aim was to determine whether in the processing of the pre-rRNA precursor molecule, 5'→3' exoribonuclease activity in addition to endonucleolytic cleavage is necessary in T. brucei as in other organisms. Our approach initiated with the bioinformatic identification of a putative 5'→3' exoribonuclease, XRNE, which is highly diverged from the XRN2/Rat1 enzyme responsible for rRNA processing in other organisms. Tagging this protein in vivo allowed us to classify XRNE as nucleolar by indirect immunofluorescence and identify by copurification interacting proteins, many of which were ribosomal proteins, ribosome biogenesis proteins, and/or RNA processing proteins. To determine whether XRNE plays a role in ribosome biogenesis in procyclic form cells, we inducibly depleted the protein by RNA interference. This resulted in the generation of aberrant preprocessed 18S rRNA and 5' extended 5.8S rRNA, implicating XRNE in rRNA processing. Polysome profiles of XRNE-depleted cells demonstrated abnormal features including an increase in ribosome small subunit abundance, a decrease in large subunit abundance, and defects in polysome assembly. Furthermore, the 5' extended 5.8S rRNA in XRNE-depleted cells was observed in the large subunit, monosomes, and polysomes in this gradient. Therefore, the function of XRNE in rRNA processing, presumably due to exonucleolytic activity very early in ribosome biogenesis, has consequences that persist throughout all biogenesis stages.

  6. Heat-induced ribosome pausing triggers mRNA co-translational decay in Arabidopsis thaliana.

    PubMed

    Merret, Rémy; Nagarajan, Vinay K; Carpentier, Marie-Christine; Park, Sunhee; Favory, Jean-Jacques; Descombin, Julie; Picart, Claire; Charng, Yee-Yung; Green, Pamela J; Deragon, Jean-Marc; Bousquet-Antonelli, Cécile

    2015-04-30

    The reprogramming of gene expression in heat stress is a key determinant to organism survival. Gene expression is downregulated through translation initiation inhibition and release of free mRNPs that are rapidly degraded or stored. In mammals, heat also triggers 5'-ribosome pausing preferentially on transcripts coding for HSC/HSP70 chaperone targets, but the impact of such phenomenon on mRNA fate remains unknown. Here, we provide evidence that, in Arabidopsis thaliana, heat provokes 5'-ribosome pausing leading to the XRN4-mediated 5'-directed decay of translating mRNAs. We also show that hindering HSC/HSP70 activity at 20°C recapitulates heat effects by inducing ribosome pausing and co-translational mRNA turnover. Strikingly, co-translational decay targets encode proteins with high HSC/HSP70 binding scores and hydrophobic N-termini, two characteristics that were previously observed for transcripts most prone to pausing in animals. This work suggests for the first time that stress-induced variation of translation elongation rate is an evolutionarily conserved process leading to the polysomal degradation of thousands of 'non-aberrant' mRNAs. PMID:25845591

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

    PubMed

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

    2015-11-24

    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

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

    PubMed

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

    2015-11-24

    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.

  11. Hierarchical recruitment into nascent ribosomes of assembly factors required for 27SB pre-rRNA processing in Saccharomyces cerevisiae

    PubMed Central

    Talkish, Jason; Zhang, Jingyu; Jakovljevic, Jelena; Horsey, Edward W.; Woolford, John L.

    2012-01-01

    To better define the roles of assembly factors required for eukaryotic ribosome biogenesis, we have focused on one specific step in maturation of yeast 60 S ribosomal subunits: processing of 27SB pre-ribosomal RNA. At least 14 assembly factors, the ‘B-factor’ proteins, are required for this step. These include most of the major functional classes of assembly factors: RNA-binding proteins, scaffolding protein, DEAD-box ATPases and GTPases. We have investigated the mechanisms by which these factors associate with assembling ribosomes. Our data establish a recruitment model in which assembly of the B-factors into nascent ribosomes ultimately leads to the recruitment of the GTPase Nog2. A more detailed analysis suggests that this occurs in a hierarchical manner via two largely independent recruiting pathways that converge on Nog2. Understanding recruitment has allowed us to better determine the order of association of all assembly factors functioning in one step of ribosome assembly. Furthermore, we have identified a novel subcomplex composed of the B-factors Nop2 and Nip7. Finally, we identified a means by which this step in ribosome biogenesis is regulated in concert with cell growth via the TOR protein kinase pathway. Inhibition of TOR kinase decreases association of Rpf2, Spb4, Nog1 and Nog2 with pre-ribosomes. PMID:22735702

  12. Lateral transfer of eukaryotic ribosomal RNA genes: an emerging concern for molecular ecology of microbial eukaryotes.

    PubMed

    Yabuki, Akinori; Toyofuku, Takashi; Takishita, Kiyotaka

    2014-07-01

    Ribosomal RNA (rRNA) genes are widely utilized in depicting organismal diversity and distribution in a wide range of environments. Although a few cases of lateral transfer of rRNA genes between closely related prokaryotes have been reported, it remains to be reported from eukaryotes. Here, we report the first case of lateral transfer of eukaryotic rRNA genes. Two distinct sequences of the 18S rRNA gene were detected from a clonal culture of the stramenopile, Ciliophrys infusionum. One was clearly derived from Ciliophrys, but the other gene originated from a perkinsid alveolate. Genome-walking analyses revealed that this alveolate-type rRNA gene is immediately adjacent to two protein-coding genes (ubc12 and usp39), and the origin of both genes was shown to be a stramenopile (that is, Ciliophrys) in our phylogenetic analyses. These findings indicate that the alveolate-type rRNA gene is encoded on the Ciliophrys genome and that eukaryotic rRNA genes can be transferred laterally.

  13. 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

  14. A group-I intron in the mitochondrial small subunit ribosomal RNA gene of Sclerotinia sclerotiorum.

    PubMed

    Carbone, I; Anderson, J B; Kohn, L M

    1995-01-01

    A 1,380-bp intervening sequence within the mitochondrial small subunit ribosomal RNA (mt SSU rRNA) gene of the fungus Sclerotinia sclerotiorum has been sequenced and identified as a group-I intron. This is the first report of an intron in the mt SSU rRNA gene. The intron shows close similarity in secondary structure to the subgroup-IC2 introns from Podospora (ND3i1, ND5i2, and COIi5) and Neurospora (ND5i1). The intron has an open reading frame (ORF) that encodes a putative protein of 420 amino acids which contains two copies of the LAGLI-DADG motif. The ORF belongs to a family of ORFs identified in Podospora (ND3i1, ND4Li1, ND4Li2, ND5i2, and COIi5) and Neurospora (ND5i1). The putative 420-aa polypeptide is also similar to a site-specific endonuclease in the chloroplast large subunit ribosomal RNA (LSU rRNA) gene of the green alga Chlamydomonas eugametos. In each clone of S. sclerotiorum examined, including several clones which were sampled over a 3-year period from geographically separated sites, all isolates either had the intron or lacked the intron within the mt SSU rRNA gene. Screening by means of Southern hybridization and PCR amplification detected the intron in the mt SSU rRNA genes of S. minor, S. trifoliorum and Sclerotium cepivorum, but not in other members of the Sclerotiniaceae, such as Botrytis anamorphs of Botryotinia spp., or in other ascomycetous and basidiomycetous fungi. PMID:7788720

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

    PubMed

    Yoshikawa, Masaru; Fujii, Yoichi Robertus

    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

  16. 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

  17. Phylogeny of lobose amoebae based on actin and small-subunit ribosomal RNA genes.

    PubMed

    Fahrni, José F; Bolivar, Ignacio; Berney, Cédric; Nassonova, Elena; Smirnov, Alexey; Pawlowski, Jan

    2003-11-01

    Lobose amoebae are abundant free-living protists and important pathogenic agents, yet their evolutionary history and position in the universal tree of life are poorly known. Molecular data for lobose amoebae are limited to a few species, and all phylogenetic studies published so far lacked representatives of many of their taxonomic groups. Here we analyze actin and small-subunit ribosomal RNA (SSU rRNA) gene sequences of a broad taxon sampling of naked, lobose amoebae. Our results support the existence of a monophyletic Amoebozoa clade, which comprises all lobose amoebae examined so far, the amitochondriate pelobionts and entamoebids, and the slime molds. Both actin and SSU rRNA phylogenies distinguish two well-defined clades of amoebae, the "Gymnamoebia sensu stricto" and the Archamoebae (pelobionts + entamoebids), and one weakly supported and ill-resolved group comprising some naked, lobose amoebae and the Mycetozoa.

  18. In vivo disruption of Xenopus U3 snRNA affects ribosomal RNA processing.

    PubMed Central

    Savino, R; Gerbi, S A

    1990-01-01

    DNA oligonucleotide complementary to sequences in the 5' third of U3 snRNA were injected into Xenopus oocyte nuclei to disrupt endogenous U3 snRNA. The effect of this treatment on rRNA processing was examined. We found that some toads have a single rRNA processing pathway, whereas in other toads, two rRNA processing pathways can coexist in a single oocyte. U3 snRNA disruption in toads with the single rRNA processing pathway caused a reduction in 20S and '32S' pre-rRNA. In addition, in toads with two rRNA processing pathways, an increase in '36S' pre-rRNA of the second pathway is observed. This is the first in vivo demonstration that U3 snRNA plays a role in rRNA processing. Cleavage site #3 is at the boundary of ITS 1 and 5.8S and links all of the affected rRNA intermediates: 20S and '32S' are the products of site #3 cleavage in the first pathway and '36S' is the substrate for cleavage at site #3 in the second pathway. We postulate that U3 snRNP folds pre-rRNA into a conformation dictating correct cleavage at processing site #3. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. Fig. 8. PMID:2357971

  19. 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

  20. [Effect of blue and red light on the synthesis of ribosomal RNA in Chlorella].

    PubMed

    Steup, M

    1975-10-27

    In autotrophic cultures of Chlorella pyrenoidosa (strain 211-8b) incorporation of tritiated guanosine and uridine into ribosomal RNA is stimulated by light. Blue light of wavelengths around 457 nm is considerably more effective than red light around 679 nm (5-10(-10) Einstein cm-2 sec-1 for both). This effect can be demonstrated for young daughter cells (at the end of the dark period) and for older cells (at the end of the light period). It is shown to depend on a regulation of rRNA-synthesis. The blue light dependent enhancement of incorporation is more pronounced in the cytoplasmic rRNA (25 and 18 s) than in the chloroplast rRNA (23 and 16 s). Blue light of low intensity (1-10(-10) Einstein cm-2 sec-1) has nearly the same effectivity as the fivefold intensity, whereas red light of equal quantum fluxes enhances incorporation only slightly compared with the dark control. The blue light dependent enhancement of rRNA-synthesis continues in the following darkness in contrary to that caused by red light. This enhancement is also found in DCMU-poisened cultures. In contrast to this, is red light in presence of DCMU, incorporation into rRNA is nearly the same as in dark. It is concluded that the regulation of rRNA-synthesis in red light is closely connected to complete photosynthesis, while in blue light an additional regulation takes place independent of photosynthesis.

  1. How does a scanning ribosomal particle move along the 5'-untranslated region of eukaryotic mRNA? Brownian Ratchet model.

    PubMed

    Spirin, Alexander S

    2009-11-17

    A model of the ATP-dependent unidirectional movement of the 43S ribosomal initiation complex (=40S ribosomal subunit + eIF1 + eIF1A + eIF2.GTP.Met-tRNA(i) + eIF3) during scanning of the 5'-untranslated region of eukaryotic mRNA is proposed. The model is based on the principles of molecular Brownian ratchet machines and explains several enigmatic data concerning the scanning complex. In this model, the one-dimensional diffusion of the ribosomal initiation complex along the mRNA chain is rectified into the net-unidirectional 5'-to-3' movement by the Feynman ratchet-and-pawl mechanism. The proposed mechanism is organized by the heterotrimeric protein eIF4F (=eIF4A + eIF4E + eIF4G), attached to the scanning ribosomal particle via eIF3, and the RNA-binding protein eIF4B that is postulated to play the role of the pawl. The energy for the useful work of the ratchet-and-pawl mechanism is supplied from ATP hydrolysis induced by the eIF4A subunit: ATP binding and its hydrolysis alternately change the affinities of eIF4A for eIF4B and for mRNA, resulting in the restriction of backward diffusional sliding of the 43S ribosomal complex along the mRNA chain, while stochastic movements ahead are allowed.

  2. 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.

  3. 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

  4. Phylogenetic position of Cryothecomonas inferred from nuclear-encoded small subunit ribosomal RNA.

    PubMed

    Kühn, S; Lange, M; Medlin, L K

    2000-12-01

    The systematic position of the genus Cryothecomonas has been determined from an analysis of the nuclear-encoded small subunit ribosomal RNA gene of Cryothecomonas longipes and two strains of Cryothecomonas aestivalis. Our phylogenetic trees inferred from maximum likelihood, distance and maximum parsimony methods robustly show that the genus Cryothecomonas clusters within the phylum Cercozoa, and is related to the sarcomonad flagellate Heteromita globosa. Morphological data supporting the taxonomic placement of Cryothecomonas near the sarcomonad flagellates has been compiled from the literature. The high number of nucleotide substitutions found between two morphologically indistinguishable strains of Cryothecomonas aestivalis suggests the possibility of cryptic species within Cryothecomonas aestivalis. PMID:11212894

  5. Mutation of the mitochondrial large ribosomal RNA can provide pentamidine resistance to Saccharomyces cerevisiae.

    PubMed

    Örs, Ş Tomris; Akdoğan, Emel; Dunn, Cory D

    2014-09-01

    Pentamidine is used to treat several trypanosomal diseases, as well as opportunistic infection by pathogenic fungi. However, the relevant targets of this drug are unknown. We isolated dominant mutations providing pentamidine resistance to Saccharomyces cerevisiae, one of which was localized to mitochondrial DNA. Next-generation sequencing revealed alteration of a widely conserved base at the peptidyl transferase center of the mitochondrial 21S ribosomal RNA. Our results provide a potential rationale for the toxicity of this drug to patients, and we discuss whether blockade of mitochondrial translation is the mechanism by which pathogenic fungi or protists are killed by pentamidine.

  6. A tandem array of 5S ribosomal RNA genes in Pythium irregulare.

    PubMed

    Belkhiri, A; Intengan, H; Klassen, G R

    1997-02-28

    The 5S ribosomal RNA genes of the oomycete Pythium irregulare exist in tandem arrays unlinked to the rDNA repeat unit. A clone with a 9.2-kb insert containing an array of 5S genes was identified in a lambda genomic library and was characterized by restriction mapping and partial sequencing. The array consisted of 9 apparently identical 5S genes and their spacers in tandem, followed by a diverged 5S-like sequence that is likely to be a pseudogene. This gene arrangement, although almost universal in plants and animals, is rare in fungi and protists.

  7. 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.

  8. Structural arrangement of tRNA binding sites on Escherichia coli ribosomes, as revealed from data on affinity labelling with photoactivatable tRNA derivatives.

    PubMed

    Graifer, D M; Babkina, G T; Matasova, N B; Vladimirov, S N; Karpova, G G; Vlassov, V V

    1989-07-01

    A systematic study of protein environment of tRNA in ribosomes in model complexes representing different translation steps was carried out using the affinity labelling of the ribosomes with tRNA derivatives bearing aryl azide groups scattered statistically over tRNA guanine residues. Analysis of the proteins crosslinked to tRNA derivatives showed that the location of the derivatives in the aminoacyl (A) site led to the labelling of the proteins S5 and S7 in all complexes studied, whereas the labelling of the proteins S2, S8, S9, S11, S14, S16, S17, S18, S19, S21 as well as L9, L11, L14, L15, L21, L23, L24, L29 depended on the state of tRNA in A site. Similarly, the location of tRNA derivatives in the peptidyl (P) site resulted in the labelling of the proteins L27, S11, S13 and S19 in all states, whereas the labelling of the proteins S5, S7, S9, S12, S14, S20, S21 as well as L2, L13, L14, L17, L24, L27, L31, L32, L33 depended on the type of complex. The derivatives of tRNA(fMet) were found to crosslink to S1, S3, S5, S7, S9, S14 and L1, L2, L7/L12, L27. Based on the data obtained, a general principle of the dynamic functioning of ribosomes has been proposed: (i) the formation of each type of ribosomal complex is accompanied by changes in mutual arrangement of proteins - 'conformational adjustment' of the ribosome - and (ii) a ribosome can dynamically change its internal structure at each step of initiation and elongation; on the 70 S ribosome there are no rigidly fixed structures forming tRNA-binding sites (primarily A and P sites).

