Roles of helix H69 of 23S rRNA in translation initiation.
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ABSTRACT:
SUBMITTER: Liu Q
PROVIDER: S-EPMC4577190 | biostudies-other | 2015 Sep
REPOSITORIES: biostudies-other
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Project description:The molecular mechanisms that govern translation initiation to ensure accuracy remain unclear. Here, we provide evidence that the subunit-joining step of initiation is controlled in part by a conformational change in the 1408 region of helix h44. First, chemical probing of 30S initiation complexes formed with either a cognate (AUG) or near-cognate (AUC) start codon shows that an IF1-dependent enhancement at A1408 is reduced in the presence of AUG. This change in reactivity is due to a conformational change rather than loss of IF1, because other portions of the IF1 footprint are unchanged and high concentrations of IF1 fail to diminish the reactivity difference seen at A1408. Second, mutations in h44 such as A1413C stimulate 50S docking and cause reduced reactivity at A1408. Third, streptomycin, which has been shown by Rodnina and coworkers to stimulate 50S docking by reversing the inhibitory effects of IF1, also causes reduced reactivity at A1408. Collectively, these data support a model in which IF1 alters the A1408 region of h44 in a way that makes 50S docking unfavorable, and canonical codon-anticodon pairing in the P site restores h44 to a docking-favorable conformation. We also find that, in the absence of factors, the cognate 30S•AUG•fMet-tRNA ternary complex is >1000-fold more stable than the near-cognate 30S•AUC•fMet-tRNA complex. Hence, the selectivity of ternary complex formation is inherently high, exceeding that of initiation in vivo by more than 10-fold.
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Project description:Aminoglycosides antibiotics negate dissociation and recycling of the bacterial ribosome's subunits by binding to Helix 69 (H69) of 23S rRNA. The differential binding of various aminoglycosides to the chemically synthesized terminal domains of the Escherichia coli and human H69 has been characterized using spectroscopy, calorimetry and NMR. The unmodified E. coli H69 hairpin exhibited a significantly higher affinity for neomycin B and tobramycin than for paromomycin (K(d)s = 0.3 +/- 0.1, 0.2 +/- 0.2 and 5.4 +/- 1.1 microM, respectively). The binding of streptomycin was too weak to assess. In contrast to the E. coli H69, the human 28S rRNA H69 had a considerable decrease in affinity for the antibiotics, an important validation of the bacterial target. The three conserved pseudouridine modifications (Psi1911, Psi1915, Psi1917) occurring in the loop of the E. coli H69 affected the dissociation constant, but not the stoichiometry for the binding of paromomycin (K(d) = 2.6 +/- 0.1 microM). G1906 and G1921, observed by NMR spectrometry, figured predominantly in the aminoglycoside binding to H69. The higher affinity of the E. coli H69 for neomycin B and tobramycin, as compared to paromomycin and streptomycin, indicates differences in the efficacy of the aminoglycosides.
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Project description:In most bacteria, the three ribosomal RNAs (rRNAs) are encoded together in each of several near-identical operons. As soon as the nascent precursor rRNA emerges from RNA polymerase, ribosome assembly begins. This process entails ribosomal protein binding, rRNA folding, rRNA modification, and rRNA processing. In the model organisms Escherichia coli and Bacillus subtilis, rRNA processing results in similar mature rRNAs, despite substantial differences in the cohort of RNAses involved. A recent study of Flavobacterium johnsoniae, a member of the phylum Bacteroidota (formerly Bacteroidetes), revealed that helix H1 of 23S rRNA is absent from ribosomes, apparently a consequence of rRNA maturation. In this work, we mined RNA-seq data from 19 individual organisms and ocean metatranscriptomic samples to compare rRNA processing across diverse bacterial lineages. We found that mature ribosomes from multiple clades lack H1, and typically these ribosomes also lack an encoded H98. For all groups analysed, H1 is predicted to form in precursor rRNA as part of a longer leader-trailer helix. Hence, we infer that evolutionary loss of H98 sets the stage for H1 removal during 50S subunit maturation.
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Project description:Ribosome biogenesis is a complex process, and dozens of factors are required to facilitate and regulate the subunit assembly in bacteria. The 2'-O-methylation of U2552 in 23S rRNA by methyltransferase RrmJ is a crucial step in late-stage assembly of the 50S subunit. Its absence results in severe growth defect and marked accumulation of pre50S assembly intermediates. In the present work, we employed cryoelectron microscopy to characterize a set of late-stage pre50S particles isolated from an Escherichia coli ΔrrmJ strain. These assembly intermediates (solved at 3.2 to 3.8 Å resolution) define a collection of late-stage particles on a progressive assembly pathway. Apart from the absence of L16, L35, and L36, major structural differences between these intermediates and the mature 50S subunit are clustered near the peptidyl transferase center, such as H38, H68-71, and H89-93. In addition, the ribosomal A-loop of the mature 50S subunit from ΔrrmJ strain displays large local flexibility on nucleotides next to unmethylated U2552. Fast kinetics-based biochemical assays demonstrate that the ΔrrmJ 50S subunit is only 50% active and two times slower than the WT 50S subunit in rapid subunit association. While the ΔrrmJ 70S ribosomes show no defect in peptide bond formation, peptide release, and ribosome recycling, they translocate with 20% slower rate than the WT ribosomes in each round of elongation. These defects amplify during synthesis of the full-length proteins and cause overall defect in protein synthesis. In conclusion, our data reveal the molecular roles of U2552 methylation in both ribosome biogenesis and protein translation.
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Project description: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.
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Project description: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.
