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Rôle du facteur d'initiation e/aIF2 dans le démarrage de la traduction chez les Eucaryotes et chez les archées.

Abstract : Eukaryotic and archaeal initiation factors 2 (e/aIF2) are heterotrimeric proteins (αβγ) that supply the small ribosomal subunit with methionylated initiator tRNA, therefore insuring specific recognition of the start codon on mRNA. The structure of aIF2γfrom the archaeon P. abyssi was previously solved in our laboratory. The γsubunit, which forms the core of the heterotrimer, is a close structural homologue of bacterial elongation factor EF1A. However, it displays specific features that could account for its specific role in translation initiation. One of the main goals of this work has been to design an in vitro assay to follow the association between aIF2 and Met-tRNAi Met. This test enabled us to identify determinants of Met-tRNAi Met important for its recognition by aIF2 and to investigate the role of each subunit of aIF2 in tRNA binding. On the one hand, the methionine moiety of initiator tRNA is a crucial determinant for Met-tRNAi Met recognition by aIF2 while the other nucleotidic determinants have only a minor role. On the other hand, the γsubunit alone is able to bind Met-tRNAi Met in a manner similar to that of EF1A. But its affinity for the tRNA molecule is strongly reduced in comparison to the affinity of the intact heterotrimer. Therefore, at least one other subunit must play a role in this interaction. Indeed, the αsubunit is required to have a full tRNA binding while βhas no role in this interaction. Within the αsubunit, domain 3 binds the γsubunit via an idiosyncratic loop located in domain 2 of aIF2γ. Moreover, the αD3γ heterodimer is necessary and sufficient to have an optimal affinity for Met-tRNAi Met. In a second step, we obtained crystals of the intact aIF2αand of a truncated version of αformed of only domains 2 and 3. The structures ofαD2-3 and αwere solved at 2.26 and 3.37 Å resolution, respectively. The three-domain organisation of αis conserved in Eukarya and Archaea. Domains 1 and 2 form a rigid body which is linked to a third mobile domain. Sequence comparisons established that the most conserved regions in aIF2αlie at opposite sides of the protein, within domain 1 and domain 3. Both domains show general RNA binding properties. Thereby, domain 1 could interact with either rRNA or mRNA on the ribosome. Finally, crystals of aIF2αγfrom Sulfolobus solftaricus were obtained and the structure of the heterodimer was solved and refined to 3.0 Å resolution. This structure confirmed biochemical data previously obtained: αD3 connects domain 2 of the γsubunit via the L1 loop of γ. For the first time in an aIF2γstructure, conformation of the two switch regions of γ involved in GTP binding are similar to those encountered in the EF1A:GTP:Phe-tRNAPhe complex. Comparison with the EF1A structure suggests that only the γsubunit of the aIF2αγ heterodimer contacts tRNA. Because the αsubunit markedly reinforces the affinity of the γ subunit for tRNA, a contribution of αto the switch movements observed in the γstructure is considered.
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Submitted on : Tuesday, July 27, 2010 - 3:38:35 PM
Last modification on : Wednesday, July 29, 2020 - 4:10:05 PM
Long-term archiving on: : Thursday, October 28, 2010 - 4:36:20 PM


  • HAL Id : pastel-00001539, version 1



Laure Yatime. Rôle du facteur d'initiation e/aIF2 dans le démarrage de la traduction chez les Eucaryotes et chez les archées.. Génétique des populations [q-bio.PE]. Ecole Polytechnique X, 2005. Français. ⟨pastel-00001539⟩



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