  9. 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

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

    PubMed

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

    2000-11-01

    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.

  11. Studies on the control of ribosomal RNA synthesis in HeLa cells.

    PubMed

    Chesterton, C J; Coupar, B E; Butterworth, P H; Green, M H

    1975-09-01

    In many eucaryotic systems protein synthesis is coupled to ribosomal RNA synthesis such that shut-down of the former causes inhibition of the latter. We have investigated this stringency phenomenon in HeLa cells. The protein synthesis inhibitors cycloheximide and puromycin cause inactivation of both processes but valine starvation totally inhibits only the processing of 45-S RNA. DNA-dependent RNA polymerases from A, B and C (or I, II and III respectively) were extracted, separated partially by DEAE-cellulose chromatography and their activity levels determined. These do not decrease significantly during inhibition of protein synthesis. To find out whether or not form A is bound to its template under these conditions, proteins were removed from chromatin with the detergent sarkosyl. This does not affect bound RNA polymerase. Inhibition of protein synthesis caused up to 50% reduction in endogenous alpha-amanitin-insensitive chromatin-RNA-synthesising activity. This reduced level of activity was not affected by sarkosyl treatment. Levels in normal cells were stimulated. This result indicates that the form A RNA polymerase is not bound to its template when protein synthesis is inhibited.

  12. 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.

  13. 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

  14. [Study of the photoaffinity modification of Escherichia coli ribosomes near the donor tRNA-binding center].

    PubMed

    Bausk, E V; Graĭfer, D M; Karpova, G G

    1985-01-01

    Affinity labelling of E. coli ribosomes near the donor tRNA-binding (P) site was studied with the use of photoreactive derivatives of tRNAPhe bearing arylazidogroups on N7 atoms of guanine residues (azido-tRNA). UV-irradiation of complexes 70S ribosome.poly(U).azido- tRNA(P-site) and 70S ribosome.poly(U).azido-tRNA(P-site).Phe- tRNAPhe(A-site) resulted in covalent attachment of azido-tRNA to ribosomes, both subunits being labelled. In both cases modification extent of 30S subunit was two-fold than that of the 50S one. It was shown that when the A-site was free the azido-tRNA located in P-site labelled proteins S9, S11, S12, S13, S21 and L14, L27, L31. Azido-tRNA located in P-site when the A-site was occupied with Phe-tRNAPhe labelled proteins S11, S12, S13, S14, S19, L32/L33 and possibly L23, L25. From the comparison of the sets of proteins labelled when A-site was free or occupied a conclusion was drawn that aminoacyl-tRNA located in ribosomal A-site affects the arrangement of deacylated tRNA in P-site. Data obtained allow to propose that proteins S5, S19, S20 and L24, L33 interact with guanine residues important for the tRNA tertiary structure formation.

  15. 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. PMID:27288410

  16. Cell-Type-Specific mRNA Purification by Translating Ribosome Affinity Purification (TRAP)

    PubMed Central

    Heiman, Myriam; Kulicke, Ruth; Fenster, Robert J.; Greengard, Paul; Heintz, Nathaniel

    2014-01-01

    Cellular diversity and architectural complexity create barriers to understanding the function of the mammalian central nervous system (CNS) at a molecular level. To address this problem, we recently developed a methodology that provides the ability to profile the entire translated mRNA complement of any genetically defined cell population. This methodology, which we termed translating ribosome affinity purification, or TRAP, combines cell-type-specific transgene expression with affinity purification of translating ribosomes. TRAP can be used to study the cell-type-specific mRNA profiles of any genetically defined cell type, and has been successfully used to date in organisms ranging from D. melanogaster to mice and human cultured cells. Unlike other methodologies that rely upon micro-dissection, cell panning, or cell sorting, the TRAP methodology bypasses the need for tissue fixation or single-cell suspensions (and potential artifacts these treatments introduce), and reports on mRNAs in the entire cell body. This protocol provides a step-by-step guide to implementing the TRAP methodology, which takes two days to complete once all materials are in hand. PMID:24810037

  17. Evidence for tertiary structural RNA-RNA interactions within the protein S4 binding site at the 5'-end of 16S ribosomal RNA of Escherichia coli.+.

    PubMed Central

    Ungewickell, E; Ehresmann, C; Stiegler, P; Garrett, R

    1975-01-01

    Evidence is presented for tertiary structural interaction(s) (interactions(s) between two regions of an RNA molecule that are widely separated in the RNA sequence) within the 5'-one third of the 16S ribosomal RNA of Escherichia coli that constitutes the binding site of protein S4. The two main interacting RNA regions were separated by about 120 nucleotides (sections Q to M) of the 16S RNA sequence. A second, smaller gap, of 13 nucleotides, occurred within section C". The two main interacting regions contain about 150 nucleotides (sections H" to Q) and 160 nucleotides (sections M to C"). They are folded back on one another and, especially in the presence of protein S4, are strongly protected against ribonuclease digestion. The intermediate region (sections Q to M), however, is relatively accessible to ribonucleases in the S4-RNP. By partial removal of subfragments from the RNA complex it was possible to localise the two main interacting sites within sections H" - H and sections I" - C". Three main criteria for the specificity of the RNA-RNA interactions were invoked and satisfied. The possibility of other tertiary structural RNA-RNA interactions occurring in other regions of the 16S RNA is discussed. Finally, all the structural information on the S4-RNP is summarised and a tentative model is proposed. Images PMID:1103089

  18. SINA: Accurate high-throughput multiple sequence alignment of ribosomal RNA genes

    PubMed Central

    Pruesse, Elmar; Peplies, Jörg; Glöckner, Frank Oliver

    2012-01-01

    Motivation: In the analysis of homologous sequences, computation of multiple sequence alignments (MSAs) has become a bottleneck. This is especially troublesome for marker genes like the ribosomal RNA (rRNA) where already millions of sequences are publicly available and individual studies can easily produce hundreds of thousands of new sequences. Methods have been developed to cope with such numbers, but further improvements are needed to meet accuracy requirements. Results: In this study, we present the SILVA Incremental Aligner (SINA) used to align the rRNA gene databases provided by the SILVA ribosomal RNA project. SINA uses a combination of k-mer searching and partial order alignment (POA) to maintain very high alignment accuracy while satisfying high throughput performance demands. SINA was evaluated in comparison with the commonly used high throughput MSA programs PyNAST and mothur. The three BRAliBase III benchmark MSAs could be reproduced with 99.3, 97.6 and 96.1 accuracy. A larger benchmark MSA comprising 38 772 sequences could be reproduced with 98.9 and 99.3% accuracy using reference MSAs comprising 1000 and 5000 sequences. SINA was able to achieve higher accuracy than PyNAST and mothur in all performed benchmarks. Availability: Alignment of up to 500 sequences using the latest SILVA SSU/LSU Ref datasets as reference MSA is offered at http://www.arb-silva.de/aligner. This page also links to Linux binaries, user manual and tutorial. SINA is made available under a personal use license. Contact: epruesse@mpi-bremen.de Supplementary information: Supplementary data are available at Bioinformatics online. PMID:22556368

  19. Ribosomal RNA gene diversity, effective population size, and evolutionary longevity in asexual glomeromycota.

    PubMed

    Vankuren, Nicholas W; den Bakker, Henk C; Morton, Joseph B; Pawlowska, Teresa E

    2013-01-01

    Arbuscular mycorrhizal fungi (phylum Glomeromycota) are among the oldest and most successful symbionts of land plants. With no evidence of sexual reproduction, their evolutionary success is inconsistent with the prediction that asexual taxa are vulnerable to extinction due to accumulation of deleterious mutations. To explore why Glomeromycota defy this prediction, we studied ribosomal RNA (rRNA) gene evolution in the Claroideoglomus lineage and estimated effective population size, N(e) , in C. etunicatum. We found that rRNA genes of these fungi exhibit unusual and complex patterns of molecular evolution. In C. etunicatum, these patterns can be collectively explained by an unexpectedly large N(e) combined with imperfect genome-wide and population-level rRNA gene repeat homogenization. The mutations accumulated in rRNA gene sequences indicate that natural selection is effective at purging deleterious mutations in the Claroideoglomus lineage, which is also consistent with the large N(e) of C. etunicatum. We propose that in the near absence of recombination, asexual reproduction involving massively multinucleate spores typical for Glomeromycota is responsible for the improved efficacy of selection relative to drift. We postulate that large effective population sizes contribute to the evolutionary longevity of Glomeromycota.

  20. Label- and amplification-free electrochemical detection of bacterial ribosomal RNA.

    PubMed

    Henihan, Grace; Schulze, Holger; Corrigan, Damion K; Giraud, Gerard; Terry, Jonathan G; Hardie, Alison; Campbell, Colin J; Walton, Anthony J; Crain, Jason; Pethig, Ronald; Templeton, Kate E; Mount, Andrew R; Bachmann, Till T

    2016-07-15

    Current approaches to molecular diagnostics rely heavily on PCR amplification and optical detection methods which have restrictions when applied to point of care (POC) applications. Herein we describe the development of a label-free and amplification-free method of pathogen detection applied to Escherichia coli which overcomes the bottleneck of complex sample preparation and has the potential to be implemented as a rapid, cost effective test suitable for point of care use. Ribosomal RNA is naturally amplified in bacterial cells, which makes it a promising target for sensitive detection without the necessity for prior in vitro amplification. Using fluorescent microarray methods with rRNA targets from a range of pathogens, an optimal probe was selected from a pool of probe candidates identified in silico. The specificity of probes was investigated on DNA microarray using fluorescently labeled 16S rRNA target. The probe yielding highest specificity performance was evaluated in terms of sensitivity and a LOD of 20 pM was achieved on fluorescent glass microarray. This probe was transferred to an EIS end point format and specificity which correlated to microarray data was demonstrated. Excellent sensitivity was facilitated by the use of uncharged PNA probes and large 16S rRNA target and investigations resulted in an LOD of 50 pM. An alternative kinetic EIS assay format was demonstrated with which rRNA could be detected in a species specific manner within 10-40min at room temperature without wash steps. PMID:27016627

  1. Label- and amplification-free electrochemical detection of bacterial ribosomal RNA.

    PubMed

    Henihan, Grace; Schulze, Holger; Corrigan, Damion K; Giraud, Gerard; Terry, Jonathan G; Hardie, Alison; Campbell, Colin J; Walton, Anthony J; Crain, Jason; Pethig, Ronald; Templeton, Kate E; Mount, Andrew R; Bachmann, Till T

    2016-07-15

    Current approaches to molecular diagnostics rely heavily on PCR amplification and optical detection methods which have restrictions when applied to point of care (POC) applications. Herein we describe the development of a label-free and amplification-free method of pathogen detection applied to Escherichia coli which overcomes the bottleneck of complex sample preparation and has the potential to be implemented as a rapid, cost effective test suitable for point of care use. Ribosomal RNA is naturally amplified in bacterial cells, which makes it a promising target for sensitive detection without the necessity for prior in vitro amplification. Using fluorescent microarray methods with rRNA targets from a range of pathogens, an optimal probe was selected from a pool of probe candidates identified in silico. The specificity of probes was investigated on DNA microarray using fluorescently labeled 16S rRNA target. The probe yielding highest specificity performance was evaluated in terms of sensitivity and a LOD of 20 pM was achieved on fluorescent glass microarray. This probe was transferred to an EIS end point format and specificity which correlated to microarray data was demonstrated. Excellent sensitivity was facilitated by the use of uncharged PNA probes and large 16S rRNA target and investigations resulted in an LOD of 50 pM. An alternative kinetic EIS assay format was demonstrated with which rRNA could be detected in a species specific manner within 10-40min at room temperature without wash steps.

  2. Novel extraction strategy of ribosomal RNA and genomic DNA from cheese for PCR-based investigations.

    PubMed

    Bonaïti, Catherine; Parayre, Sandrine; Irlinger, Françoise

    2006-03-15

    Cheese microorganisms, such as bacteria and fungi, constitute a complex ecosystem that plays a central role in cheeses ripening. The molecular study of cheese microbial diversity and activity is essential but the extraction of high quality nucleic acid may be problematic: the cheese samples are characterised by a strong buffering capacity which negatively influenced the yield of the extracted rRNA. The objective of this study is to develop an effective method for the direct and simultaneous isolation of yeast and bacterial ribosomal RNA and genomic DNA from the same cheese samples. DNA isolation was based on a protocol used for nucleic acids isolation from anaerobic digestor, without preliminary washing step with the combined use of the action of chaotropic agent (acid guanidinium thiocyanate), detergents (SDS, N-lauroylsarcosine), chelating agent (EDTA) and a mechanical method (bead beating system). The DNA purification was carried out by two washing steps of phenol-chloroform. RNA was isolated successfully after the second acid extraction step by recovering it from the phenolic phase of the first acid extraction. The novel method yielded pure preparation of undegraded RNA accessible for reverse transcription-PCR. The extraction protocol of genomic DNA and rRNA was applicable to complex ecosystem of different cheese matrices.

  3. Stimulation of -1 programmed ribosomal frameshifting by a metabolite-responsive RNA pseudoknot.

    PubMed

    Chou, Ming-Yuan; Lin, Szu-Chieh; Chang, Kung-Yao

    2010-06-01

    Specific recognition of metabolites by functional RNA motifs within mRNAs has emerged as a crucial regulatory strategy for feedback control of biochemical reactions. Such riboswitches have been demonstrated to regulate different gene expression processes, including transcriptional termination and translational initiation in prokaryotic cells, as well as splicing in eukaryotic cells. The regulatory process is usually mediated by modulating the accessibility of specific sequence information of the expression platforms via metabolite-induced RNA conformational rearrangement. In eukaryotic systems, viral and the more limited number of cellular decoding -1 programmed ribosomal frameshifting (PRF) are commonly promoted by a 3' mRNA pseudoknot. In addition, such -1 PRF is generally constitutive rather than being regulatory, and usually results in a fixed ratio of products. We report here an RNA pseudoknot capable of stimulating -1 PRF whose efficiency can be tuned in response to the concentration of S-adenosylhomocysteine (SAH), and the improvement of its frameshifting efficiency by RNA engineering. In addition to providing an alternative approach for small-molecule regulation of gene expression in eukaryotic cells, such a metabolite-responsive pseudoknot suggests a plausible mechanism for metabolite-driven translational regulation of gene expression in eukaryotic systems. PMID:20435898

  4. [Study of the mRNA-binding region of ribosomes at different steps of translation. II. Affinity modification of Escherichia coli ribosomes by benzylidene derivative of AUGU6 in the 70S initiation complex].

    PubMed

    Babkina, G T; Karpova, G G; Matasova, N B; Berzin', V M; Gren, E Ia

    1985-01-01

    2',3'-O-(4-[N-(2-chloroethyl)-N-methylamino]) benzylidene derivative of AUGU6 was used for identification of the proteins in the region of the mRNA-binding centre of E. coli ribosomes. This derivative alkylated ribosomes (preferentially 30S ribosomal) with high efficiency within the 70S initiation complex. In both 30S and 50S ribosomal subunits proteins and rRNA were modified. Specificity of the alkylation of ribosomal proteins and rRNA with the reagent was proved by the inhibitory action of AUGU6. Using the method of two-dimensional electrophoresis in polyacrylamide gel the proteins S4, S12, S13, S14, S15, S18, S19 and S20/L26 which are labelled by the analog of mRNA were identified.

  5. 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. PMID:26003987

  6. 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.

  7. Thermotoga maritima ribonuclease III. Characterization of thermostable biochemical behavior and analysis of conserved base pairs that function as reactivity epitopes for the Thermotoga 23S rRNA precursor.