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Project description:A total of 25 modifications were mapped to 16S and 23S RNA in B. subtilis ribosome, however most of the genes responsible for these modification sites remain unknown. In this work, bottom-up oligonucleotide LC-MS/MS was used to detect rRNA modifications in five single gene knock-out strains: yydA, yhcT, yjbO, ylyB, yqxC. By comparing oligonucleotide modification states between the background 168 strain (annotated as WT) and the mutants: ylyB was confirmed to be a pseudouridine synthase at positions 1940, 1944, 1946 of 23S; yydA is a N3-pseudouridine methyltransferase at 1944 of 23S; and yqxC is a dual 2’O-cytidine methyltransferase at 1417 of 16S and 1949 of 23S. Deletion of yhcT or yjbO does not change rRNA modification pattern.
2024-08-06 | PXD051518 | Pride
Project description:We provide experimental evidence for RNase III-dependent processing in helix 9 of the 23S rRNA as a general feature of many species in the alpha subclass of Proteobacteria (alpha-Proteobacteria). We investigated 12 Rhodobacter, Rhizobium, Sinorhizobium, Rhodopseudomonas, and Bartonella strains. The processed region is characterized by the presence of intervening sequences (IVSs). The 23S rDNA sequences between positions 109 and 205 (Escherichia coli numbering) were determined, and potential secondary structures are proposed. Comparison of the IVSs indicates very different evolutionary rates in some phylogenetic branches, lateral genetic transfer, and evolution by insertion and/or deletion. We show that the IVS processing in Rhodobacter capsulatus in vivo is RNase III-dependent and that RNase III cleaves additional sites in vitro. While all IVS-containing transcripts tested are processed in vitro by RNase III from R. capsulatus, E. coli RNase III recognizes only some of them as substrates and in these substrates frequently cleaves at different scissile bonds. These results demonstrate the different substrate specificities of the two enzymes. Although RNase III plays an important role in the rRNA, mRNA, and bacteriophage RNA maturation, its substrate specificity is still not well understood. Comparison of the IVSs of helix 9 does not hint at sequence motives involved in recognition but reveals that the "antideterminant" model, which represents the most recent attempt to explain the E. coli RNase III specificity in vitro, cannot be applied to substrates derived from alpha-Proteobacteria.
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Project description:Histophilus somni is a Gram-negative bacterial organism that acts as an opportunistic pathogen and is a fastidious member of the Pasteurellaceae family associated with diseases of respiratory, reproductive, cardiac, and other tissues of ruminants. We identified an intervening sequence (IVS) embedded in all five copies of the 23S rRNA gene in the closed genome sequence of the H. somni isolate USDA-ARS-USMARC-63250 that may play an important role in affecting the biology of the organism. Sequencing the RNA from this isolate shows that most of the IVS is cleaved from the transcript, resulting in independent fragments of this structural rRNA that remain functional within the bacterial ribosome. The IVS lies between positions 1170 and 1278 bp of the 3,017-bp gene and exhibits self-complementarity between its 5' and 3' ends that predicts a stem-loop structure interrupting helix-45 in the transcribed 23S rRNA. Excision removes a 94-nucleotide (nt) stem-loop structure that displays an unusual 1-nt 3' end overhang instead of the more typical 2-nt overhang commonly observed at the ends of other excised IVS stem-loops. A comparison with genomes of other H. somni isolates indicates that this IVS is highly conserved, with 31 of 32 complete genomes having similar interruptions of canonical 23S rRNA genes. The potential biological effects of either the released IVS or the fragmentation of the functional 23S rRNA are unknown, but fragmentation may enhance rRNA degradation in ways that contribute to the regulation of gene expression. IMPORTANCE The genome biology underlying H. somni virulence, pathogenicity, environmental adaptability, and broad tissue tropism is understood poorly. We identified a novel H. somni 109-nt IVS stem-loop structure, of which the central portion is excised from the 23S rRNA transcript, resulting in the fragmentation of this rRNA in the H. somni isolate USDA-ARS-USMARC-63250 and the release of a 94-nt structured RNA of unknown function. We determined that this peculiar rRNA biology is widespread among sequenced H. somni isolates, suggesting it has importance to organism biology. The fragmented 23S rRNA molecules remain functional in the ribosome, given that the isolate grows in culture. The structured excised portion of the IVS, presumably due to the action of the endoribonuclease III, has an unusual 1-nt 3' end overhang. This newly discovered H. somni 23S rRNA fragmentation may enhance rRNA degradation providing a previously unrecognized avenue for regulating H. somni biological processes.
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Project description:Peptide bond formation and peptidyl-tRNA hydrolysis are the two elementary chemical reactions of protein synthesis catalyzed by the ribosomal peptidyl transferase ribozyme. Due to the combined effort of structural and biochemical studies, details of the peptidyl transfer reaction have become increasingly clearer. However, significantly less is known about the molecular events that lead to peptidyl-tRNA hydrolysis at the termination phase of translation. Here we have applied a recently introduced experimental system, which allows the ribosomal peptidyl transferase center (PTC) to be chemically engineered by the introduction of non-natural nucleoside analogs. By this approach single functional group modifications are incorporated, thus allowing their functional contributions in the PTC to be unravelled with improved precision. We show that an intact ribose sugar at the 23S rRNA residue A2602 is crucial for efficient peptidyl-tRNA hydrolysis, while having no apparent functional relevance for transpeptidation. Despite the fact that all investigated active site residues are universally conserved, the removal of the complete nucleobase or the ribose 2'-hydroxyl at A2602, U2585, U2506, A2451 or C2063 has no or only marginal inhibitory effects on the overall rate of peptidyl-tRNA hydrolysis. These findings underscore the exceptional functional importance of the ribose moiety at A2602 for triggering peptide release.
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