Isolée d'une bactérie, Streptomyces alboniger, la puromycine est un nucléoside antibiotique naturel présentant une analogie structurale avec l'adénosine terminale de l'extrémité 3' de l'ARNt aminoacylé. Cette similarité confère à cette molécule la faculté de pouvoir s'insérer dans le site A (actif) du ribosome et d'inhiber la synthèse des protéines. Cependant, du fait de la formation d'un produit toxique lors de sa métabolisation, la puromycine n'a jamais été employée à des fins thérapeutiques chez l'homme. Néanmoins, utilisée en tant qu'outil synthétique, elle a largement contribué à une meilleure compréhension du mécanisme du transfert peptidique. Au travers de cette thèse, six analogues carbobicycliques (deux en série ribo et quatre en série 2'-désoxy), mimant de façon optimale les conformations extrêmes nord ou sud de la puromycine, ont été synthétisés puis testés dans le ribosome. Outre confirmer que la présence d'un groupement 2'-hydroxyle améliorait l'activité inhibitrice, ces expériences in vitro ont apporté une preuve que, dans le site actif, le déplacement de l'équilibre conformationnel du ribofuranose de l'adénosine terminale de l'ARNt aminoacylé - analogue structural de la puromycine - en faveur de son conformère nord pourrait être directement impliqué dans la catalyse ribosomale du transfert peptidique. Par ailleurs, un projet annexe sur le développement de nouveaux antipaludiques potentiels a permis la synthèse, en série xylo, de la puromycine et de son métabolite naturel le puromycine aminonucléoside. Ces composés ont été testés sur les souches 3D7 et Dd2 du parasite Plasmodium falciparum.

<p>The stringent response plays a significant role in the survival of bacteria during different environmental conditions. It is activated by the binding of stringent factor (SF) to stalled ribosomes that have an unacylated tRNA in the ribosomal A-site which leads to the synthesis of (p)ppGpp. ppGpp binds to the RNA polymerase, resulting in a rapid down-regulation of rRNA and tRNA transcription and up-regulation of mRNAs coding for enzymes involved in amino acid biosynthesis. The importance of the A-site and unacylated tRNA in the activation of SF was confirmed by chemical modification and subsequent primer extension experiments (footprinting experiments) which showed that binding of SF to ribosomes resulted in the protection of regions in 23S rRNA, the A-loop and helix 89 that are involved in the binding of the A-site tRNA. An in vitro assay showed that the ribosomal protein L11 and its flexible N-terminal part was important in the activation of SF. Interestingly the N-terminal part of L11 was shown to activate SF on its own and this activation was dependent on both ribosomes and an unacylated tRNA in the A-site. The N-terminal part of L11 was suggested to mediate an interaction between ribosome-bound SF and the unacylated tRNA in the A-site or interact with SF and the unacylated tRNA independently of each other. Footprinting experiments showed that SF bound to the ribosome protected bases in the L11 binding domain of the ribosome that were not involved in an interaction with ribosomal protein L11. The sarcin/ricin loop, in close contact with the L11 binding domain on the ribosome and essential for the binding and activation of translation elongation factors was also found to be protected by the binding of SF. Altogether the presented results suggest that SF binds to the factor-binding stalk of the ribosome and that activation of SF is dependent on the flexible N-terminal domain of L11 and an interaction of SF with the unacylated tRNA in the A-site of the 50S subunit.</p>

<p>This thesis addresses different aspects of the question about accuracy of protein synthesis: i) the mechanism of tRNA selection during translation ii) study of ribosomal mutations that affect accuracy and iii) the choice of aminoacyl-tRNA isoacceptors on synonymous codons.</p><p>By measuring the codon reading efficiencies of cognate and near-cognate ternary complexes we demonstrate that in optimal physiological conditions accuracy of substrate selection is much higher than previously reported; that during translation the ribosomal A site is not blocked by unspecific binding of the non-cognate tRNAs which would inhibit the speed of protein synthesis. Our results suggest that there is an asymmetry between initial selection and proofreading step concerning the wobble position, and that binding of non-cognate substrate does not induce GTP hydrolysis on the ribosome.</p><p>The knowledge obtained from the ribosomal mutant strains can be used to explain the general relation between the structure of the ribosome and the mechanism of codon recognition, as well as the streptomycin resistance or dependence phenomenon.</p><p>Our work showed experimentally that the probability for binding certain tRNA to the A site of the ribosome is not based on the simple codon-anticodon base pair matching. In the living cell the availability of cognate tRNAs versus the demand for them (the frequency of codon usage) is finely balanced to ensure critical protein synthesis in stress conditions. We have also discovered a new codon assignment for a specific tRNALeu isoacceptor and detected a base modification in its anticodon, which has not been previously observed. The motivation for the later findings comes from a system biology modeling and the results are an example of an interdisciplinary collaboration.</p>