    PubMed

    Nathania, Lilian; Nicholson, Allen W

    2010-08-24

    The cleavage of double-stranded (ds) RNA by ribonuclease III is a conserved early step in bacterial rRNA maturation. Studies on the mechanism of dsRNA cleavage by RNase III have focused mainly on the enzymes from mesophiles such as Escherichia coli. In contrast, neither the catalytic properties of extremophile RNases III nor the structures and reactivities of their cognate substrates have been described. The biochemical behavior of RNase III of the hyperthermophilic bacterium Thermotoga maritima was analyzed using purified recombinant enzyme. T. maritima (Tm) RNase III catalytic activity exhibits a broad optimal temperature range of approximately 40-70 degrees C, with significant activity at 95 degrees C. Tm-RNase III cleavage of substrate is optimally supported by Mg(2+) at >or=1 mM concentrations. Mn(2+), Co(2+), and Ni(2+) also support activity but with reduced efficiencies. The enzyme functions optimally at pH 8 and approximately 50-80 mM salt concentrations. Small RNA hairpins that incorporate the 16S and 23S pre-rRNA stem sequences are efficiently cleaved by Tm-RNase III at sites that are consistent with production in vivo of the immediate precursors to the mature rRNAs. Analysis of pre-23S substrate variants reveals a dependence of reactivity on the base-pair (bp) sequence in the proximal box (pb), a site of protein contact that functions as a positive recognition determinant for Escherichia coli (Ec) RNase III substrates. The dependence of reactivity on the pb sequence is similar to that observed with Ec-RNase III substrates. In fact, Tm-RNase III cleaves an Ec-RNase III substrate with identical specificity and is inhibited by antideterminant bp that also inhibit Ec-RNase III. These results indicate the conservation, across a broad phylogenetic distance, of positive and negative determinants of reactivity of bacterial RNase III substrates.

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

    PubMed Central

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

    2013-01-01

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

  9. Comparison of sequence differences in a variable 23S rRNA domain among sets of cryptic species of ciliated protozoa.

    PubMed

    Nanney, D L; Park, C; Preparata, R; Simon, E M

    1998-01-01

    Studies were undertaken to discover the relative molecular distances separating some familiar forms of ciliated protozoa, and the genetic species they include. Sequences of 190 bases of the D2 domain of the large ribosomal nucleic acid molecule were obtained by polymerase chain reaction from protists of three distinctive groups of ciliated protozoa-Colpoda, Paramecium and Tetrahymena. Evolutionary trees were constructed for each set of sequences using the PHYLOGEN 1.0 string programs. All three groups of ciliates manifested large molecular diversity among strains difficult or impossible to distinguish morphologically. The largest single evolutionary distance within a group was the 75 differences separating Tetrahymena paravorax from the other tetrahymenids. The largest mean distance for a group was the 21.2 for the colpodids. In all the protist groups the large molecular diversity is obscured by morphological conservatism associated with constraints of ancient designs. The molecular diversity within morphotypes argues for long evolutionary coexistence of species differentiated from each other in significant physiological, ecological, or nutritional ways.

  10. Bacteria evade immune recognition via TLR13 and binding of their 23S rRNA by MLS antibiotics by the same mechanisms

    PubMed Central

    Hochrein, Hubertus; Kirschning, Carsten J.

    2013-01-01

    The immune system recognizes pathogens and other danger by means of pattern recognition receptors. Recently, we have demonstrated that the orphan Toll-like receptor 13 (TLR13) senses a defined sequence of the bacterial rRNA and that bacteria use specific mechanisms to evade macrolide lincosamide streptogramin (MLS) antibiotics detection via TLR13. PMID:23802068

  11. Partial methylation at Am100 in 18S rRNA of baker's yeast reveals ribosome heterogeneity on the level of eukaryotic rRNA modification.

    PubMed

    Buchhaupt, Markus; Sharma, Sunny; Kellner, Stefanie; Oswald, Stefanie; Paetzold, Melanie; Peifer, Christian; Watzinger, Peter; Schrader, Jens; Helm, Mark; Entian, Karl-Dieter

    2014-01-01

    Ribosome heterogeneity is of increasing biological significance and several examples have been described for multicellular and single cells organisms. In here we show for the first time a variation in ribose methylation within the 18S rRNA of Saccharomyces cerevisiae. Using RNA-cleaving DNAzymes, we could specifically demonstrate that a significant amount of S. cerevisiae ribosomes are not methylated at 2'-O-ribose of A100 residue in the 18S rRNA. Furthermore, using LC-UV-MS/MS of a respective 18S rRNA fragment, we could not only corroborate the partial methylation at A100, but could also quantify the methylated versus non-methylated A100 residue. Here, we exhibit that only 68% of A100 in the 18S rRNA of S.cerevisiae are methylated at 2'-O ribose sugar. Polysomes also contain a similar heterogeneity for methylated Am100, which shows that 40S ribosome subunits with and without Am100 participate in translation. Introduction of a multicopy plasmid containing the corresponding methylation guide snoRNA gene SNR51 led to an increased A100 methylation, suggesting the cellular snR51 level to limit the extent of this modification. Partial rRNA modification demonstrates a new level of ribosome heterogeneity in eukaryotic cells that might have substantial impact on regulation and fine-tuning of the translation process.

  12. RNA–DNA differences in human mitochondria restore ancestral form of 16S ribosomal RNA

    PubMed Central

    Bar-Yaacov, Dan; Avital, Gal; Levin, Liron; Richards, Allison L.; Hachen, Naomi; Rebolledo Jaramillo, Boris; Nekrutenko, Anton; Zarivach, Raz; Mishmar, Dan

    2013-01-01

    RNA transcripts are generally identical to the underlying DNA sequences. Nevertheless, RNA–DNA differences (RDDs) were found in the nuclear human genome and in plants and animals but not in human mitochondria. Here, by deep sequencing of human mitochondrial DNA (mtDNA) and RNA, we identified three RDD sites at mtDNA positions 295 (C-to-U), 13710 (A-to-U, A-to-G), and 2617 (A-to-U, A-to-G). Position 2617, within the 16S rRNA, harbored the most prevalent RDDs (>30% A-to-U and ∼15% A-to-G of the reads in all tested samples). The 2617 RDDs appeared already at the precursor polycistrone mitochondrial transcript. By using traditional Sanger sequencing, we identified the A-to-U RDD in six different cell lines and representative primates (Gorilla gorilla, Pongo pigmaeus, and Macaca mulatta), suggesting conservation of the mechanism generating such RDD. Phylogenetic analysis of more than 1700 vertebrate mtDNA sequences supported a thymine as the primate ancestral allele at position 2617, suggesting that the 2617 RDD recapitulates the ancestral 16S rRNA. Modeling U or G (the RDDs) at position 2617 stabilized the large ribosomal subunit structure in contrast to destabilization by an A (the pre-RDDs). Hence, these mitochondrial RDDs are likely functional. PMID:23913925

  13. One step engineering of the small-subunit ribosomal RNA using CRISPR/Cas9

    PubMed Central

    Kannan, Krishna; Tsvetanova, Billyana; Chuang, Ray-Yuan; Noskov, Vladimir N.; Assad-Garcia, Nacyra; Ma, Li; Hutchison III, Clyde A.; Smith, Hamilton O.; Glass, John I.; Merryman, Chuck; Venter, J. Craig; Gibson, Daniel G.

    2016-01-01

    Bacteria are indispensable for the study of fundamental molecular biology processes due to their relatively simple gene and genome architecture. The ability to engineer bacterial chromosomes is quintessential for understanding gene functions. Here we demonstrate the engineering of the small-ribosomal subunit (16S) RNA of Mycoplasma mycoides, by combining the CRISPR/Cas9 system and the yeast recombination machinery. We cloned the entire genome of M. mycoides in yeast and used constitutively expressed Cas9 together with in vitro transcribed guide-RNAs to introduce engineered 16S rRNA genes. By testing the function of the engineered 16S rRNA genes through genome transplantation, we observed surprising resilience of this gene to addition of genetic elements or helix substitutions with phylogenetically-distant bacteria. While this system could be further used to study the function of the 16S rRNA, one could envision the “simple” M. mycoides genome being used in this setting to study other genetic structures and functions to answer fundamental questions of life. PMID:27489041

  14. One step engineering of the small-subunit ribosomal RNA using CRISPR/Cas9.

    PubMed

    Kannan, Krishna; Tsvetanova, Billyana; Chuang, Ray-Yuan; Noskov, Vladimir N; Assad-Garcia, Nacyra; Ma, Li; Hutchison Iii, Clyde A; Smith, Hamilton O; Glass, John I; Merryman, Chuck; Venter, J Craig; Gibson, Daniel G

    2016-01-01

    Bacteria are indispensable for the study of fundamental molecular biology processes due to their relatively simple gene and genome architecture. The ability to engineer bacterial chromosomes is quintessential for understanding gene functions. Here we demonstrate the engineering of the small-ribosomal subunit (16S) RNA of Mycoplasma mycoides, by combining the CRISPR/Cas9 system and the yeast recombination machinery. We cloned the entire genome of M. mycoides in yeast and used constitutively expressed Cas9 together with in vitro transcribed guide-RNAs to introduce engineered 16S rRNA genes. By testing the function of the engineered 16S rRNA genes through genome transplantation, we observed surprising resilience of this gene to addition of genetic elements or helix substitutions with phylogenetically-distant bacteria. While this system could be further used to study the function of the 16S rRNA, one could envision the "simple" M. mycoides genome being used in this setting to study other genetic structures and functions to answer fundamental questions of life. PMID:27489041

  15. Evolutionarily conserved autoregulation of alternative pre-mRNA splicing by ribosomal protein L10a

    PubMed Central

    Takei, Satomi; Togo-Ohno, Marina; Suzuki, Yutaka; Kuroyanagi, Hidehito

    2016-01-01

    Alternative splicing of pre-mRNAs can regulate expression of protein-coding genes by generating unproductive mRNAs rapidly degraded by nonsense-mediated mRNA decay (NMD). Many of the genes directly regulated by alternative splicing coupled with NMD (AS-NMD) are related to RNA metabolism, but the repertoire of genes regulated by AS-NMD in vivo is to be determined. Here, we analyzed transcriptome data of wild-type and NMD-defective mutant strains of the nematode worm Caenorhabditis elegans and demonstrate that eight of the 82 cytoplasmic ribosomal protein (rp) genes generate unproductively spliced mRNAs. Knockdown of any of the eight rp genes exerted a dynamic and compensatory effect on alternative splicing of its own transcript and inverse effects on that of the other rp genes. A large subunit protein L10a, termed RPL-1 in nematodes, directly and specifically binds to an evolutionarily conserved 39-nt stretch termed L10ARE between the two alternative 5′ splice sites in its own pre-mRNA to switch the splice site choice. Furthermore, L10ARE-mediated splicing autoregulation of the L10a-coding gene is conserved in vertebrates. These results indicate that L10a is an evolutionarily conserved splicing regulator and that homeostasis of a subset of the rp genes are regulated at the level of pre-mRNA splicing in vivo. PMID:26961311

  16. The human insulin receptor mRNA contains a functional internal ribosome entry segment

    PubMed Central

    Spriggs, Keith A.; Cobbold, Laura C.; Ridley, Simon H.; Coldwell, Mark; Bottley, Andrew; Bushell, Martin; Willis, Anne E.; Siddle, Kenneth

    2009-01-01

    Regulation of mRNA translation is an important mechanism determining the level of expression of proteins in eukaryotic cells. Translation is most commonly initiated by cap-dependent scanning, but many eukaryotic mRNAs contain internal ribosome entry segments (IRESs), providing an alternative means of initiation capable of independent regulation. Here, we show by using dicistronic luciferase reporter vectors that the 5′-UTR of the mRNA encoding human insulin receptor (hIR) contains a functional IRES. RNAi-mediated knockdown showed that the protein PTB was required for maximum IRES activity. Electrophoretic mobility shift assays confirmed that PTB1, PTB2 and nPTB, but not unr or PTB4, bound to hIR mRNA, and deletion mapping implicated a CCU motif 448 nt upstream of the initiator AUG in PTB binding. The IR-IRES was functional in a number of cell lines, and most active in cells of neuronal origin, as assessed by luciferase reporter assays. The IRES was more active in confluent than sub-confluent cells, but activity did not change during differentiation of 3T3-L1 fibroblasts to adipocytes. IRES activity was stimulated by insulin in sub-confluent cells. The IRES may function to maintain expression of IR protein in tissues such as the brain where mRNA translation by cap-dependent scanning is less effective. PMID:19654240

  17. 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.

  18. Cyanobacterial Ecotypes in Different Optical Microenvironments of a 68°C Hot Spring Mat Community Revealed by 16S-23S rRNA Internal Transcribed Spacer Region Variation†

    PubMed Central

    Ferris, Mike J.; Kühl, Michael; Wieland, Andrea; Ward, David M.

    2003-01-01

    We examined the population of unicellular cyanobacteria (Synechococcus) in the upper 3-mm vertical interval of a 68°C region of a microbial mat in a hot spring effluent channel (Yellowstone National Park, Wyoming). Fluorescence microscopy and microsensor measurements of O2 and oxygenic photosynthesis demonstrated the existence of physiologically distinct Synechococcus populations at different depths along a light gradient quantified by scalar irradiance microprobes. Molecular methods were used to evaluate whether physiologically distinct populations could be correlated with genetically distinct populations over the vertical interval. We were unable to identify patterns in genetic variation in Synechococcus 16S rRNA sequences that correlate with different vertically distributed populations. However, patterns of variation at the internal transcribed spacer locus separating 16S and 23S rRNA genes suggested the existence of closely related but genetically distinct populations corresponding to different functional populations occurring at different depths. PMID:12732563

  19. Human NAT10 Is an ATP-dependent RNA Acetyltransferase Responsible for N4-Acetylcytidine Formation in 18 S Ribosomal RNA (rRNA)*

    PubMed Central

    Ito, Satoshi; Horikawa, Sayuri; Suzuki, Tateki; Kawauchi, Hiroki; Tanaka, Yoshikazu; Suzuki, Takeo; Suzuki, Tsutomu

    2014-01-01

    Human N-acetyltransferase 10 (NAT10) is known to be a lysine acetyltransferase that targets microtubules and histones and plays an important role in cell division. NAT10 is highly expressed in malignant tumors, and is also a promising target for therapies against laminopathies and premature aging. Here we report that NAT10 is an ATP-dependent RNA acetyltransferase responsible for formation of N4-acetylcytidine (ac4C) at position 1842 in the terminal helix of mammalian 18 S rRNA. RNAi-mediated knockdown of NAT10 resulted in growth retardation of human cells, and this was accompanied by high-level accumulation of the 30 S precursor of 18 S rRNA, suggesting that ac4C1842 formation catalyzed by NAT10 is involved in rRNA processing and ribosome biogenesis. PMID:25411247

  20. Ribosome regulation by the nascent peptide.

    PubMed Central

    Lovett, P S; Rogers, E J

    1996-01-01

    Studies of bacterial and eukaryotic systems have identified two-gene operons in which the translation product of the upstream gene influences translation of the downstream gene. The upstream gene, referred to as a leader (gene) in bacterial systems or an upstream open reading frame (uORF) in eukaryotes, encodes a peptide that interferes with a function(s) of its translating ribosome. The peptides are therefore cis-acting negative regulators of translation. The inhibitory peptides typically consist of fewer than 25 residues and function prior to emergence from the ribosome. A biological role for this class of translation inhibitor is demonstrated in translation attenuation, a form or regulation that controls the inducible translation of the chloramphenicol resistance genes cat and cmlA in bacteria. Induction of cat or cmlA requires ribosome stalling at a particular codon in the leader region of the mRNA. Stalling destabilizes an adjacent, downstream mRNA secondary structure that normally sequesters the ribosome-binding site for the cat or cmlA coding regions. Genetic studies indicate that the nascent, leader-encoded peptide is the selector of the site of ribosome stalling in leader mRNA by cis interference with translation. Synthetic leader peptides inhibit ribosomal peptidyltransferase in vitro, leading to the prediction that this activity is the basis for stall site selection. Recent studies have shown that the leader peptides are rRNA-binding peptides with targets at the peptidyl transferase center of 23S rRNA. uORFs associated with several eukaryotic genes inhibit downstream translation. When inhibition depends on the specific codon sequence of the uORF, it has been proposed that the uORF-encoded nascent peptide prevents ribosome release from the mRNA at the uORF stop codon. This sets up a blockade to ribosome scanning which minimizes downstream translation. Segments within large proteins also appear to regulate ribosome activity in cis, although in most of the

  1. 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

  2. The effect of aminoacyl- or peptidyl-tRNA at the A-site on the arrangement of deacylated tRNA at the ribosomal P-site.

