Supramolecular complexes of aminoacyl-tRNA synthetases in translation machinery (from nematode to human)

Au cours de ce travail de thèse, nous avons étudié l'organisation structurale des aminoacyl-ARNt synthétases (aaRSs), enzymes responsables de la lecture du code génétique, chez l'homme et chez le nématode Caenorhabditis elegans. Ces études ont porté sur l'analyse d'une synthétase individuelle, la méthionyl-ARNt synthétase de C. elegans, sur la caractérisation des complexes multisynthétasiques du nématode, et sur l'organisation subcellulaire des aaRSs dans la cellule humaine. La MetRS-Ce possède une longue extension polypeptidique C-terminale. Elle représente la fusion entre une MetRS minimale bactérienne et une composante du complexe multienzymatiques d'aaRSs (MARS) humain, la protéine p43. Pour déterminer le rôle de ce domaine, deux formes enzymatiques, surproduites chez E. coli, ont été purifiées. Les paramètres cinétiques et j'affinité pour l'ARNt de la forme native et d'une forme délétée de son domaine C-terminal ont été déterminés. L'extension C-terminale de la MetRS-Ce, est un domaine fonctionnel de liaison des ARNt (tRBD). Bien que les tRBD des MetRS-Ce et MetRS-Hs ne soient pas orthologues, ils confèrent à la MetRS les mêmes propriétés séquestrantes de l'aa-ARNt, révélant un phénomène d'évolution convergente. Nous avons recherché l'existence de complexes multisynthétasiques chez le nématode C. elegans. Sur l'arbre phylogénétique des métazoaires, C. elegans appartient à l'embranchement des protostomes, distinct de la branche des deutérostomes conduisant à l'homme. L'analyse d'extraits de C. elegans sur tamis moléculaire a montré que certaines aaRSs, dont la MetRS, se comportent comme des protéines de haute masse moléculaire. Nous avons isolé les partenaires de la MetRS par immunoprécipitation à l'aide d'anticorps dirigés contre la forme tronquée. Ils ont été identifiés par LC-MS/MS. Le complexe MARS-Ce comprend 8 aaRS. Ces résultats permettent de proposer un schéma d'évolution des structures multisynthétasiques, partant de la plus simple, le complexe à deux aaRS de la levure S. cerevisiae, pour aboutir à la plus complexe connue à ce jour, le complexe MARS humain à neuf aaRS. Parmi les protéines associées à MARS-Ce, nous avons identifié le produit du gène mrsp-38, jusqu'à présent sans fonction connue, à la protéine d'échafaudage du complexe. L'analyse d'une souche mutante de C. elegans, portant une délétion au niveau de ce gène, a révélé l'importance de l'intégrité de cette protéine pour l'association des composantes de MARS-Ce. Ce travail avait été initié chez C. elegans car ce ver permet des études fonctionnelles in vivo dans un organisme multicellulaire. En combinant les outils d'extinction du gène endogène par RNAi et d'expression de transgènes après transformation par biolistique, nous avons pu tester in vivo si l'organisation en complexe MARS est essentielle au développement d'un organisme multicellulaire. Nous avons montré que seules les copies de MetRS qui s'associent à MARS-Ce complémentent une inactivation de la copie endogène. Des protéines ribosomales ont été copurifiées avec MARS-Ce. Chez l'homme, MARS-Hs est également associé de façon transitoire à d'autres composants cellulaires, les polysomes et les filaments d'actine. Nous avons montré que l'association aux polysomes n'est observable que dans un contexte de synthèse protéique active, lorsque des cellules HeLa sont en phase exponentielle de croissance. De même, la désorganisation du complexe MARS-Hs par siRNA s'accompagne d'un phénotype de retard de croissance, lié à une diminution de l'efficacité traductionnelle. Ces données révèlent l'importance de l'organisation subcellulaire de l'appareil de traduction des eucaryotes supérieurs.This thesis is dedicated to the study of structural organization of aminoacyl-tRNA synthetases (aaRSs), a family of enzymes responsible for establishing the genetic code, in human and in the nematode Caenorhabditis elegans. ln particular, our studies concerned the analysis of structure-function relationships of MetRS of C. elegans, the characterization of multisynthetase complexes in the nematode, and the deciphering of subcellular organization of aaRS in human cells. MetRS of C. elegans (MetRS-Ce) possesses a long C-terminal polypeptide extension. This enzyme represents the fusion between a minimal bacterial MetRS and a component of human MARS complex, the p43 protein. ln order to determine the role of this domain, we purified two forms of the protein, the full-Iength species and a C-truncated derivative, to determine their catalytic parameters in tRNA aminoacylation reaction and their relative affinity for tRNA. The C-terminal extension of MetRS-Ce is a functional tRNA-binding domain (tRBD). The tRBDs of human and nematode MetRS have distinct structural folds. They are not orthologs but they provide MetRS with similar tRNA-sequestering properties. Thus, functional convergence of human and nematode MetRS is the result of parallel and convergent evolution that might have been triggered by the selective pressure to invent processivity of tRNA handling in translation in higher eukaryotes. We searched for the presence of supramolecular complexes of aaRS in the nematode C. elegans. According to metazoan phylogeny, C. elegans belongs to protostomes, a phylum distinct from the deuterostome branch leading to H. sapiens. The analysis of C. elegans extracts by gel-filtration showed that some aaRS, among which MetRS, behave as high-molecular mass entities. We isolated the partners of MetRS-Ce by immunoprecipitation. They were identified by LC-MS/MS. MARS-Ce complex contains 8 aaRS. Its distinct composition gives insight into possible evolution pathways that led to the emergence of the MARS complex containing 9 aaRS in human, statting From the two-component complex in the yeast S. cerevisiae. Among the proteins associated with MARS-Ce, we identified the product of the mrsp-38 gene of unknown function, to the scaffold protein of the complex. The analysis of a mutant strain carrying a deletion in the 3'end of this gene revealed the importance of integrity of this protein for association of MARS-Ce components. This work was initiated in C. elegans in part due to the feasibility of functional studies in vivo in a multicellular organism. By combining tools of extinction of the endogenous gene by RNAi and ectopic expression of transgenic variants of MetRS, we couId test in vivo if the assembly into the MARS complex is essential for growth of a multicellular organism. We found that only copies of MetRS-Ce that associate with MARS-Ce complement the inactivation of the endogenous copy. Ribosomal proteins were copurified with MARS-Ce. ln human, MARS-Hs is also transiently associated with other subcellular components, polysomes and actin filaments. We showed that association with polysomes can only be observed in exponentially growing HeLa cells, in the context of active protein synthesis. Also, disruption of the MARS complex by siRNA is accompanied with a growth retardation phenotype, which is related to a decreased rate of protein synthesis. These data reveal the importance of subcellular organization of translation machinery in higher eukaryotes.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