Ce travail porte sur l'étude de la fidélité de la traduction chez les eucaryotes d'un point de vue mécanistique et génomique. Au cours de ma thèse j'ai développé trois approches :Le premier projet porte sur l'étude du rôle du facteur de l'élongation eEF2 dans le maintien du cadre de lecture. La stratégie associe une mutagénèse aléatoire du gène EFT2 à un criblage phénotypique, elle permet d'isoler des mutants capables d'augmenter ou diminuer l'efficacité de recodage d'une séquence de décalage du cadre de lecture en -1.Le second projet décrit la mise au point d'un système de traduction en molécule unique qui permet d'étudier le ribosome eucaryote. La traduction est initiée grâce à l'IRES CrPV qui a pour caractéristique d'être totalement indépendante des facteurs d'initiation et de l'ARNt initiateur. L'élongation de la traduction est détectée grâce au départ d'un oligonucléotide fluorescent qui est décroché par l'activité hélicase du ribosome. Les résultats de ces expériences constituent une preuve de principe démontrant que l'étude de la traduction eucaryote en molécule unique est possible.Le troisième projet est une étude de génomique comparative qui permet de rechercher des événements de recodage ainsi que d'autres événements non-conventionnels de la traduction dans le génome de la levure Saccharomyces cerevisiae. L'approche est basée sur une recherche d'organisations génomiques conservées au sein de 19 génomes de levures. Les gènes candidats sont testés in vivo grâce à un vecteur double rapporteur. Cette étude a permis de mettre en évidence le gène VOA1 qui a été ensuite caractérisé plus en détails.

The stringent response plays a significant role in the survival of bacteria during different environmental conditions. It is activated by the binding of stringent factor (SF) to stalled ribosomes that have an unacylated tRNA in the ribosomal A-site which leads to the synthesis of (p)ppGpp. ppGpp binds to the RNA polymerase, resulting in a rapid down-regulation of rRNA and tRNA transcription and up-regulation of mRNAs coding for enzymes involved in amino acid biosynthesis. The importance of the A-site and unacylated tRNA in the activation of SF was confirmed by chemical modification and subsequent primer extension experiments (footprinting experiments) which showed that binding of SF to ribosomes resulted in the protection of regions in 23S rRNA, the A-loop and helix 89 that are involved in the binding of the A-site tRNA. An in vitro assay showed that the ribosomal protein L11 and its flexible N-terminal part was important in the activation of SF. Interestingly the N-terminal part of L11 was shown to activate SF on its own and this activation was dependent on both ribosomes and an unacylated tRNA in the A-site. The N-terminal part of L11 was suggested to mediate an interaction between ribosome-bound SF and the unacylated tRNA in the A-site or interact with SF and the unacylated tRNA independently of each other. Footprinting experiments showed that SF bound to the ribosome protected bases in the L11 binding domain of the ribosome that were not involved in an interaction with ribosomal protein L11. The sarcin/ricin loop, in close contact with the L11 binding domain on the ribosome and essential for the binding and activation of translation elongation factors was also found to be protected by the binding of SF. Altogether the presented results suggest that SF binds to the factor-binding stalk of the ribosome and that activation of SF is dependent on the flexible N-terminal domain of L11 and an interaction of SF with the unacylated tRNA in the A-site of the 50S subunit.

This thesis addresses different aspects of the question about accuracy of protein synthesis: i) the mechanism of tRNA selection during translation ii) study of ribosomal mutations that affect accuracy and iii) the choice of aminoacyl-tRNA isoacceptors on synonymous codons. By measuring the codon reading efficiencies of cognate and near-cognate ternary complexes we demonstrate that in optimal physiological conditions accuracy of substrate selection is much higher than previously reported; that during translation the ribosomal A site is not blocked by unspecific binding of the non-cognate tRNAs which would inhibit the speed of protein synthesis. Our results suggest that there is an asymmetry between initial selection and proofreading step concerning the wobble position, and that binding of non-cognate substrate does not induce GTP hydrolysis on the ribosome. The knowledge obtained from the ribosomal mutant strains can be used to explain the general relation between the structure of the ribosome and the mechanism of codon recognition, as well as the streptomycin resistance or dependence phenomenon. Our work showed experimentally that the probability for binding certain tRNA to the A site of the ribosome is not based on the simple codon-anticodon base pair matching. In the living cell the availability of cognate tRNAs versus the demand for them (the frequency of codon usage) is finely balanced to ensure critical protein synthesis in stress conditions. We have also discovered a new codon assignment for a specific tRNALeu isoacceptor and detected a base modification in its anticodon, which has not been previously observed. The motivation for the later findings comes from a system biology modeling and the results are an example of an interdisciplinary collaboration.