    PubMed

    Babkina, G T; Bausk, E V; Graifer, D M; Karpova, G G; Matasova, N B

    1984-05-21

    Photoreactive derivatives of E. coli tRNAPhe bearing arylazido groups on guanine residues (azido-tRNA) were used for affinity labelling of E. coli ribosomes in the region of the P-site when the A-site was either free or occupied by aminoacyl- or peptidyl-tRNA. Corresponding complexes of azido-tRNA with ribosomes and poly(U) were obtained both nonenzymatically and with the use of elongation factors. UV-irradiation of the complexes resulted in labelling of ribosomal proteins (preferentially of 30 S subunit). Proteins S9 and S21 were labelled only when the A-site was free; S14 - only when it was occupied; S11, S13, S19 - in both cases; S5, S7, S12, S20 - in some states.

  3. 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

  4. Genetic Characterization of Clinical Acanthamoeba Isolates from Japan using Nuclear and Mitochondrial Small Subunit Ribosomal RNA

    PubMed Central

    Rahman, Md Moshiur; Yagita, Kenji; Kobayashi, Akira; Oikawa, Yosaburo; Hussein, Amjad I.A.; Matsumura, Takahiro

    2013-01-01

    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. PMID:24039282

  5. Basonuclin Regulates a Subset of Ribosomal RNA Genes in HaCaT Cells

    PubMed Central

    Zhang, Shengliang; Wang, Junwen; Tseng, Hung

    2007-01-01

    Basonuclin (Bnc1), a cell-type-specific ribosomal RNA (rRNA) gene regulator, is expressed mainly in keratinocytes of stratified epithelium and gametogenic cells of testis and ovary. Previously, basonuclin was shown in vitro to interact with rRNA gene (rDNA) promoter at three highly conserved sites. Basonuclin's high affinity binding site overlaps with the binding site of a dedicated and ubiquitous Pol I transcription regulator, UBF, suggesting that their binding might interfere with each other if they bind to the same promoter. Knocking-down basonuclin in mouse oocytes eliminated approximately one quarter of RNA polymerase I (Pol I) transcription foci, without affecting the BrU incorporation of the remaining ones, suggesting that basonuclin might regulate a subset of rDNA. Here we show, via chromatin immunoprecipitation (ChIP), that basonuclin is associated with rDNA promoters in HaCaT cells, a spontaneously established human keratinocyte line. Immunoprecipitation data suggest that basonuclin is in a complex that also contains the subunits of Pol I (RPA194, RPA116), but not UBF. Knocking-down basonuclin in HaCaT cells partially impairs the association of RPA194 to rDNA promoter, but not that of UBF. Basonuclin-deficiency also reduces the amount of 47S pre-rRNA, but this effect can be seen only after cell-proliferation related rRNA synthesis has subsided at a higher cell density. DNA sequence of basonuclin-bound rDNA promoters shows single nucleotide polymorphisms (SNPs) that differ from those associated with UBF-bound promoters, suggesting that basonuclin and UBF interact with different subsets of promoters. In conclusion, our results demonstrate basonuclin's functional association with rDNA promoters and its interaction with Pol I in vivo. Our data also suggest that basonuclin-Pol I complex transcribes a subset of rDNA. PMID:17878937

  6. Connecting the kinetics and energy landscape of tRNA translocation on the ribosome.

    PubMed

    Whitford, Paul C; Blanchard, Scott C; Cate, Jamie H D; Sanbonmatsu, Karissa Y

    2013-01-01

    Functional rearrangements in biomolecular assemblies result from diffusion across an underlying energy landscape. While bulk kinetic measurements rely on discrete state-like approximations to the energy landscape, single-molecule methods can project the free energy onto specific coordinates. With measures of the diffusion, one may establish a quantitative bridge between state-like kinetic measurements and the continuous energy landscape. We used an all-atom molecular dynamics simulation of the 70S ribosome (2.1 million atoms; 1.3 microseconds) to provide this bridge for specific conformational events associated with the process of tRNA translocation. Starting from a pre-translocation configuration, we identified sets of residues that collectively undergo rotary rearrangements implicated in ribosome function. Estimates of the diffusion coefficients along these collective coordinates for translocation were then used to interconvert between experimental rates and measures of the energy landscape. This analysis, in conjunction with previously reported experimental rates of translocation, provides an upper-bound estimate of the free-energy barriers associated with translocation. While this analysis was performed for a particular kinetic scheme of translocation, the quantitative framework is general and may be applied to energetic and kinetic descriptions that include any number of intermediates and transition states.

  7. Mutant forms of Escherichia coli protein L25 unable to bind to 5S rRNA are incorporated efficiently into the ribosome in vivo.

    PubMed

    Anikaev, A Y; Korepanov, A P; Korobeinikova, A V; Kljashtorny, V G; Piendl, W; Nikonov, S V; Garber, M B; Gongadze, G M

    2014-08-01

    5S rRNA-binding ribosomal proteins of the L25 family are an evolutional acquisition of bacteria. Earlier we showed that (i) single replacements in the RNA-binding module of the protein of this family result in destabilization or complete impossibility to form a complex with 5S rRNA in vitro; (ii) ΔL25 ribosomes of Escherichia coli are less efficient in protein synthesis in vivo than the control ribosomes. In the present work, the efficiency of incorporation of the E. coli protein L25 with mutations in the 5S rRNA-binding region into the ribosome in vivo was studied. It was found that the mutations in L25 that abolish its ability to form the complex with free 5S rRNA do not prevent its correct and efficient incorporation into the ribosome. This is supported by the fact that even the presence of a very weakly retained mutant form of the protein in the ribosome has a positive effect on the activity of the translational machinery in vivo. All this suggests the existence of an alternative incorporation pathway for this protein into the ribosome, excluding the preliminary formation of the complex with 5S rRNA. At the same time, the stable L25-5S rRNA contact is important for the retention of the protein within the ribosome, and the conservative amino acid residues of the RNA-binding module play a key role in this.

  8. Small-subunit ribosomal RNA gene sequences of Phaeodarea challenge the monophyly of Haeckel's Radiolaria.

    PubMed

    Polet, Stephane; Berney, Cédric; Fahrni, José; Pawlowski, Jan

    2004-03-01

    In his grand monograph of Radiolaria, Ernst Haeckel originally included Phaeodarea together with Acantharea and Polycystinea, all three taxa characterized by the presence of a central capsule and the possession of axopodia. Cytological and ultrastructural studies, however, questioned the monophyly of Radiolaria, suggesting an independent evolutionary origin of the three taxa, and the first molecular data on Acantharea and Polycystinea brought controversial results. To test further the monophyly of Radiolaria, we sequenced the complete small subunit ribosomal RNA gene of three phaeodarians and three polycystines. Our analyses reveal that phaeodarians clearly branch among the recently described phylum Cercozoa, separately from Acantharea and Polycystinea. This result enhances the morphological variability within the phylum Cercozoa, which already contains very heterogeneous groups of protists. Our study also confirms the common origin of Acantharea and Polycystinea, which form a sister-group to the Cercozoa, and allows a phylogenetic reinterpretation of the morphological features of the three radiolarian groups. PMID:15144058

  9. Sequence and structure of the extrachromosomal palindrome encoding the ribosomal RNA genes in Dictyostelium.

    PubMed

    Sucgang, Richard; Chen, Guokai; Liu, Wen; Lindsay, Ryan; Lu, Jing; Muzny, Donna; Shaulsky, Gad; Loomis, William; Gibbs, Richard; Kuspa, Adam

    2003-05-01

    Ribosomal RNAs (rRNAs) are encoded by multicopy families of identical genes. In Dictyostelium and other protists, the rDNA is carried on extrachromosomal palindromic elements that comprise up to 20% of the nuclear DNA. We present the sequence of the 88 kb Dictyostelium rDNA element, noting that the rRNA genes are likely to be the only transcribed regions. By interrogating a library of ordered YAC clones, we provide evidence for a chromosomal copy of the rDNA on chromosome 4. This locus may provide master copies for the stable transmission of the extrachromosomal elements. The extrachromosomal elements were also found to form chromosome-sized clusters of DNA within nuclei of nocodazole-treated cells arrested in mitosis. These clusters resemble true chromosomes and may allow the efficient segregation of the rDNA during mitosis. These rDNA clusters may also explain the cytological observations of a seventh chromosome in this organism.

  10. Small-subunit ribosomal RNA gene sequences of Phaeodarea challenge the monophyly of Haeckel's Radiolaria.

    PubMed

    Polet, Stephane; Berney, Cédric; Fahrni, José; Pawlowski, Jan

    2004-03-01

    In his grand monograph of Radiolaria, Ernst Haeckel originally included Phaeodarea together with Acantharea and Polycystinea, all three taxa characterized by the presence of a central capsule and the possession of axopodia. Cytological and ultrastructural studies, however, questioned the monophyly of Radiolaria, suggesting an independent evolutionary origin of the three taxa, and the first molecular data on Acantharea and Polycystinea brought controversial results. To test further the monophyly of Radiolaria, we sequenced the complete small subunit ribosomal RNA gene of three phaeodarians and three polycystines. Our analyses reveal that phaeodarians clearly branch among the recently described phylum Cercozoa, separately from Acantharea and Polycystinea. This result enhances the morphological variability within the phylum Cercozoa, which already contains very heterogeneous groups of protists. Our study also confirms the common origin of Acantharea and Polycystinea, which form a sister-group to the Cercozoa, and allows a phylogenetic reinterpretation of the morphological features of the three radiolarian groups.

  11. An analysis of partial 28S ribosomal RNA sequences suggests early radiations of sponges.

    PubMed

    Lafay, B; Boury-Esnault, N; Vacelet, J; Christen, R

    1992-01-01

    Sequences from the 5' end terminal part of 28S ribosomal RNA were obtained and compared for 22 animals belonging to all diploblastic phyla and for a large number of representatives of triploblastic Metazoa and protists. Phylogenetic analyses undertaken using different methods showed deep radiations of phyla such as Ctenophora, Cnidaria and Placozoa but also for groups of Porifera of low taxonomic rank. Short internodes between these radiations suggested an early rapid diversification of diploblasts. A long internal branch preceding the diversification of all triploblasts analyzed could be explained either by a long period with a single ancestor or by the extinction of the earliest triploblastic radiations. Finally some unexpected relationships were revealed among Porifera.

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

    PubMed

    Krakowetz, Chantel N; Chilton, Neil B

    2015-02-01

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

  13. 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.

  14. Reconstructing evolution from eukaryotic small-ribosomal-subunit RNA sequences: calibration of the molecular clock.

    PubMed

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

    1993-08-01

    The detailed descriptions now available for the secondary structure of small-ribosomal-subunit RNA, including areas of highly variable primary structure, facilitate the alignment of nucleotide sequences. However, for optimal exploitation of the information contained in the alignment, a method must be available that takes into account the local sequence variability in the computation of evolutionary distance. A quantitative definition for the variability of an alignment position is proposed in this study. It is a parameter in an equation which expresses the probability that the alignment position contains a different nucleotide in two sequences, as a function of the distance separating these sequences, i.e., the number of substitutions per nucleotide that occurred during their divergence. This parameter can be estimated from the distance matrix resulting from the conversion of pairwise sequence dissimilarities into pairwise distances. Alignment positions can then be subdivided into a number of sets of matching variability, and the average variability of each set can be derived. Next, the conversion of dissimilarity into distance can be recalculated for each set of alignment positions separately, using a modified version of the equation that corrects for multiple substitutions and changing for each set the parameter that reflects its average variability. The distances computed for each set are finally averaged, giving a more precise distance estimation. Trees constructed by the algorithm based on variability calibration have a topology markedly different from that of trees constructed from the same alignments in the absence of calibration. This is illustrated by means of trees constructed from small-ribosomal-subunit RNA sequences of Metazoa. A reconstruction of vertebrate evolution based on calibrated alignments matches the consensus view of paleontologists, contrary to trees based on uncalibrated alignments. In trees derived from sequences covering several metazoan

  15. 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

  16. Evidence That Antibiotics Bind to Human Mitochondrial Ribosomal RNA Has Implications for Aminoglycoside Toxicity.

    PubMed

    Hong, Seoyeon; Harris, Kimberly A; Fanning, Kathryn D; Sarachan, Kathryn L; Frohlich, Kyla M; Agris, Paul F

    2015-07-31

    Aminoglycosides are a well known antibiotic family used to treat bacterial infections in humans and animals, but which can be toxic. By binding to the decoding site of helix44 of the small subunit RNA of the bacterial ribosome, the aminoglycoside antibiotics inhibit protein synthesis, cause misreading, or obstruct peptidyl-tRNA translocation. Although aminoglycosides bind helix69 of the bacterial large subunit RNA as well, little is known about their interaction with the homologous human helix69. To probe the role this binding event plays in toxicity, changes to thermal stability, base stacking, and conformation upon aminoglycoside binding to the human cytoplasmic helix69 were compared with those of the human mitochondrial and Escherichia coli helix69. Surprisingly, binding of gentamicin and kanamycin A to the chemically synthesized terminal hairpins of the human cytoplasmic, human mitochondrial, and E. coli helix69 revealed similar dissociation constants (1.3-1.7 and 4.0-5.4 μM, respectively). In addition, aminoglycoside binding enhanced conformational stability of the human mitochondrial helix69 by increasing base stacking. Proton one-dimensional and two-dimensional NMR suggested significant and specific conformational changes of human mitochondrial and E. coli helix69 upon aminoglycoside binding, as compared with human cytoplasmic helix69. The conformational changes and similar aminoglycoside binding affinities observed for human mitochondrial helix69 and E. coli helix69, as well as the increase in structural stability shown for the former, suggest that this binding event is important to understanding aminoglycoside toxicity. PMID:26060252

  17. Wnt5a Signals through DVL1 to Repress Ribosomal DNA Transcription by RNA Polymerase I.

    PubMed

    Dass, Randall A; Sarshad, Aishe A; Carson, Brittany B; Feenstra, Jennifer M; Kaur, Amanpreet; Obrdlik, Ales; Parks, Matthew M; Prakash, Varsha; Love, Damon K; Pietras, Kristian; Serra, Rosa; Blanchard, Scott C; Percipalle, Piergiorgio; Brown, Anthony M C; Vincent, C Theresa

    2016-08-01

    Ribosome biogenesis is essential for cell growth and proliferation and is commonly elevated in cancer. Accordingly, numerous oncogene and tumor suppressor signaling pathways target rRNA synthesis. In breast cancer, non-canonical Wnt signaling by Wnt5a has been reported to antagonize tumor growth. Here, we show that Wnt5a rapidly represses rDNA gene transcription in breast cancer cells and generates a chromatin state with reduced transcription of rDNA by RNA polymerase I (Pol I). These effects were specifically dependent on Dishevelled1 (DVL1), which accumulates in nucleolar organizer regions (NORs) and binds to rDNA regions of the chromosome. Upon DVL1 binding, the Pol I transcription activator and deacetylase Sirtuin 7 (SIRT7) releases from rDNA loci, concomitant with disassembly of Pol I transcription machinery at the rDNA promoter. These findings reveal that Wnt5a signals through DVL1 to suppress rRNA transcription. This provides a novel mechanism for how Wnt5a exerts tumor suppressive effects and why disruption of Wnt5a signaling enhances mammary tumor growth in vivo. PMID:27500936

  18. Wnt5a Signals through DVL1 to Repress Ribosomal DNA Transcription by RNA Polymerase I

    PubMed Central

    Dass, Randall A.; Sarshad, Aishe A.; Feenstra, Jennifer M.; Kaur, Amanpreet; Pietras, Kristian; Serra, Rosa; Blanchard, Scott C.; Percipalle, Piergiorgio; Brown, Anthony M. C.; Vincent, C. Theresa

    2016-01-01

    Ribosome biogenesis is essential for cell growth and proliferation and is commonly elevated in cancer. Accordingly, numerous oncogene and tumor suppressor signaling pathways target rRNA synthesis. In breast cancer, non-canonical Wnt signaling by Wnt5a has been reported to antagonize tumor growth. Here, we show that Wnt5a rapidly represses rDNA gene transcription in breast cancer cells and generates a chromatin state with reduced transcription of rDNA by RNA polymerase I (Pol I). These effects were specifically dependent on Dishevelled1 (DVL1), which accumulates in nucleolar organizer regions (NORs) and binds to rDNA regions of the chromosome. Upon DVL1 binding, the Pol I transcription activator and deacetylase Sirtuin 7 (SIRT7) releases from rDNA loci, concomitant with disassembly of Pol I transcription machinery at the rDNA promoter. These findings reveal that Wnt5a signals through DVL1 to suppress rRNA transcription. This provides a novel mechanism for how Wnt5a exerts tumor suppressive effects and why disruption of Wnt5a signaling enhances mammary tumor growth in vivo. PMID:27500936

  19. Proteins associated with rRNA in the Escherichia coli ribosome.

    PubMed

    Bernabeu, C; Vazquez, D; Ballesta, J P

    1978-04-27

    Ribosomal proteins located near the rRNA have been identified by cross linking to [14C]spermine with 1,5-difluoro-2,4-dinitrobenzene. The polyamine binds to double-stranded rRNA; those proteins showing radioactivity covalently bound after treatment with the bifunctional reagent should therefore be located in the vicinity of these regions of rRNA. Six proteins from the small subunit, S4, S5, S9, S18, S19 and S20 and ten proteins from the large subunit L2, L6, L13, L14, L16, L17, L18, L19, L22 and L27 preferentially take up the label. The results obtained with three proteins from the large subunit, L6, L16 and L27, show a high degree of variability that could reflect differences of conformation in the subunit population. Several proteins were drastically modified by the cross-linking agent but were not detected in the two-dimensional gel electrophoresis (e.g., S1, S11, S21, L7, L8 and L12) and therefore could not be studied.

