Ribosomes translate the genetic information encoded by mRNA into proteins. Defective mRNAs can cause stalling of translating ribosomes. The molecule tmRNA (transfer-messenger RNA) rescues stalled ribosomes in eubacteria. Together with its protein partner SmpB, tmRNA mimics a tRNA by entering the ribosomal A site and linking an alanine residue to the growing polypeptide chain. The ribosome then abandons the defective mRNA template and resumes translation on tmRNA, adding ten more amino acids to the nascent polypeptide. As a result of tmRNA action, stalled ribosomes are released and recycled, the defective mRNA is destroyed, and the aborted protein product is tagged for destruction by proteases. It is unknown how the ribosome correctly chooses the position on tmRNA to resume translation. Previous studies implicate the sequence UAGUC found immediately upstream of the first codon in the tmRNA open reading frame. These nucleotides are highly conserved in natural tmRNA sequences. Mutations in this area cause loss of tmRNA function and improper frame choice. Using a genetic selection that ties the life of E. coli cells to the function of tmRNA, we have identified several SmpB mutants that rescue an inactive tmRNA in which this upstream sequence was altered. This links SmpB to the function of these key tmRNA nucleotides. We show that our SmpB mutants affect frame choice using an in vivo assay for tagging in the various frames. We conclude that SmpB plays a role in setting the reading frame on tmRNA.

Ribosomes stall on defective messenger RNA transcripts in eubacteria. Without a mechanism to release stalled ribosomes, these cells would die. Transfer-messenger RNA (tmRNA) and small protein B (SmpB) reactivate stalled ribosomes in a process known as trans-translation. Together, tmRNA and SmpB mimic alanyl-tRNA, entering the A site of stalled ribosomes and accepting transfer of the stalled polypeptide. A portion of tmRNA is then positioned as a template for the ribosome to resume translating. The tmRNA open reading frame encodes a proteolysis tag to mark the aberrant polypeptide for degradation and a stop codon to release the ribosome. How are tmRNA and SmpB allowed into stalled ribosomes? In normal translation, decoding mechanisms carefully monitor the anticodon of tRNAs entering the A site and select only those that are complementary to the mRNA codon. How do tmRNA and SmpB get around the decoding machinery? It appears that interactions between the SmpB C-terminal tail and the decoding center are responsible. Using an in vivo tagging assay and an in vitro peptidyl-transfer assay, we monitored the effect of mutations in the SmpB tail on trans-translation. We found that mutations in SmpB that prevent helix formation are unable to support peptidyl transfer. We also found that while mutation of key nucleotides in the ribosomal decoding center severely inhibit peptidyl transfer to normal tRNAs, these mutations do not inhibit peptidyl transfer to tmRNA. We conclude that the SmpB tail stimulates peptidyl transfer by forming a helix that interacts with the ribosome to signal decoding in a novel manner. How is the tmRNA open reading frame positioned for the ribosome to resume translating? Mutation of the tmRNA nucleotide A86 alters reading frame selection. Using a genetic selection, we identified SmpB mutants that restore normal frame selection to A86C tmRNA without altering frame selection on wild-type tmRNA. Through rational mutation of the SmpB tail we identified an SmpB mutant that supports peptidyl transfer but prevents translation of the tmRNA open reading frame. We conclude that SmpB plays a functional role in selecting the tmRNA open reading frame.

