Studies reported in this thesis address the question of how pre-termination ribosomal complexes stalled during translation of mRNA are recycled. The process of recycling of the stalled ribosomes involves many translational factors. During the course of my studies, I have uncovered new roles of SsrA (tmRNA), IF3 and ribosome recycling factor (RRF) in recycling stalled ribosomes. These findings are summarized as follows: (i) A physiological connection between tmRNA and peptidyl-tRNA hydrolase functions in Escherichia coli The bacterial ssrA gene codes for a dual function RNA, tmRNA, which possesses tRNA-like and mRNA-like regions. The tmRNA appends an oligopeptide tag to the polypeptide on the P-site tRNA by a trans-translation process that rescues ribosomes stalled on mRNAs and targets the aberrant protein for degradation. In cells, processing of the stalled ribosomes is also pioneered by drop-off of peptidyl-tRNAs. The ester bond linking the peptide to tRNA is hydrolyzed by peptidyl-tRNA hydrolase (Pth), an essential enzyme, which releases the tRNA and the aberrant peptide. As the trans-translation mechanism utilizes the peptidyl-transferase activity of the stalled ribosomes to free the tRNA (as opposed to peptidyl-tRNA drop-off), the need for Pth to recycle such tRNAs is bypassed. Thus, we hypothesized that tmRNA may rescue a defect in Pth. The findings of the experiments detailed in this thesis show that SsrA rescues a defect in Pth by reducing the peptidyl-tRNA load on Pth. (ii) Evidence for a role of initiation factor 3 in recycling ribosomal complexes stalled on mRNAs in Escherichia coli. Specific interactions between ribosome recycling factor (RRF) and EF-G mediate disassembly of post-termination ribosomal complexes for new rounds of initiation. The interactions between RRF and EF-G are also important in peptidyl-tRNA release from pre-termination complexes. Unlike the post-termination complexes (harboring tRNA), the pre-termination complexes (harboring peptidyl-tRNA) are not recycled by RRF and EF-G in vitro, suggesting participation of additional factor(s) in the process. Using a combination of biochemical and genetic approaches, we show that, 1. Inclusion of IF3 with RRF and EF-G results in recycling of the pre-termination complexes; 2. IF3 overexpression in Escherichia coli LJ14 rescues its temperature sensitive phenotype for RRF; (3) Transduction of infC135 (encoding functionally compromised IF3) in E. coli LJ14 generates a ‘synthetic severe’ phenotype; (4) The infC135 and frr1 (a promoter down RRF gene) alleles synergistically rescue a temperature sensitive mutation in peptidyl-tRNA hydrolase in E. coli; and (5) IF3 facilitates ribosome recycling by Thermus thermophilus RRF and E. coli EFG in vivo and in vitro. These lines of evidence clearly demonstrate the physiological importance of IF3 in the overall mechanism of ribosome recycling in E. coli. (iii) The role of RRF in dissociating of pre-termination ribosomal complexes stalled during elongation Translating ribosomes often stall during the repetitive steps of elongation for various reasons. The stalled ribosomes are rescued by the process of trans-translation involving tmRNA (SsrA) or by a factor mediated dissociation of the stalled ribosome into its subunits leading to the drop-off of the peptidyl-tRNA. The mechanistic details of how the factor mediated dissociation is carried out, is not well studied. Studies described in the above section have highlighted the role of RRF in dissociating stalled pre-termination complexes. However, the in vivo studies in this area have been limited for lack of defined pre-termination complexes. Two in vivo systems based on translation of AGA minigene and the ung gene (EcoUngstopless) transcripts were designed. Evidence is presented to show that translation of both of these transcripts is toxic to E. coli because of the accumulation of the transcript specific stalled pre-termination complexes. Availability of these model systems has allowed us to address the role of RRF in dissociating stalled ribosomes. We show that RRF rescues stalled ribosomes on these constructs and its overexpression can rescue the toxicity. The physiological importance of this observation is highlighted by the rescue of AGA minigene inhibitory effect on λimmP22 hybrid phage growth upon RRF overexpression.

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.