  20. End-targeting proteomics of isolated chromatin segments of a mammalian ribosomal RNA gene promoter.

    PubMed

    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

  1. HCV IRES interacts with the 18S rRNA to activate the 40S ribosome for subsequent steps of translation initiation.

    PubMed

    Malygin, Alexey A; Kossinova, Olga A; Shatsky, Ivan N; Karpova, Galina G

    2013-10-01

    Previous analyses of complexes of 40S ribosomal subunits with the hepatitis C virus (HCV) internal ribosome entry site (IRES) have revealed contacts made by the IRES with ribosomal proteins. Here, using chemical probing, we show that the HCV IRES also contacts the backbone and bases of the CCC triplet in the 18S ribosomal RNA (rRNA) expansion segment 7. These contacts presumably provide interplay between IRES domain II and the AUG codon close to ribosomal protein S5, which causes a rearrangement of 18S rRNA structure in the vicinity of the universally conserved nucleotide G1639. As a result, G1639 becomes exposed and the corresponding site of the 40S subunit implicated in transfer RNA discrimination can select . These data are the first demonstration at nucleotide resolution of direct IRES-rRNA interactions and how they induce conformational transition in the 40S subunit allowing the HCV IRES to function without AUG recognition initiation factors.

  2. 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.

  3. Molecular identification of nanoplanktonic protists based on small subunit ribosomal RNA gene sequences for ecological studies.

    PubMed

    Lim, E L

    1996-01-01

    Nanoplanktonic protists are comprised of a diverse assemblage of species which are responsible for a variety of trophic processes in marine and freshwater ecosystems. Current methods for identifying small protists by electron microscopy do not readily permit both identification and enumeration of nanoplanktonic protists in field samples. Thus, one major goal in the application of molecular approaches in protistan ecology has been the detection and quantification of individual species in natural water samples. Sequences of small subunit ribosomal RNA (SSU rRNA) genes have proven to be useful towards achieving this goal. Comparison of sequences from clone libraries of protistan SSU rRNA genes amplified from natural assemblages of protists by the polymerase chain reaction (PCR) can be used to examine protistan diversity. Furthermore, oligonucleotide probes complementary to short sequence regions unique to species of small protists can be designed by comparative analysis of rRNA gene sequences. These probes may be used to either detect the RNA of particular species of protists in total nucleic acid extracts immobilized on membranes, or the presence of target species in water samples via in situ hybridization of whole cells. Oligonucleotide probes may also serve as primers for the selective amplification of target sequences from total population DNA by PCR. Thus, molecular sequence information is becoming increasingly useful for identifying and enumerating protists, and for studying their spatial and temporal distribution in nature. Knowledge of protistan species composition, abundance and variability in an environment can ultimately be used to relate community structure to various aspects of community function and biogeochemical activity.

  4. [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.

  5. Next-generation Sequencing of 16S Ribosomal RNA Gene Amplicons

    PubMed Central

    Sanschagrin, Sylvie; Yergeau, Etienne

    2014-01-01

    One of the major questions in microbial ecology is “who is there?” This question can be answered using various tools, but one of the long-lasting gold standards is to sequence 16S ribosomal RNA (rRNA) gene amplicons generated by domain-level PCR reactions amplifying from genomic DNA. Traditionally, this was performed by cloning and Sanger (capillary electrophoresis) sequencing of PCR amplicons. The advent of next-generation sequencing has tremendously simplified and increased the sequencing depth for 16S rRNA gene sequencing. The introduction of benchtop sequencers now allows small labs to perform their 16S rRNA sequencing in-house in a matter of days. Here, an approach for 16S rRNA gene amplicon sequencing using a benchtop next-generation sequencer is detailed. The environmental DNA is first amplified by PCR using primers that contain sequencing adapters and barcodes. They are then coupled to spherical particles via emulsion PCR. The particles are loaded on a disposable chip and the chip is inserted in the sequencing machine after which the sequencing is performed. The sequences are retrieved in fastq format, filtered and the barcodes are used to establish the sample membership of the reads. The filtered and binned reads are then further analyzed using publically available tools. An example analysis where the reads were classified with a taxonomy-finding algorithm within the software package Mothur is given. The method outlined here is simple, inexpensive and straightforward and should help smaller labs to take advantage from the ongoing genomic revolution. PMID:25226019

  6. Re-analysis of cryoEM data on HCV IRES bound to 40S subunit of human ribosome integrated with recent structural information suggests new contact regions between ribosomal proteins and HCV RNA

    PubMed Central

    Joseph, Agnel Praveen; Bhat, Prasanna; Das, Saumitra; Srinivasan, Narayanaswamy

    2014-01-01

    In this study, we combine available high resolution structural information on eukaryotic ribosomes with low resolution cryo-EM data on the Hepatitis C Viral RNA (IRES) human ribosome complex. Aided further by the prediction of RNA-protein interactions and restrained docking studies, we gain insights on their interaction at the residue level. We identified the components involved at the major and minor contact regions, and propose that there are energetically favorable local interactions between 40S ribosomal proteins and IRES domains. Domain II of the IRES interacts with ribosomal proteins S5 and S25 while the pseudoknot and the downstream domain IV region bind to ribosomal proteins S26, S28 and S5. We also provide support using UV cross-linking studies to validate our proposition of interaction between the S5 and IRES domains II and IV. We found that domain IIIe makes contact with the ribosomal protein S3a (S1e). Our model also suggests that the ribosomal protein S27 interacts with domain IIIc while S7 has a weak contact with a single base RNA bulge between junction IIIabc and IIId. The interacting residues are highly conserved among mammalian homologs while IRES RNA bases involved in contact do not show strict conservation. IRES RNA binding sites for S25 and S3a show the best conservation among related viral IRESs. The new contacts identified between ribosomal proteins and RNA are consistent with previous independent studies on RNA-binding properties of ribosomal proteins reported in literature, though information at the residue level is not available in previous studies. PMID:25268799

  7. 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.

  8. Bacterial clade with the ribosomal RNA operon on a small plasmid rather than the chromosome

    PubMed Central

    Anda, Mizue; Ohtsubo, Yoshiyuki; Okubo, Takashi; Sugawara, Masayuki; Nagata, Yuji; Tsuda, Masataka; Minamisawa, Kiwamu; Mitsui, Hisayuki

    2015-01-01

    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. PMID:26534993

  9. The human 18S rRNA base methyltransferases DIMT1L and WBSCR22-TRMT112 but not rRNA modification are required for ribosome biogenesis

    PubMed Central

    Zorbas, Christiane; Nicolas, Emilien; Wacheul, Ludivine; Huvelle, Emmeline; Heurgué-Hamard, Valérie; Lafontaine, Denis L. J.

    2015-01-01

    At the heart of the ribosome lie rRNAs, whose catalytic function in translation is subtly modulated by posttranscriptional modifications. In the small ribosomal subunit of budding yeast, on the 18S rRNA, two adjacent adenosines (A1781/A1782) are N6-dimethylated by Dim1 near the decoding site, and one guanosine (G1575) is N7-methylated by Bud23-Trm112 at a ridge between the P- and E-site tRNAs. Here we establish human DIMT1L and WBSCR22-TRMT112 as the functional homologues of yeast Dim1 and Bud23-Trm112. We report that these enzymes are required for distinct pre-rRNA processing reactions leading to synthesis of 18S rRNA, and we demonstrate that in human cells, as in budding yeast, ribosome biogenesis requires the presence of the modification enzyme rather than its RNA-modifying catalytic activity. We conclude that a quality control mechanism has been conserved from yeast to human by which binding of a methyltransferase to nascent pre-rRNAs is a prerequisite to processing, so that all cleaved RNAs are committed to faithful modification. We further report that 18S rRNA dimethylation is nuclear in human cells, in contrast to yeast, where it is cytoplasmic. Yeast and human ribosome biogenesis thus have both conserved and distinctive features. PMID:25851604

  10. Thalamic WNT3 Secretion Spatiotemporally Regulates the Neocortical Ribosome Signature and mRNA Translation to Specify Neocortical Cell Subtypes

    PubMed Central

    Kraushar, Matthew L.; Viljetic, Barbara; Wijeratne, H. R. Sagara; Thompson, Kevin; Jiao, Xinfu; Pike, Jack W.; Medvedeva, Vera; Groszer, Matthias; Kiledjian, Megerditch; Hart, Ronald P.

    2015-01-01

    Neocortical development requires tightly controlled spatiotemporal gene expression. However, the mechanisms regulating ribosomal complexes and the timed specificity of neocortical mRNA translation are poorly understood. We show that active mRNA translation complexes (polysomes) contain ribosomal protein subsets that undergo dynamic spatiotemporal rearrangements during mouse neocortical development. Ribosomal protein specificity within polysome complexes is regulated by the arrival of in-growing thalamic axons, which secrete the morphogen Wingless-related MMTV (mouse mammary tumor virus) integration site 3 (WNT3). Thalamic WNT3 release during midneurogenesis promotes a change in the levels of Ribosomal protein L7 in polysomes, thereby regulating neocortical translation machinery specificity. Furthermore, we present an RNA sequencing dataset analyzing mRNAs that dynamically associate with polysome complexes as neocortical development progresses, and thus may be regulated spatiotemporally at the level of translation. Thalamic WNT3 regulates neocortical translation of two such mRNAs, Foxp2 and Apc, to promote FOXP2 expression while inhibiting APC expression, thereby driving neocortical neuronal differentiation and suppressing oligodendrocyte maturation, respectively. This mechanism may enable targeted and rapid spatiotemporal control of ribosome composition and selective mRNA translation in complex developing systems like the neocortex. SIGNIFICANCE STATEMENT The neocortex is a highly complex circuit generating the most evolutionarily advanced complex cognitive and sensorimotor functions. An intricate progression of molecular and cellular steps during neocortical development determines its structure and function. Our goal is to study the steps regulating spatiotemporal specificity of mRNA translation that govern neocortical development. In this work, we show that the timed secretion of Wingless-related MMTV (mouse mammary tumor virus) integration site 3 (WNT3) by

  11. The ribosomal RNA transcription unit of Entamoeba invadens: accumulation of unprocessed pre-rRNA and a long non coding RNA during encystation.

    PubMed

    Ojha, Sandeep; Singh, Nishant; Bhattacharya, Alok; Bhattacharya, Sudha

    2013-01-01

    The ribosomal RNA genes in Entamoeba spp. are located on extrachromosomal circular molecules. Unlike model organisms where rRNA transcription stops during growth stress, Entamoeba histolytica continues transcription; but unprocessed pre-rRNA accumulates during stress, along with a novel class of circular transcripts from the 5'-external transcribed spacer (ETS). To determine the fate of rRNA transcription during stage conversion between trophozoite to cyst we analyzed Entamoeba invadens, a model system for differentiation studies in Entamoeba. We characterized the complete rDNA transcription unit by mapping the ends of pre-rRNA and mature rRNAs. The 3' end of mature 28S rRNA was located 321 nt downstream of the end predicted by sequence homology with E. histolytica. The major processing sites were mapped in external and internal transcribed spacers. The promoter located within 146 nt upstream of 5' ETS was used to transcribe the pre-rRNA. On the other hand, a second promoter located at the 3' end of 28S rDNA was used to transcribe almost the entire intergenic spacer into a long non coding (nc) RNA (>10 kb). Interestingly we found that the levels of pre-rRNA and long ncRNA, measured by northern hybridization, decreased initially in cells shifted to encystation medium, after which they began to increase and reached high levels by 72 h when mature cysts were formed. Unlike E. histolytica, no circular transcripts were found in E. invadens. E. histolytica and E. invadens express fundamentally different ncRNAs from the rDNA locus, which may reflect their adaptation to different hosts (human and reptiles, respectively). This is the first description of rDNA organization and transcription in E. invadens, and provides the framework for further studies on regulation of rRNA synthesis during cyst formation.

  12. Time-Dependent Decay of mRNA and Ribosomal RNA during Platelet Aging and Its Correlation with Translation Activity

    PubMed Central

    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

  13. Photoinduced cross-linkage, in situ, of Escherichia coli 30S ribosomal proteins to 16S rRNA: identification of cross-linked proteins and relationships between reactivity and ribosome structure.

    PubMed

    Gorelic, L

    1976-08-10

    The kinetics of photoinduced cross-linkage of Escherichia coli 30S ribosomal proteins to the 16S-rRNA molecule in the intact Escherichia coli 30S ribosomal subunit was studied in this report. All of the 30S ribosomal proteins become cross-linked to the 16S rRNA before changes in the sedimentation characteristics of the 30S ribosomal subunit can be detected. The proteins exhibit different reactivities in the cross-linkage reaction. One group of proteins-S3, S7-S9, S11, S12, and S15-S19-is cross-linked to the 16S rRNA by single-hit kinetics, or by photoprocesses of nonunity but low multiplicities. A second group of proteins--S1, S2, S4-S6, S10, S13, S14, and S21--is cross-linked to the 16S rRNA by photoprocesses of a complex nature. A comparison of these data with other properties of the individual 30S ribosomal proteins related to ribosome structure indicated that most of the 30S ribosomal proteins cross-linked to the 16S rRNA by photoprocesses of low multiplicities had been classified rRNA-binding proteins by nonphotochemical methods, and most of the proteins cross-linked to the 16S rRNA by photoprocesses of large multiplicities had been classified as nonbinding proteins. There were certain exceptions to these correlations. Proteins S4 and S20, both RNA-binding proteins, become cross-linked to the 16S rRNA by photoprocessses of large multiplicities, and proteins S3, S11, S12, and S18, none of which have been classified RNA-binding proteins, exhibited low multiplicities in the cross-linkage reaction. All of these exceptions could be explained in terms of limitations inherent in the photochemical methods used in this study and in other types of methods that have been used to study RNA-protein interactions in the 30S ribosomal subunit. The data presented here also suggest that labile RNA-protein cross-links are present in the uv-irradiated 30S ribosomal subunits, and that neither peptide-bond cleavage nor photoinduced modification of the charged side-chain groups in

  14. Isolation of ribosome bound nascent polypeptides in vitro to identify translational pause sites along mRNA.

    PubMed

    Jha, Sujata S; Komar, Anton A

    2012-01-01

    The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the message(for review see 1). However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates(1). Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others(2). Codon bias is organism as well as tissue specific(2,3). Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs(4). Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes(5-8). These pause sites can have functional impact on the protein expression, mRNA stability and protein folding(for review see 9). Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding(1,7,10,11). To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation. Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems(6-8). This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the

  15. Molecular phylogenetic analysis of Vibrio cholerae O1 El Tor strains isolated before, during and after the O 139 outbreak based on the inter-genomic heterogeneity of the 16S-23S rRNA intergenic spacer regions.