<p>Ribosomes stalled on problematic mRNAs can be rescued by a mechanism called <i>trans</i>-translation. This mechanism employs a dual transfer-messenger RNA molecule (tmRNA) together with a helper protein (SmpB). </p><p>In this work we have used an <i>in vitro</i> translation system with pure components to further clarify the roles of tmRNA and SmpB in <i>trans-</i>translation. </p><p>We found that SmpB binds ribosomes <i>in vivo</i> and <i>in vitro</i> independently of tmRNA presence and is essential for tmRNA binding and <i>trans-</i>peptidation. We show that two SmpB molecules can bind per ribosome, that SmpB does not leave the ribosome after <i>trans-</i>peptidation and that SmpB pre-bound to the ribosome can trigger <i>trans-</i>translation. </p><p>We demonstrated that the rate of <i>trans-</i>transfer of a peptide from the P-site tRNA to Ala-tmRNA and the efficiency by which Ala-tmRNA competes with peptide release factors decrease with increasing the mRNA length downstream from the P site of the ribosome. We showed that <i>trans-</i>translation is strongly stimulated by RelE cleavage of A-site mRNA. We concluded that tmRNA action<i> in vivo</i> must always be preceded by mRNA truncation.</p><p>We showed that rapid release of truncated mRNAs from the ribosome requires translocation of the peptidyl-tmRNA into the ribosomal P site, which is strictly EF-G dependent. mRNA release is slowed down by strong Shine and Dalgarno like sequences upstream the A site and by long 3’-extensions downstream from the P-site codon. </p><p>Footprinting was used to monitor SmpB binding to tmRNA, ribosomes and subunits and to study tmRNA interactions with the ribosome at distinct <i>trans-</i>translation stages. We confirmed that two SmpB molecules bind per ribosome and interact with nucleotides below the L7/L12-stalk on the 50S subunit and near the subunit interface on the 30S. We showed that tmRNA is mostly in complex with SmpB <i>in vivo</i> and during <i>trans-</i>translation. Specific cleavage patterns of tmRNA were observed at different stages of <i>trans-</i>translation, but the overall tmRNA conformation seems to be maintained during the whole process.</p>

Ribosomes stalled on problematic mRNAs can be rescued by a mechanism called trans-translation. This mechanism employs a dual transfer-messenger RNA molecule (tmRNA) together with a helper protein (SmpB). In this work we have used an in vitro translation system with pure components to further clarify the roles of tmRNA and SmpB in trans-translation. We found that SmpB binds ribosomes in vivo and in vitro independently of tmRNA presence and is essential for tmRNA binding and trans-peptidation. We show that two SmpB molecules can bind per ribosome, that SmpB does not leave the ribosome after trans-peptidation and that SmpB pre-bound to the ribosome can trigger trans-translation. We demonstrated that the rate of trans-transfer of a peptide from the P-site tRNA to Ala-tmRNA and the efficiency by which Ala-tmRNA competes with peptide release factors decrease with increasing the mRNA length downstream from the P site of the ribosome. We showed that trans-translation is strongly stimulated by RelE cleavage of A-site mRNA. We concluded that tmRNA action in vivo must always be preceded by mRNA truncation. We showed that rapid release of truncated mRNAs from the ribosome requires translocation of the peptidyl-tmRNA into the ribosomal P site, which is strictly EF-G dependent. mRNA release is slowed down by strong Shine and Dalgarno like sequences upstream the A site and by long 3’-extensions downstream from the P-site codon. Footprinting was used to monitor SmpB binding to tmRNA, ribosomes and subunits and to study tmRNA interactions with the ribosome at distinct trans-translation stages. We confirmed that two SmpB molecules bind per ribosome and interact with nucleotides below the L7/L12-stalk on the 50S subunit and near the subunit interface on the 30S. We showed that tmRNA is mostly in complex with SmpB in vivo and during trans-translation. Specific cleavage patterns of tmRNA were observed at different stages of trans-translation, but the overall tmRNA conformation seems to be maintained during the whole process.

In eubacteria, stalled ribosomes are rescued by a conserved quality-control mechanism involving transfer-messenger RNA (tmRNA) and its protein partner SmpB. Mimicking a tRNA, tmRNA enters stalled ribosomes, adds Ala to the nascent polypeptide, and serves as a template to encode a short peptide that tags the nascent protein for destruction. To further characterize the tagging process, we developed two genetic selections that link tmRNA activity to cell death. These negative selections can be used to identify inhibitors of tagging or to identify mutations in key residues essential for ribosome rescue. Little is known about which ribosomal elements are specifically required for tmRNA activity. Using these selections, we isolated ribosomal RNA mutations that block the rescue of ribosomes stalled at rare Arg codons or at the inefficient termination signal Pro-opal. We find that deletion of A1150 in the 16S rRNA blocks tagging regardless of the stalling sequence, suggesting that it inhibits tmRNA activity directly. The C889U mutation in 23S rRNA, however, lowers tagging levels at Pro-opal and rare Arg codons but not at the 3'-end of an mRNA lacking a stop codon. We conclude that the C889U mutation does not inhibit tmRNA activity per se but interferes with an upstream step intermediate between stalling and tagging.