Combinatorial protein libraries based on a protein template offer a vast potential for deriving protein variants harboring new receptor specificity while retaining other tem-plate functions to serve as library search-engines, cell-routing sequences and therapeutic domains. This concept was tested with the design and synthesis of protein libraries where short random peptide motifs were embedded directly within the catalytic A subunit of the bacterial ribosome-inactivating protein (RIP) known as Shiga-like toxin 1 (SLT-1). More precisely, a seven amino acid peptide epitope (PDTRPAP) was inserted between residues 245-246 of its A subunit (SLT-1APDTRPAP) and shown to preserve catalytic function while exposing the epitope. SLT-1 A chain libraries harboring tripep-tide and heptapeptide random elements were subsequently constructed, screened and shown to express more than 90% of expected cytotoxic A chain variants. Finally, more than 9,000 purified SLT-1 A chain variants were screened using their ribosome-inactivating function in a cell-based assay to identify mutants that are able to kill human melanoma 518-A2 cells. This search led to the striking discovery of a single chain RIP that displays selectivity for a panel of human melanoma cell lines as well as minimal immunogenicity when injected repeatedly into mice. This directed evolution of a RIP template provides a broad platform for identifying cell type specific cytotoxic agents.

Bacterial cells were once treated as membrane-enclosed bags of cytoplasm: a homogeneous, undifferentiated suspension in which polymers (proteins, nucleic acids, etc.) and small molecules diffused freely to interact with each other. Biochemical studies have determined the molecular mechanisms underlying the biological processes of metabolism, replication and transcription-translation, etc. However, recent advancements in optical techniques armed with fluorescent tags for proteins and nucleic acids have increased our ability to peer into the interior of live bacterial cells. This has revealed an organized layout of multi-protein complexes, or molecular machines, dedicated to specific functions at defined sub-cellular locations; the timing of their assembly and/or rates of their activity being determined by available nutrition and environmental signals from the niche occupied by the organism. In the present study, we have attempted to identify the intracellular location and organization of the molecular machines assembled for protein synthesis (ribosomes), DNA replication (replisomes) and cell division (divisome) in different bacteria. We have used the model system Escherichia coli as well as Helicobacter pylori and mycobacterial strains (Mycobacterium marinum and Mycobacterium smegmatis), which grow at different rates and move to dormancy late into stationary phase Bacterial nucleoid plays a major role in organizing the location and movement of active ribosomes, replisomes and placement of divisome. While the active ribosomes appear to follow the dynamic folds of the bacterial nucleoid during cell growth in E. coli, inactive ribosomes appear to accumulate near the periphery. The replisome in H. pylori was visualized as a sharp, single focus upon SSB and DnaB co-localization in growing helical rods but disassembled into diffused fluorescence when the cells attained non-replicative coccoid stage. Our investigation into mycobacterial life-cycle revealed unique features such as an absence of a dedicated mid-cell site for divisome assembly and endosporulation upon entry into stationary phase. In brief, we present the cell cycle-dependent subcellular organization of molecular machines in bacteria.

Combinatorial protein libraries based on a protein template offer a vast potential for deriving protein variants harboring new receptor specificity while retaining other tem-plate functions to serve as library search-engines, cell-routing sequences and therapeutic domains. This concept was tested with the design and synthesis of protein libraries where short random peptide motifs were embedded directly within the catalytic A subunit of the bacterial ribosome-inactivating protein (RIP) known as Shiga-like toxin 1 (SLT-1). More precisely, a seven amino acid peptide epitope (PDTRPAP) was inserted between residues 245-246 of its A subunit (SLT-1APDTRPAP) and shown to preserve catalytic function while exposing the epitope. SLT-1 A chain libraries harboring tripep-tide and heptapeptide random elements were subsequently constructed, screened and shown to express more than 90% of expected cytotoxic A chain variants. Finally, more than 9,000 purified SLT-1 A chain variants were screened using their ribosome-inactivating function in a cell-based assay to identify mutants that are able to kill human melanoma 518-A2 cells. This search led to the striking discovery of a single chain RIP that displays selectivity for a panel of human melanoma cell lines as well as minimal immunogenicity when injected repeatedly into mice. This directed evolution of a RIP template provides a broad platform for identifying cell type specific cytotoxic agents.

The purpose of this thesis is to analyze the variation in the maximal discrimination of the interaction between cognate and a non-cognate codon and anti-codon (also called the d-value). The variation was analyzed with a multiple regression model with the d-value as the dependent variable and with the codon position and the different mRNA and tRNA bases as independent variables. The result of the analysis not only confirmed earlier studies that the maximal accuracy was highest in the second codon position and lowest in the third codon position but we also found significant relationships and interaction effects.