    PubMed

    Ghatak, Atreyi; Majumdar, Anasuya; Ghosh, Ranajit K

    2005-12-01

    We have cloned, sequenced and analysed all the five classes of the intergenic (16S-23S rRNA) spacer region (ISR) associated with the eight rrn operons (rrna-rrnh) of Vibrio cholerae serogroup O1 El Tor strains isolated before, during and after the O 139 outbreak. ISR classes 'a' and 'g' were found to be invariant, ISR-B (ISRb and ISRe) exhibited very little variation, whereas ISR-C (ISRc, ISRd, and ISRf) and ISRh showed the maximum variation. Phylogenetic analysis conducted with all three ISR classes (ISR-B, ISR-C and ISRh) showed that the pre-O 139 serogroup and post-O 139 serogroup O1 El Tor strains arose out of two independent clones, which was congruent with the observation made by earlier workers suggesting that analyses of ISR-C and ISR-h, instead of all five ISR classes, could be successfully used to study phylogeny in this organism.

  16. 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

  17. Stability of the 'L12 stalk' in ribosomes from mesophilic and (hyper)thermophilic Archaea and Bacteria.

    PubMed

    Shcherbakov, D; Dontsova, M; Tribus, M; Garber, M; Piendl, W

    2006-01-01

    The ribosomal stalk complex, consisting of one molecule of L10 and four or six molecules of L12, is attached to 23S rRNA via protein L10. This complex forms the so-called 'L12 stalk' on the 50S ribosomal subunit. Ribosomal protein L11 binds to the same region of 23S rRNA and is located at the base of the 'L12 stalk'. The 'L12 stalk' plays a key role in the interaction of the ribosome with translation factors. In this study stalk complexes from mesophilic and (hyper)thermophilic species of the archaeal genus Methanococcus and from the Archaeon Sulfolobus solfataricus, as well as from the Bacteria Escherichia coli, Geobacillus stearothermophilus and Thermus thermophilus, were overproduced in E.coli and purified under non-denaturing conditions. Using filter-binding assays the affinities of the archaeal and bacterial complexes to their specific 23S rRNA target site were analyzed at different pH, ionic strength and temperature. Affinities of both archaeal and bacterial complexes for 23S rRNA vary by more than two orders of magnitude, correlating very well with the growth temperatures of the organisms. A cooperative effect of binding to 23S rRNA of protein L11 and the L10/L12(4) complex from mesophilic and thermophilic Archaea was shown to be temperature-dependent.

  18. 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.

  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. PMID:26038763

  20. Assessment of phylogenetic relationships among ciliated protists using partial ribosomal RNA sequences derived from reverse transcripts.

    PubMed

    Lynn, D H; Sogin, M L

    1988-01-01

    Partial sequences were derived for the 16S-like ribosomal RNA of Halteria grandinella, Glaucoma chattoni, Colpidium campylum, Opisthonecta henneguyi and Colpoda inflata. Using isolated bulk nucleic acid preparations, partial rRNA sequences were determined using a series of oligonucleotide primers complementary to conserved sequence elements of the molecule and a dideoxynucleotide sequencing reaction using reverse transcriptase. Sequences were aligned and an evolutionary tree was generated by a distance-matrix method, comparing these partial sequences to the complete sequences of species of Tetrahymena, Paramecium, Euplotes, Oxytricha and Stylonychia. Halteria shows greater similarity to the stichotrichs Oxytricha and Stylonychia than to the hypotrich Euplotes, while these four genera share the same branch. Glaucoma and Colpidium appear closely related to Tetrahymena, with all three being more closely related to Opisthonecta than to Paramecium. Colpoda diverges near the base of the branch shared by Paramecium and the four oligohymenophorean genera. These results are discussed with reference to published revisions of the systematics of the phylum Ciliophora.

  1. Probing functions of the ribosomal peptidyl transferase center by nucleotide analog interference.

    PubMed

    Erlacher, Matthias D; Polacek, Norbert

    2012-01-01

    The ribosome is a huge ribonucleoprotein complex in charge of protein synthesis in every living cell. The catalytic center of this dynamic molecular machine is entirely built up of 23S ribosomal RNA and therefore the ribosome can be referred to as the largest natural ribozyme known so far. The in vitro reconstitution approach of large ribosomal subunits described herein allows nucleotide analog interference studies to be performed. The approach is based on the site-specific introduction of nonnatural nucleotide analogs into the peptidyl transferase center, the active site located on the interface side of the large ribosomal subunit. This method combined with standard tests of ribosomal functions broadens the biochemical repertoire to investigate the mechanism of diverse aspects of translation considerably and adds another layer of molecular information on top of structural and mutational studies of the ribosome. PMID:22315072

  2. Ribosome Shut-Down by 16S rRNA Fragmentation in Stationary-Phase Escherichia coli.

    PubMed

    Luidalepp, Hannes; Berger, Stefan; Joss, Oliver; Tenson, Tanel; Polacek, Norbert

    2016-05-22

    Stationary-phase bacterial cells are characterized by vastly reduced metabolic activities yielding a dormant-like phenotype. Several hibernation programs ensure the establishment and maintenance of this resting growth state. Some of the stationary phase-specific modulations affect the ribosome and its translational activity directly. In stationary-phase Escherichia coli, we observed the appearance of a 16S rRNA fragmentation event at the tip of helix 6 within the small ribosomal subunit (30S). Stationary-phase 30S subunits showed markedly reduced activities in protein biosynthesis. On the other hand, the functional performance of stationary-phase large ribosomal subunits (50S) was indistinguishable from particles isolated from exponentially growing cells. Introduction of the 16S rRNA cut in vitro at helix 6 of exponential phase 30S subunits renders them less efficient in protein biosynthesis. This indicates that the helix 6 fragmentation is necessary and sufficient to attenuate translational activities of 30S ribosomal subunits. These results suggest that stationary phase-specific cleavage of 16S rRNA within the 30S subunit is an efficient means to reduce global translation activities under non-proliferating growth conditions. PMID:27067112

  3. 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. PMID:22967708

  4. A programmed –1 ribosomal frameshift signal can function as a cis-acting mRNA destabilizing element

    PubMed Central

    Plant, Ewan P.; Wang, Pinger; Jacobs, Jonathan L.; Dinman, Jonathan D.

    2004-01-01

    Nonsense-mediated mRNA decay (NMD) directs rapid degradation of premature termination codon (PTC)-containing mRNAs, e.g. those containing frameshift mutations. Many viral mRNAs encode polycistronic messages where programmed –1 ribosomal frameshift (–1 PRF) signals direct ribosomes to synthesize polyproteins. A previous study, which identified consensus –1 PRF signals in the yeast genome, found that, in contrast to viruses, the majority of predicted –1 PRF events would direct translating ribosomes to PTCs. Here we tested the hypothesis that a –1 PRF signal can function as a cis-acting mRNA destabilizing element by inserting an L-A viral –1 PRF signal into a PGK1 reporter construct in the ‘genomic’ orientation. The results show that even low levels of –1 PRF are sufficient to target the reporter mRNA for degradation via the NMD pathway, with half-lives similar to messages containing in-frame PTCs. The demonstration of an inverse correlation between frameshift efficiency and mRNA half-lives suggests that modulation of –1 PRF frequencies can be used to post-transcriptionally regulate gene expression. Analysis of the mRNA decay profiles of the frameshift-signal- containing reporter mRNAs also supports the notion that NMD remains active on mRNAs beyond the ‘pioneer round’ of translation in yeast. PMID:14762205

  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. Yeast ribosomal protein L32 recognizes an RNA G:U juxtaposition.

    PubMed Central

    White, S A; Li, H

    1996-01-01

    Yeast ribosomal protein L32, RPL32, specifically represses splicing by binding to a purine-rich asymmetric loop adjacent to the 5' splice site of its own transcript. A potential G:U pair closes the internal loop and the goal of the present study is to understand what features of the putative G:U pair are recognized by RPL32. Two RNA oligomers containing 10 and 13 nt were annealed to form a bimolecular stem-loop-stem protein-binding site. Protein binding to each of 16 sequence variants was examined using electrophoretic bandshift and filter-binding experiments. The proteins binds to only the duplex RNA and not to the individual oligomers, and the G:U pair is critical for full-strength binding. Mutational studies show that the duplex having a G:U has the highest protein affinity (Kd = 10 nM), followed by RNAs bearing G:A, C:C, U:A, U:C, or G:G. Duplexes containing the other possible pairs bind very weakly and Watson-Crick pairing does not favor protein binding. The G of the G:U is required for strong protein binding, but replacement by inosine reduces binding only modestly. Therefore, the minor groove guanine amino group is not a key protein recognition element. Both nucleotides of the pair influence the binding strength, but their contributions are in general not additive. These data imply that the G:U is probably paired and influences binding indirectly through its effect on the conformation of the RNA. PMID:8608446

  7. Cryo-EM structure and rRNA model of a translating eukaryotic 80S ribosome at 5.5-Å resolution

    PubMed Central

    Armache, Jean-Paul; Jarasch, Alexander; Anger, Andreas M.; Villa, Elizabeth; Becker, Thomas; Bhushan, Shashi; Jossinet, Fabrice; Habeck, Michael; Dindar, Gülcin; Franckenberg, Sibylle; Marquez, Viter; Mielke, Thorsten; Thomm, Michael; Berninghausen, Otto; Beatrix, Birgitta; Söding, Johannes; Westhof, Eric; Wilson, Daniel N.; Beckmann, Roland

    2010-01-01

    Protein biosynthesis, the translation of the genetic code into polypeptides, occurs on ribonucleoprotein particles called ribosomes. Although X-ray structures of bacterial ribosomes are available, high-resolution structures of eukaryotic 80S ribosomes are lacking. Using cryoelectron microscopy and single-particle reconstruction, we have determined the structure of a translating plant (Triticum aestivum) 80S ribosome at 5.5-Å resolution. This map, together with a 6.1-Å map of a Saccharomyces cerevisiae 80S ribosome, has enabled us to model ∼98% of the rRNA. Accurate assignment of the rRNA expansion segments (ES) and variable regions has revealed unique ES–ES and r-protein–ES interactions, providing insight into the structure and evolution of the eukaryotic ribosome. PMID:20980660

  8. A Ribosome-Binding, 3′ Translational Enhancer Has a T-Shaped Structure and Engages in a Long-Distance RNA-RNA Interaction

    PubMed Central

    Gao, Feng; Kasprzak, Wojciech; Stupina, Vera A.

    2012-01-01

    Many plant RNA viruses contain elements in their 3′ untranslated regions (3′ UTRs) that enhance translation. The PTE (Panicum mosaic virus-like translational enhancer) of Pea enation mosaic virus (PEMV) binds to eukaryotic initiation factor 4E (eIF4E), but how this affects translation from the 5′ end is unknown. We have discovered a three-way branched element just upstream of the PEMV PTE that engages in a long-distance kissing-loop interaction with a coding sequence hairpin that is critical for the translation of a reporter construct and the accumulation of the viral genome in vivo. Loss of the long-distance interaction was more detrimental than elimination of the adjacent PTE, indicating that the RNA-RNA interaction supports additional translation functions besides relocating the PTE to the 5′ end. The branched element is predicted by molecular modeling and molecular dynamics to form a T-shaped structure (TSS) similar to the ribosome-binding TSS of Turnip crinkle virus (TCV). The PEMV element binds to plant 80S ribosomes with a Kd (dissociation constant) of 0.52 μM and to 60S subunits with a Kd of 0.30 μM. Unlike the TCV TSS, the PEMV element also binds 40S subunits (Kd, 0.36 μM). Mutations in the element that suppressed translation reduced either ribosome binding or the RNA-RNA interaction, suggesting that ribosome binding is important for function. This novel, multifunctional element is designated a kl-TSS (kissing-loop T-shaped structure) to distinguish it from the TCV TSS. The kl-TSS has sequence and structural features conserved with the upper portion of most PTE-type elements, which, with the exception of the PEMV PTE, can engage in similar long-distance RNA-RNA interactions. PMID:22761367

  9. 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.

  10. Erythromycin and 5S rRNA binding properties of the spinach chloroplast ribosomal protein CL22.

    PubMed Central

    Carol, P; Rozier, C; Lazaro, E; Ballesta, J P; Mache, R

    1993-01-01

    The spinach chloroplast ribosomal protein (r-protein) CL22 contains a central region homologous to the Escherichia coli r-protein L22 plus long N- and C-terminal extensions. We show in this study that the CL22 combines two properties which in E. coli ribosome are split between two separate proteins. The CL22 which binds to the 5S rRNA can also be linked to an erythromycin derivative added to the 50S ribosomal subunit. This latter property is similar to that of the E. coli L22 and suggests a similar localization in the 50S subunit. We have overproduced the r-protein CL22 and deleted forms of this protein in E. coli. We show that the overproduced CL22 binds to the chloroplast 5S rRNA and that the deleted protein containing the N- and C-terminal extensions only has lost the 5S rRNA binding property. We suggest that the central homologous regions of the CL22 contains the RNA binding domain. Images PMID:8441674

  11. 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

  12. 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.

  13. AtPPR2, an Arabidopsis pentatricopeptide repeat protein, binds to plastid 23S rRNA and plays an important role in the first mitotic division during gametogenesis and in cell proliferation during embryogenesis

    PubMed Central

    Lu, Yuqing; Li, Cong; Wang, Hai; Chen, Hao; Berg, Howard; Xia, Yiji

    2011-01-01

    SUMMARY Pentatricopeptide repeat (PPR) proteins are mainly involved in regulating post-transcriptional processes in mitochondria and plastids, including chloroplasts. Mutations in the Arabidopsis PPR2 gene have previously been found to cause defects in seed development and reduced transmission through male and female gametophytes. However, the exact function of AtPPR2 has not been defined. We found that a loss-of-function mutation of AtPPR2 leads to arrest of the first mitotic division during both male and female gametogenesis. In addition, the Atppr2 mutation causes delayed embryogenesis, leading to embryonic lethality. Mutation in emb2750, which appears to be a weak mutant allele of the AtPPR2 locus, also results in defective seeds. However, a majority of emb2750 seeds were able to germinate, but their cotyledons were albino and often deformed, and growth of the emb2750 seedlings were arrested after germination. AtPPR2 is mainly expressed in plant parts that undergo cell division, and AtPPR2 protein was localized to chloroplasts. RNA immunoprecipitation and protein gel mobility shift assays showed that AtPPR2 binds to plastid 23S rRNA. Our study adds to a growing body of evidence that plastids and/or chloroplasts play a key role in cell division. AtPPR2 may modulate the translational process to fine-tune plastid function, thereby regulating cell division. PMID:21435048

  14. 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. PMID:21102526

  15. Large-subunit ribosomal RNA systematics of symbiotic dinoflagellates: morphology does not recapitulate phylogeny.

    PubMed

    Wilcox, T P

    1998-12-01

    Biochemical, histological, physiological, and genetic evidence indicates that dinoflagellates symbiotic with marine invertebrates are a heterogeneous complex of taxa, representing at least five genera in three orders. Despite a wealth of data regarding morphological, biochemical, and behavioral differences among symbiotic dinoflagellates, knowledge concerning patterns of diversification is limited. I analyzed approximately 900 bp of the 5' end of the large-subunit ribosomal RNA gene from 14 dinoflagellate isolates: six cultured Symbiodinium specimens, two cultured symbiotic Gymnodinium, two algal samples isolated from reef-building corals, an algal sample obtained from cultures of the jellyfish Cassiopea xamachana, and three free-living Gymnodinium isolates. Results show that morphological similarities among the examined symbiotic taxa do not necessarily correspond with molecular phylogeny. The included Symbiodinium taxa represent a paraphyletic assemblage while Gymnodinium is reconstructed as a polyphyletic assemblage. Analysis indicates that all the included symbiotic dinoflagellates descended from a common, symbiotic ancestor (though within the dinoflagellates, symbiosis is a polyphyletic trait). Additionally, two free-living dinoflagellates emerge within the symbiotic clade, suggesting that the symbiotic lifestyle has been lost at least once in this group. It has been hypothesized that rates of evolution within mutualistic endosymbioses should be reduced relative to free-living taxa. However, results indicate that rates of molecular, morphological, biochemical and behavioral change are similar among branches leading to symbiotic and free-living dinoflagellates. PMID:10051396

  16. 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.