Résumé en français confidentiel / Résumé en anglais confidentiel

In eubacteria, ribosome stalling on broken messenger RNA transcripts can lead to cell death. The trans-translation quality control mechanism rescues many of these stalled ribosomes. In this process, tmRNA enters stalled ribosomes by mimicking a transfer RNA, accepting the stalled nascent peptide. The ribosome then releases the broken mRNA and resumes translation on a coding region within tmRNA itself. Translation of tmRNA marks the nascent peptide for destruction by the addition of a short proteolysis tag and the ribosome is released at a stop codon within the tmRNA open reading frame. An intriguing aspect of trans-translation is that the ribosome synthesizes one protein from two RNA templates. How is the proper site chosen on tmRNA to resume translation? Do the conserved pseudoknot structures help set the reading frame? Using a genetic selection to assay libraries of tmRNA mutants, we found that stable hairpin structures can functionally replace pseudoknot 1. We conclude that the role of pseudoknot 1 in tmRNA function is purely structural. Our results demonstrate that the inactivity of an RNA mutant designed to destroy a given structure should not be interpreted as proof that the structure is necessary for RNA function. Such mutations may only destabilize a global fold that could be formed equally well by an entirely different, stable structure. Broken mRNAs are not the only cause of ribosome stalling; stalling can also result from nascent peptide interactions with the ribosomal exit tunnel that inhibit peptidyl-transferase activity. SecM, TnaC, and ErmCL all stall ribosomes to regulate the expression of downstream genes. What other peptide sequences can cause ribosome stalling? We modified our tmRNA-based selection to screen libraries of random peptides and identified a number of novel stalling peptides, including the sequence FxxYxIWPP. This sequence interacts with the exit tunnel differently than SecM and TnaC as seen in studies using mutant ribosomes. Like SecM, stalling occurs on this sequence with the next aminoacyl tRNA trapped in the A site but unable to react with the nascent peptide. These results show that a variety of peptides can interact in the exit tunnel and peptidyl-transferase center to regulate ribosome activity.

Le travail retranscrit dans cette thèse regroupe l'étude de différents processus biologiques impliqués dans la synthèse protéique bactérienne. Dans un premier chapitre, les origines de la synthèse protéique au temps du monde ARN sont traitées en guise d'introduction. Ce travail théorique se poursuit par la présentation d'une structure à haute résolution du facteur d'élongation G (EF-G) en complexe avec le ribosome par cryo-microscopie électronique à transmission (cryo-MET). Grâce aux avancées techniques de la cryo-MET, nous avons observé pour la première fois EF-G lié au ribosome en l'absence de tout inhibiteur. Cet état particulièr d'EF-G permet de visualiser une flexibilité de son doamine III. Cette étude permet aussi de rationaliser le fonctionnement de l'antibiotique acide fusidique. Nous nous sommes ensuite intéressés aux voies de sauvetage de la synthèse protéique et plus particulièrement de la trans-traduction. Ce mécanisme fascinant permet le recyclage des ribosomes bloqués sur un ARN messager défectueux. Cette voie de sauvetage est généralement vitale ou alors indispensable pour la virulence bactérienne. Nous avons réalisé une étude structurale préliminaire de la dégradation de l'ARNm défectueux durant ce processus. Après une revue traitant du sujet, nous présentons une étude de la trans-traduction comme cible pour le développement de nouveaux antibiotiques. Pour cela, nous avons mis au point un système rapporteur avec contrôle interne de l'activité trans-traductionnelle bactérienne. Après avoir mis au point ce système et validé son utilisation, nous l'avons exploité en testant des molécules ciblant la trans-traduction. / The current PhD work brings together various studies linked to bacterial protein synthesis. The first chapter is about the origins of protein synthesis at the time of the RNA world. This theoretical work continues with the presentation of a high-resolution structure of the elongation factor G (EF-G) in complex with the ribosome by cryo-electron transmission microscopy (cryo-TEM). We describe for the first time EF-G bound to the ribosome in the absence of any inhibitor. This particular structure of EF-G displays a yet unseen positioning of its third domain, which becomes very flexible. This study helps to understand the way the antibiotic fusidic acid blocks translation. The work then switches to a study of trans-translation, the main rescuing system of stalled ribosomes in bacteria. Trans-translation is generally vital or at least necessary for bacterial virulence. We conducted a preliminary structural study on the way faulty mRNAs are degraded during this process. This is why we present a study of trans-translation as a target for the development of new antibiotics. For this we developed and validated a reporter system for trans-translation, which is used to screen molecules targeting trans-translation.