Mécanisme de formation du complexe de démarrage de la traduction chez les Archées

Abstract : A cell is subjected to different internal and external stimuli and must adapt quickly to fulfill its functions. This involves a fine regulation of gene expression. This occurs at the transcriptional level, but also at the translational level. Translation has three phases: initiation, elongation and termination. Selection of the start codon and therefore the choice of the mRNA reading frame is performed during translation initiation. From a kinetic point of view, translation initiation is the rate limiting step. Thus, it appears as a prime target for translational control.In archaea, initiation of translation involves a macromolecular complex containing the small subunit of the ribosome, mRNA, an initiator methionyl tRNA (Met-tRNAiMet) and three initiation factors (aIF1, aIF1A and aIF2). Interestingly, each of three initiation factors has an ortholog in eukaryotes.Archaeal and eukaryotic tRNAi have highly conserved bases A1-U72, at the extremity of the acceptor stem. This base pair was shown to be important for discrimination of initiator tRNAs from elongator tRNAs. In addition, other studies suggest the importance of the geometry of the A1-U72 pair for the initiator identity of those tRNAs. At the beginning of my thesis, no structural information was available to explain how the characteristics of the A1-U72 pair were involved in the selection of the initiator tRNA. At first, my thesis work involved the construction of an E. coli strain using as only source of tRNAi, a bacterial variant of tRNA initiator (tRNAfMet) having a base pair A1-U72 (tRNAfMetA1-U72). The use of this strain allowed us to obtain large quantities of purified tRNAfMetA1-U72. In addition, the crystal structure of this tRNAfMetA1-U72 has been determined at 2.8 Å of resolution. An unusual arrangement of bases A1 and U72 was observed.All archaeal translation initiation actors being available in the laboratory, a study of the archeal translation initiation complex by cryo-electron microscopy was achieved. The study identified two conformations of the tRNAi. In the first complex, both conformations (IC0-Premote (5.3 Å resolution) and IC1-Pin (7.5 Å resolution)) allowed us to propose a model for the accommodation of the initiator tRNA during start codon recognition.Finally, I was also interested in the role of the aIF1 factor. Availability of 3D structures, assembly models and alignments of the archeal aIF1 sequences allowed us to identify amino acids or regions that could be involved in ribosome binding and/or in the selection of initiator tRNA. In order to study the involvement of these regions, I have developed a method to study the binding of aIF1 to the small ribosomal subunit using fluorescence anisotropy. This study highlights two basic residues of aIF1 involved in binding to the ribosome. On the other hand, the roles of aIF1 in the selection of the start codon and in the stabilization of initiation complex on the mRNA were studied by the ribosome footprint method also called « toeprint ».
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Auriane Monestier. Mécanisme de formation du complexe de démarrage de la traduction chez les Archées. Biochimie, Biologie Moléculaire. Université Paris-Saclay, 2016. Français. ⟨NNT : 2016SACLX047⟩. ⟨tel-01513313⟩

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