  17. Nucleotide excision repair of the 5 S ribosomal RNA gene assembled into a nucleosome.

    PubMed

    Liu, X; Smerdon, M J

    2000-08-01

    A-175-base pair fragment containing the Xenopus borealis somatic 5 S ribosomal RNA gene was used as a model system to determine the effect of nucleosome assembly on nucleotide excision repair (NER) of the major UV photoproduct (cyclobutane pyrimidine dimer (CPD)) in DNA. Xenopus oocyte nuclear extracts were used to carry out repair in vitro on reconstituted, positioned 5 S rDNA nucleosomes. Nucleosome structure strongly inhibits NER at many CPD sites in the 5 S rDNA fragment while having little effect at a few sites. The time course of CPD removal at 35 different sites indicates that >85% of the CPDs in the naked DNA fragment have t(12) values <2 h, whereas <26% of the t(12) values in nucleosomes are <2 h, and 15% are >8 h. Moreover, removal of histone tails from these mononucleosomes has little effect on the repair rates. Finally, nucleosome inhibition of repair shows no correlation with the rotational setting of a 14-nucleotide-long pyrimidine tract located 30 base pairs from the nucleosome dyad. These results suggest that inhibition of NER by mononucleosomes is not significantly influenced by the rotational orientation of CPDs on the histone surface, and histone tails play little (or no) role in this inhibition. PMID:10821833

  18. 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.

  19. Genetic variability of Echinococcus granulosus based on the mitochondrial 16S ribosomal RNA gene.

    PubMed

    Wang, Ning; Wang, Jiahai; Hu, Dandan; Zhong, Xiuqin; Jiang, Zhongrong; Yang, Aiguo; Deng, Shijin; Guo, Li; Tsering, Dawa; Wang, Shuxian; Gu, Xiaobin; Peng, Xuerong; Yang, Guangyou

    2015-06-01

    Echinococcus granulosus is the etiological agent of cystic echinococcosis, a major zoonotic disease of both humans and animals. In this study, we assessed genetic variability and genetic structure of E. granulosus in the Tibet plateau, using the complete mitochondrial 16 S ribosomal RNA gene for the first time. We collected and sequenced 62 isolates of E. granulosus from 3 populations in the Tibet plateau. A BLAST analysis indicated that 61 isolates belonged to E. granulosus sensu stricto (genotypes G1-G3), while one isolate belonged to E. canadensis (genotype G6). We detected 16 haplotypes with a haplotype network revealing a star-like expansion, with the most common haplotype occupying the center of the network. Haplotype diversity and nucleotide diversity were low, while negative values were observed for Tajima's D and Fu's Fs. AMOVA results and Fst values revealed that the three geographic populations were not genetically differentiated. Our results suggest that a population bottleneck or population expansion has occurred in the past, and that this explains the low genetic variability of E. granulosus in the Tibet Plateau.

  20. 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

  1. Combined large and small subunit ribosomal RNA phylogenies support a basal position of the acoelomorph flatworms.

    PubMed

    Telford, Maximilian J; Lockyer, Anne E; Cartwright-Finch, Chloë; Littlewood, D Timothy J

    2003-05-22

    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.

  2. Phylogeny of the conserved 3' terminal structure of the RNA of small ribosomal subunits.

    PubMed Central

    Van Knippenberg, P H; Van Kimmenade, J M; Heus, H A

    1984-01-01

    The strongest conserved part of the RNA of small ribosomal subunits is probably located near the 3' end. This paper reviews the primary and secondary structures of some 40 sequenced 3' termini and tries to classify these structures according to common features and differences. The regions under consideration contain at the 5' side an almost universal, supposedly single-stranded stretch of nucleotides with the sequence--AAGUCGUAACAAGGU--. This is followed by a stem-loop structure. The stem always contains 9 basepairs (including U-G pairs) and no mismatches or bulged nucleotides. The loop of the hairpin is either (m2)GGm62Am62A (bacteria, chloroplasts and mitochondria) or UGm62Am62A (cytoplasm). The hairpin is, in most cases, followed at the 3' side by--GGAUCA--. Next to it bacteria and chloroplasts contain the so-called "Shine and Dalgarno" sequence --CCUCC--. The stem region of the hairpin contains a conserved A-U U-G junction. The two basepairs between this junction and the loop are either of type 1 (G-C G-C) or type 2 (C-G C-G). Classification according to type links certain bacteria with mitochondria of yeast and plants and others with chloroplasts and with animal mitochondria. PMID:6709501

  3. Identification and phylogenetic analysis of morphologically similar naked amoebae using small subunit ribosomal RNA.

    PubMed

    Sims, Gary P; Aitken, Robert; Rogerson, Andrew

    2002-01-01

    Fan-shaped, naked amoebae are commonly encountered in samples from freshwater and marine habitats suggesting that they are an important component of the microbial food web. However, there are considerable problems in both detecting these amoebae and identifying them, given their morphological similarity. In this study we used restriction analysis and partial sequence analysis of the small-subunit 18S ribosomal RNA gene to examine the phylogenetic relationships between nine "fan-shaped" Vannella and Platyamoeba species. The molecular phylogeny showed that the marine Vannella and Platyamoeba isolates are closely related, whereas the freshwater isolates are disparate. Thus, the current reliance on the fine structure of the cell coat (glycocalyx) used to separate these genera is not justified. The study also highlights sequence elements that might be targeted by fluorescent probes for the direct detection of these amoebae in field samples. The molecular data were also used to aid the identification of three unknown fan-shaped isolates. All three unknowns resembled Vannella or Platyamoeba. However, one of the strains (a small < 10 microm, benthic, fan-shaped amoeba) probably represents a new genus.

  4. Human ribosomal RNA gene cluster: Identification of the proximal end containing a novel tandem repeat sequence

    SciTech Connect

    Sakai, K.; Ohta, T.; Minoshima, S.

    1995-04-10

    Human ribosomal RNA genes (rDNA) are arranged as tandem repeat clusters on the short arms of five pairs of acrocentric chromosomes. We have demonstrated that a majority of the rDNA clusters are detected as 3-Mb DNA fragments when released from human genomic DNA by EcoRV digestion. This indicated the absence of the EcoRV restriction site within the rDNA clusters. We then screened for rDNA-positive cosmid clones using a chromosome 22-specific cosmid library that was constructed from MboI partial digests of the flow-sorted chromosomes. Three hundred twenty rDNA-positive clones negative for the previously reported distal flanking sequence (pACR1) were chosen and subjected to EcoRV digestion. Seven clones susceptible to EcoRV were further characterized as candidate clones that might have been derived from the junctions of the 3-Mb rDNA cluster. We identified one clone containing part of the rDNA unit sequence and a novel flanking sequence. Detailed analysis of this unique clone revealed that the coding region of the last rRNA gene located at the proximal end of the cluster is interrupted with a novel sequence of {approximately}147 bp that is tandemly repeated and is connected with an intervening 68-bp unique sequence. This junction sequence was readily amplified from chromosomes 21 and 15 as well as 22 using the polymerase chain reaction. Fluorescence in situ hybridization further indicated that the {approximately}147-bp sequence repeat is commonly distributed among all the acrocentric short arms. 23 refs., 5 figs.

  5. 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.

  6. A new model for the three-dimensional folding of Escherichia coli 16 S ribosomal RNA. II. The RNA-protein interaction data.

    PubMed

    Mueller, F; Brimacombe, R

    1997-08-29

    The map of the mass centres of the 21 proteins from the Escherichia coli 30 S ribosomal subunit, as determined by neutron scattering, was fitted to a cryoelectron microscopic (cryo-EM) model at a resolution of 20 A of 70 S ribosomes in the pre-translocational state, carrying tRNA molecules at the A and P sites. The fit to the 30 S moiety of the 70 S particles was accomplished with the help of the well-known distribution of the ribosomal proteins in the head, body and side lobe regions of the 30 S subunit, as determined by immuno electron microscopy (IEM). Most of the protein mass centres were found to lie close to the surface (or even outside) of the cryo-EM contour of the 30 S subunit, supporting the idea that the ribosomal proteins are arranged peripherally around the rRNA. The ribosomal protein distribution was then compared with the corresponding model for the 16 S rRNA, fitted to the same EM contour (described in an accompanying paper), in order to analyse the mutual compatibility of the arrangement of proteins and rRNA in terms of the available RNA-protein interaction data. The information taken into account included the hydroxyl radical and base foot-printing data from Noller's laboratory, and our own in situ cross-linking results. Proteins S1 and S14 were not considered, due to the lack of RNA-protein data. Among the 19 proteins analysed, 12 (namely S2, S4, S5, S7, S8, S9, S10, S11, S12, S15, S17 and S21) showed a fit to the rRNA model that varied from being excellent to at least acceptable. Of the remaining 7, S3 and S13 showed a rather poor fit, as did S18 (which is considered in combination with S6 in the foot-printing experiments). S16 was difficult to evaluate, as the foot-print data for this protein cover a large area of the rRNA. S19 and S20 showed a bad fit in terms of the neutron map, but their foot-print and cross-link sites were clustered into compact groups in the rRNA model in those regions of the 30 S subunit where these proteins have

  7. Structural features of the plastid ribosomal RNA operons of two red algae: Antithamnion sp. and Cyanidium caldarium.

    PubMed

    Maid, U; Zetsche, K

    1991-04-01

    The nucleotide sequences of the plastid 16S rDNA of the multicellular red alga Antithamnion sp. and the 16S rDNA/23S rDNA intergenic spacers of the plastid DNAs of the unicellular red alga Cyanidium caldarium and of Antithamnion sp. were determined. Sequence comparisons support the idea of a polyphyletic origin of the red algal and the higher-plant chloroplasts. Both spacer regions include the unsplit tRNA(Ile)(GAU) and tRNA(Ala)(UGC) genes and so the plastids of both algae form a homogeneous group with those of chromophytic algae and Cyanophora paradoxa characterized by 'small-sized' rDNA spacers in contrast to green algae and higher plants. Nevertheless, remarkable sequence differences within the rRNA and the tRNA genes give the plastids of Cyanidium caldarium a rather isolated position. PMID:1868197

  8. Selection of random RNA fragments as method for searching for a site of regulation of translation of E. coli streptomycin mRNA by ribosomal protein S7.

    PubMed

    Surdina, A V; Rassokhin, T I; Golovin, A V; Spiridonova, V A; Kraal, B; Kopylov, A M

    2008-06-01

    In E. coli cells ribosomal small subunit biogenesis is regulated by RNA-protein interactions involving protein S7. S7 initiates the subunit assembly interacting with 16S rRNA. During shift-down of rRNA synthesis level, free S7 inhibits self-translation by interacting with 96 nucleotides long specific region of streptomycin (str) mRNA between cistrons S12 and S7 (intercistron). Many bacteria do not have the extended intercistron challenging development of specific approaches for searching putative mRNA regulatory regions, which are able to interact with proteins. The paper describes application of SERF approach (Selection of Random RNA Fragments) to reveal regulatory regions of str mRNA. Set of random DNA fragments has been generated from str operon by random hydrolysis and then transcribed into RNA; the fragments being able to bind protein S7 (serfamers) have been selected by iterative rounds. S7 binds to single serfamer, 109 nucleotide long (RNA109), derived from the intercistron. After multiple copying and selection, the intercistronic mutant (RNA109) has been isolated; it has enhanced affinity to S7. RNA109 binds to the protein better than authentic intercistronic str mRNA; apparent dissociation constants are 26 +/- 5 and 60 +/- 8 nM, respectively. Location of S7 binding site on the mRNA, as well as putative mode of regulation of coupled translation of S12 and S7 cistrons have been hypothesized.

  9. Asaia bogorensis peritonitis identified by 16S ribosomal RNA sequence analysis in a patient receiving peritoneal dialysis.

    PubMed

    Snyder, Richard W; Ruhe, Jorg; Kobrin, Sidney; Wasserstein, Alan; Doline, Christa; Nachamkin, Irving; Lipschutz, Joshua H

    2004-08-01

    Here the authors report a case of refractory peritonitis leading to multiple hospitalizations and the loss of peritoneal dialysis access in a patient on automated peritoneal dialysis, caused by Asaia bogorensis, a bacterium not previously described as a human pathogen. This organism was identified by sequence analysis of the 16S ribosomal RNA gene. Unusual microbial agents may cause peritonitis, and molecular microbiological techniques are important tools for identifying these agents.

  10. 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. PMID:26809121

  11. A ribonucleoprotein fragment of the 30 S ribosome of E. coli containing two contiguous domains of the 16 S RNA.

    PubMed

    Spitnik-Elson, P; Elson, D; Avital, S; Abramowitz, R

    1982-08-11

    Ribonucleoprotein fragments of the 30 S ribosome of E. coli have been prepared by limited ribonuclease digestion and mild heating of the ribosome in a constant ionic environment. One such fragment has been described previously. A second electrophoretically homogeneous fragment has now been isolated and its RNA and protein moieties have been characterized. It contains the 5' half of the 16 S RNA, encompassing domains I and II except for the extreme 5' terminus and several small gaps. Seven proteins are present: S4, S5, S6, S8, S12, S15 and S20. The RNA binding sites of five of these proteins are known, and all are RNA sequences that are present in the fragment. Published neutron scattering and immuno-electron microscopic data indicate that six of the proteins are clustered together in a cross sectional slice through the center of the subunit. After deproteinization, the RNA moiety gives two bands in gel electrophoresis, one containing domains I and II and the other, essentially only domain II. The former, although larger, migrates faster in gel electrophoresis, indicating that RNA domains I and II interact with each other in such a way as to become more compact than domain II by itself.

  12. Cockayne syndrome protein A is a transcription factor of RNA polymerase I and stimulates ribosomal biogenesis and growth.

    PubMed

    Koch, Sylvia; Garcia Gonzalez, Omar; Assfalg, Robin; Schelling, Adrian; Schäfer, Patrick; Scharffetter-Kochanek, Karin; Iben, Sebastian

    2014-01-01

    Mutations in the Cockayne syndrome A (CSA) protein account for 20% of Cockayne syndrome (CS) cases, a childhood disorder of premature aging and early death. Hitherto, CSA has exclusively been described as DNA repair factor of the transcription-coupled branch of nucleotide excision repair. Here we show a novel function of CSA as transcription factor of RNA polymerase I in the nucleolus. Knockdown of CSA reduces pre-rRNA synthesis by RNA polymerase I. CSA associates with RNA polymerase I and the active fraction of the rDNA and stimulates re-initiation of rDNA transcription by recruiting the Cockayne syndrome proteins TFIIH and CSB. Moreover, compared with CSA deficient parental CS cells, CSA transfected CS cells reveal significantly more rRNA with induced growth and enhanced global translation. A previously unknown global dysregulation of ribosomal biogenesis most likely contributes to the reduced growth and premature aging of CS patients.

  13. Analysis on the preference for sequence matching between mRNA sequences and the corresponding introns in ribosomal protein genes.

    PubMed

    Zhang, Qiang; Li, Hong; Zhao, Xiaoqing; Zheng, Yan; Meng, Hu; Jia, Yun; Xue, Hui; Bo, Sulin

    2016-03-01

    Introns after splicing still play an important role. Introns can accomplish gene expression and regulation by interaction with corresponding mRNA sequences. Based on the Smith-Waterman method, local comparing makes us get the optimal matched segments between intron sequences and mRNA sequences. Analyzing the distribution regulation of the optimal matching region on mRNA sequences of ribosomal protein genes about 27 species, we find a strong interaction between UTR region sequences and introns. There are a lot of the optimal matching regions and low matching ones, and the latter are supposed to be the combined regions of protein complexes. The optimal matching frequency distributions have obvious differences nearby the mRNA functional sites such as translation initiation and termination sites, exon-exon joints and EJC regions. This conclusion shows that intron sequences and mature mRNA sequences are co-evolved and interactive to play their functions. PMID:26707402

  14. Cockayne syndrome protein A is a transcription factor of RNA polymerase I and stimulates ribosomal biogenesis and growth

    PubMed Central

    Koch, Sylvia; Garcia Gonzalez, Omar; Assfalg, Robin; Schelling, Adrian; Schäfer, Patrick; Scharffetter-Kochanek, Karin; Iben, Sebastian

    2014-01-01

    Mutations in the Cockayne syndrome A (CSA) protein account for 20% of Cockayne syndrome (CS) cases, a childhood disorder of premature aging and early death. Hitherto, CSA has exclusively been described as DNA repair factor of the transcription-coupled branch of nucleotide excision repair. Here we show a novel function of CSA as transcription factor of RNA polymerase I in the nucleolus. Knockdown of CSA reduces pre-rRNA synthesis by RNA polymerase I. CSA associates with RNA polymerase I and the active fraction of the rDNA and stimulates re-initiation of rDNA transcription by recruiting the Cockayne syndrome proteins TFIIH and CSB. Moreover, compared with CSA deficient parental CS cells, CSA transfected CS cells reveal significantly more rRNA with induced growth and enhanced global translation. A previously unknown global dysregulation of ribosomal biogenesis most likely contributes to the reduced growth and premature aging of CS patients. PMID:24781187

  15. Characterisation of RNA fragments obtained by mild nuclease digestion of 30-S ribosomal subunits from Escherichia coli.

    PubMed

    Rinke, J; Ross, A; Brimacombe, R

    1977-06-01

    When Escherichia coli 30-S ribosomal subunits are hydrolysed under mild conditions, two ribonucleoprotein fragments of unequal size are produced. Knowledge of the RNA sequences contained in these hydrolysis products was required for the experiments described in the preceding paper, and the RNA sub-fragments have therefore been examined by oligonucleotide analysis. Two well-defined small fragments of free RNA, produced concomitantly with the ribonucleoprotein fragments, were also analysed. The larger ribonucleoprotein fragment, containing predominantly proteins S4, S5, S8, S15, S16 (17) and S20, contains a complex mixture of RNA sub-fragments varying from about 100 to 800 nucleotides in length. All these fragments arose from the 5'-terminal 900 nucleotides of 16-S RNA, corresponding to the well-known 12-S fragment. No long-range interactions could be detected within this RNA region in these experiments. The RNA from the smaller ribonucleoprotein fragment (containing proteins S7, S9 S10, S14 and S19) has been described in detail previously, and consists of about 450 nucleotides near the 3' end of the 16-S RNA, but lacking the 3'-terminal 150 nucleotides. The two small free RNA fragments (above) partly account for these missing 150 nucleotides; both fragments arose from section A of the 16-S RNA, but section J (the 3'-terminal 50 nucleotides) was not found. This result suggests that the 3' region of 16-S RNA is not involved in stable interactions with protein.

  16. RMF inactivates ribosomes by covering the peptidyl transferase centre and entrance of peptide exit tunnel.

    PubMed

    Yoshida, Hideji; Yamamoto, Hiroshi; Uchiumi, Toshio; Wada, Akira

    2004-04-01

    In gram-negative bacteria such as Escherichia coli, protein synthesis is suppressed by the formation of 100S ribosomes under stress conditions. The 100S ribosome, a dimer of 70S ribosomes, is formed by ribosome modulation factor (RMF) binding to the 70S ribosomes. During the stationary phase, most of the 70S ribosomes turn to 100S ribosomes, which have lost translational activity. This 100S formation is called the hibernation process in the ribosome cycle of the stationary phase. If stationary phase cells are transferred to fresh medium, the 100S ribosomes immediately go back to active 70S ribosomes, showing that inactive 100S <--> active 70S interconversion is a major system regulating translation activity in stationary phase cells. To elucidate the mechanisms of translational inactivation, the binding sites of RMF on 23S rRNA in 100S ribosome of E. coli were examined by a chemical probing method using dimethyl sulphate (DMS). As the results, the nine bases in 23S rRNA were protected from DMS modifications and the modification of one base was enhanced. Interestingly A2451 is included among the protected bases, which is thought to be directly involved in peptidyl transferase activity. We conclude that RMF inactivates ribosomes by covering the peptidyl transferase (PTase) centre and the entrance of peptide exit tunnel. It is surprising that the cell itself produces a protein that seems to inhibit protein synthesis in a similar manner to antibiotics and that it can reversibly bind to and release from the ribosome in response to environmental conditions.

  17. Complementarity of Bacillus subtilis 16S rRNA with sites of antibiotic-dependent ribosome stalling in cat and erm leaders.

    PubMed Central

    Rogers, E J; Ambulos, N P; Lovett, P S

    1990-01-01

    Inducible cat and erm genes are regulated by translational attenuation. In this regulatory model, gene activation results from chloramphenicol- or erythromycin-dependent stalling of a ribosome at a precise site in the leader region of cat or erm transcripts. The stalled ribosome is believed to destabilize a downstream region of RNA secondary structure that sequesters the ribosome-binding site for the cat or erm coding sequence. Here we show that the ribosome stall sites in cat and erm leader mRNAs, designated crb and erb, respectively, are largely complementary to an internal sequence in 16S rRNA of Bacillus subtilis. A tetracycline resistance gene that is likely regulated by translational attenuation also contains a sequence in its leader mRNA, trb, which is complementary to a sequence in 16S rRNA that overlaps with the crb and erb complements. An in vivo assay is described which is designed to test whether 16S rRNA of a translating ribosome can interact with the crb sequence in mRNA in an inducer-dependent reaction. The assay compares the growth rate of cells expressing crb-86 with the growth rate of cells lacking crb-86 in the presence of subinhibitory levels of inducers of cat-86, chloramphenicol, fluorothiamphenicol, amicetin, or erythromycin. Under these conditions, crb-86 retarded growth. Deletion of the crb-86 sequence, insertion of ochre mutations into crb-86, or synonymous codon changes in crb-86 that decreased its complementarity with 16S rRNA all eliminated from detection inducer-dependent growth retardation. Lincomycin, a ribosomally targeted antibiotic that is not an inducer of cat-86, failed to selectively retard the growth of cells expressing crb-86. We suggest that cat-86 inducers enable the crb-86 sequence in mRNA to base pair with 16S rRNA of translating ribosome. When the base pairing is extensive, as with crb-86, ribosomes become transiently trapped on crb and are temporarily withdrawn from protein synthesis to the extent that growth rate

  18. Improved Ribosome-Footprint and mRNA Measurements Provide Insights into Dynamics and Regulation of Yeast Translation.

    PubMed

    Weinberg, David E; Shah, Premal; Eichhorn, Stephen W; Hussmann, Jeffrey A; Plotkin, Joshua B; Bartel, David P

    2016-02-23

    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 codons matching rare tRNAs are more slowly translated. They also indicate that emergent polypeptides with as few as three basic residues within a ten-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' UTRs. Collectively, our results provide a framework for executing and interpreting ribosome-profiling studies and reveal key features of translational control in yeast. PMID:26876183

  19. 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

  20. Seasonal Succession Leads to Habitat-Dependent Differentiation in Ribosomal RNA:DNA Ratios among Freshwater Lake Bacteria

    PubMed Central

    Denef, Vincent J.; Fujimoto, Masanori; Berry, Michelle A.; Schmidt, Marian L.

    2016-01-01

    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. However, the similarity between RNA and DNA measurements and taxa-specific PSPs that drive community-level similarities are conditional on spatiotemporal factors. PMID:27199936

  1. 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.; 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 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

  2. Seasonal Succession Leads to Habitat-Dependent Differentiation in Ribosomal RNA:DNA Ratios among Freshwater Lake Bacteria.

    PubMed

    Denef, Vincent J; Fujimoto, Masanori; Berry, Michelle A; Schmidt, Marian L

    2016-01-01

    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. However, the similarity between RNA and DNA measurements and taxa-specific PSPs that drive community-level similarities are conditional on spatiotemporal factors. PMID:27199936

  3. 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.

  4. Genotypic Characterization of Bradyrhizobium Strains Nodulating Endemic Woody Legumes of the Canary Islands by PCR-Restriction Fragment Length Polymorphism Analysis of Genes Encoding 16S rRNA (16S rDNA) and 16S-23S rDNA Intergenic Spacers, Repetitive Extragenic Palindromic PCR Genomic Fingerprinting, and Partial 16S rDNA Sequencing

    PubMed Central

    Vinuesa, Pablo; Rademaker, Jan L. W.; de Bruijn, Frans J.; Werner, Dietrich

    1998-01-01

    We present a phylogenetic analysis of nine strains of symbiotic nitrogen-fixing bacteria isolated from nodules of tagasaste (Chamaecytisus proliferus) and other endemic woody legumes of the Canary Islands, Spain. These and several reference strains were characterized genotypically at different levels of taxonomic resolution by computer-assisted analysis of 16S ribosomal DNA (rDNA) PCR-restriction fragment length polymorphisms (PCR-RFLPs), 16S-23S rDNA intergenic spacer (IGS) RFLPs, and repetitive extragenic palindromic PCR (rep-PCR) genomic fingerprints with BOX, ERIC, and REP primers. Cluster analysis of 16S rDNA restriction patterns with four tetrameric endonucleases grouped the Canarian isolates with the two reference strains, Bradyrhizobium japonicum USDA 110spc4 and Bradyrhizobium sp. strain (Centrosema) CIAT 3101, resolving three genotypes within these bradyrhizobia. In the analysis of IGS RFLPs with three enzymes, six groups were found, whereas rep-PCR fingerprinting revealed an even greater genotypic diversity, with only two of the Canarian strains having similar fingerprints. Furthermore, we show that IGS RFLPs and even very dissimilar rep-PCR fingerprints can be clustered into phylogenetically sound groupings by combining them with 16S rDNA RFLPs in computer-assisted cluster analysis of electrophoretic patterns. The DNA sequence analysis of a highly variable 264-bp segment of the 16S rRNA genes of these strains was found to be consistent with the fingerprint-based classification. Three different DNA sequences were obtained, one of which was not previously described, and all belonged to the B. japonicum/Rhodopseudomonas rDNA cluster. Nodulation assays revealed that none of the Canarian isolates nodulated Glycine max or Leucaena leucocephala, but all nodulated Acacia pendula, C. proliferus, Macroptilium atropurpureum, and Vigna unguiculata. PMID:9603820

  5. 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.

  6. Identification by affinity chromatography of the eukaryotic ribosomal proteins that bind to 5.8 S ribosomal ribonucleic acid.

    PubMed

    Ulbrich, N; Lin, A; Wool, I G

    1979-09-10

    The proteins that bind to rat liver 5.8 S ribosomal ribonucleic acid were identified by affinity chromatography. The nucleic acid was oxidized with periodate and coupled by its 3'-terminus to Sepharose 4B through and adipic acid dihydrazide spacer. The ribosomal proteins that associate with the immobilized 5.8 S rRNA were identified by polyacrylamide gel electrophoresiss: they were L19, L8, and L6 from the 60 S subunit; and S13 and S9 from the small subparticle. Small amounts of L14, L17', L18, L27/L27', and L35', and of S11, S15, S23/S24, and S26 also were bound to the affinity column, but whether they associate directly and specifically with 5.8 S rRNA is not known. Escherichia coli ribosomal proteins did not bind to the rat liver 5.8 S rRNA affinity column. PMID:468846

  7. Linezolid-resistant Staphylococcus haemolyticus and Staphylococcus hominis: single and double mutations at the domain V of 23S rRNA among isolates from a Rio de Janeiro hospital.

    PubMed

    Chamon, Raiane Cardoso; Iorio, Natalia Lopes Pontes; Cavalcante, Fernanda Sampaio; Teodoro, Cristiane R S; de Oliveira, Ana Paula Chaves; Maia, Fernanda; dos Santos, Kátia Regina Netto

    2014-12-01

    In this work, the molecular and phenotypic antimicrobial resistance and clonal diversity of 10 linezolid-resistant Staphylococcus spp. isolates were investigated. The 7 Staphylococcus haemolyticus isolates presented Staphylococcal cassete chromosome mec (SCCmec) V and belonged to the same pulsed-field gel electrophoresis pulsotype. Their MICs for oxacillin, vancomycin, and linezolid were ≥ 256 μg/mL, 1-4 μg/mL, and 8-16 μg/mL, respectively. The 3 S. hominis presented MIC values 32 to >256 μg/mL, 2-4 μg/mL, and 12-24 μg/mL, and all carried the nontypeable SCCmec (ccr1 + mecA class) and belonged to 2 different genotypes. The cfr gene was not found, but the mutation G2603T was detected in S. haemolyticus and C2190T and G2603T in Staphylococcus hominis in 23S rRNA. This study demonstrates the spread of a linezolid-resistant S. haemolyticus genotype and, for the first time, describes the mutation C2190T among S. hominis isolates with a double mutation in Brazil.

  8. Evaluation of a fluorescence-labelled oligonucleotide probe targeting 23S rRNA for in situ detection of Salmonella serovars in paraffin-embedded tissue sections and their rapid identification in bacterial smears.

    PubMed Central

    Nordentoft, S; Christensen, H; Wegener, H C

    1997-01-01

    A method for the detection of Salmonella based on fluorescence in situ hybridization (FISH) has been developed and applied for the direct detection of Salmonella in pure cultures and in formalin-fixed, paraffin-embedded tissue sections. On the basis of the 23S rRNA gene sequences representing all of the S. enterica subspecies and S. bongori, an 18-mer oligonucleotide probe was selected. The specificity of the probe was tested by in situ hybridization to bacterial cell smears of pure cultures. Forty-nine of 55 tested Salmonella serovars belonging to subspecies I, II, IIIb, IV, and VI hybridized with the probe. The probe did not hybridize to serovars from subspecies IIIa (S. arizonae) or to S. bongori. No cross-reaction to 64 other strains of the family Enterobacteriaceae or 18 other bacterial strains outside this family was observed. The probe was tested with sections of formalin-fixed, paraffin-embedded tissue from experimentally infected mice or from animals with a history of clinical salmonellosis. In these tissue sections the probe hybridized specifically to Salmonella serovars, allowing for the detection of single bacterial cells. The development of a fluorescence-labelled specific oligonucleotide probe makes the FISH technique a promising tool for the rapid identification of S. enterica in bacterial smears, as well as for the detection of S. enterica in histological tissue sections. PMID:9316923

  9. A Novel SimpleProbe PCR Assay for Detection of Mutations in the 23S rRNA Gene Associated with Macrolide Resistance in Mycoplasma genitalium in Clinical Samples

    PubMed Central

    Lysvand, Hilde; Pukstad, Brita; Nordbø, Svein Arne

    2016-01-01

    Macrolide-resistant strains of Mycoplasma genitalium are an increasing problem throughout the world, and the implementation of a rapid and sensitive assay for mutation detection to guide treatment is needed. Macrolide-resistant strains have been shown to contain base substitutions in positions 2058 and 2059 (Escherichia coli numbering) in region V of the 23S rRNA gene. In this study, we present a SimpleProbe PCR followed by melting curve analysis to differentiate between macrolide-resistant mutants and wild types. The assay was performed on 159 Mycoplasma genitalium-positive samples, and the results were compared with DNA sequencing. We also looked at the prevalence of macrolide-resistant strains in a Norwegian population. Of 139 samples characterized successfully by sequencing, 54 (39%) were wild types and 85 (61%) were mutants, consisting of 59 (42%) A2059G, 24 (17%) A2058G, 1 (1%) A2058T, and 1 (1%) A2059C mutation. The melting curve analysis correctly differentiated between wild-type and mutant strains in all cases, but it could not identify the different mutant types. The SimpleProbe PCR proved to be a simple, rapid, and reliable method for the detection of macrolide-resistant isolates of Mycoplasma genitalium in a clinical setting. PMID:27487958

  10. Stoichiometry and Change of the mRNA Closed-Loop Factors as Translating Ribosomes Transit from Initiation to Elongation

    PubMed Central

    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