A2 isoform of mammalian translation factor eEF1A displays increased tyrosine phosphorylation and ability to interact with different signalling molecules

The eEF1A1 and eEF1A2 isoforms of translation elongation factor 1A have 98% similarity and perform the same protein synthesis function catalyzing codon-dependent binding of aminoacyl-tRNA to 80S ribosome. However, the isoforms apparently play different non-canonical roles in apoptosis and cancer development which are awaiting further investigations. We hypothesize that the difference in non-translational functions could be caused, in particular, by differential ability of the isoforms to be involved in phosphotyrosine-mediated signalling

Mammalian translation elongation factor eEF1A2: X-ray structure and new features of GDP/GTP exchange mechanism in higher eukaryotes

International audienceEukaryotic elongation factor eEF1A transits between the GTP- and GDP-bound conformations during the ribosomal polypeptide chain elongation. eEF1A*GTP establishes a complex with the aminoacyl-tRNA in the A site of the 80S ribosome. Correct codon-anticodon recognition triggers GTP hydrolysis, with subsequent dissociation of eEF1A*GDP from the ribosome. The structures of both the 'GTP'- and 'GDP'-bound conformations of eEF1A are unknown. Thus, the eEF1A-related ribosomal mechanisms were anticipated only by analogy with the bacterial homolog EF-Tu. Here, we report the first crystal structure of the mammalian eEF1A2*GDP complex which indicates major differences in the organization of the nucleotide-binding domain and intramolecular movements of eEF1A compared to EF-Tu. Our results explain the nucleotide exchange mechanism in the mammalian eEF1A and suggest that the first step of eEF1A*GDP dissociation from the 80S ribosome is the rotation of the nucleotide-binding domain observed after GTP hydrolysis

Evidence for the formation of an unusual ternary complex of rabbit liver EF-1α with GDP and deacylated tRNA

AbstractEukaryotic translation elongation factor1α is known to interact in GTP-bound form with aminoacyl-tRNA promoting its binding to the ribosome. In this paper another ternary complex [EF-1α*GDP*deacylated tRNA], never considered in widely accepted elongation schemes, is reported for the first time. The formation of this unusual complex, postulated earlier (FEBS Lett. (1996) 382, 18–20), has been detected by four independent methods. [EF-1α*GDP]-interacting sites are located in the acceptor stem, TψC stem and TψC loop of tRNAPhe and tRNALeu molecules. Both tRNA and EF-1α are found to undergo certain conformational changes during their interaction. The ability of EF-1α to form a complex with deacylated tRNA indicates that the factor may perform an important role in tRNA and aminoacyl-tRNA channeling in higher eukaryotic cells

Conservation of exposed residues in putative calmodulin binding domain of the eEF1A homologues

<p><b>Copyright information:</b></p><p>Taken from "Multiple molecular dynamics simulation of the isoforms of human translation elongation factor 1A reveals reversible fluctuations between "open" and "closed" conformations and suggests specific for eEF1A1 affinity for Ca-calmodulin"</p><p>http://www.biomedcentral.com/1472-6807/8/4</p><p>BMC Structural Biology 2008;8():4-4.</p><p>Published online 25 Jan 2008</p><p>PMCID:PMC2275276.</p><p></p

Multiple molecular dynamics simulation of the isoforms of human translation elongation factor 1A reveals reversible fluctuations between "open" and "closed" conformations and suggests specific for eEF1A1 affinity for Ca-calmodulin-1

Ctory 9, cyan – trajectory 10, magenta – trajectory 11, yellow – trajectory 12, orange – trajectory 13. Black – average curves.<p><b>Copyright information:</b></p><p>Taken from "Multiple molecular dynamics simulation of the isoforms of human translation elongation factor 1A reveals reversible fluctuations between "open" and "closed" conformations and suggests specific for eEF1A1 affinity for Ca-calmodulin"</p><p>http://www.biomedcentral.com/1472-6807/8/4</p><p>BMC Structural Biology 2008;8():4-4.</p><p>Published online 25 Jan 2008</p><p>PMCID:PMC2275276.</p><p></p

Multiple molecular dynamics simulation of the isoforms of human translation elongation factor 1A reveals reversible fluctuations between "open" and "closed" conformations and suggests specific for eEF1A1 affinity for Ca-calmodulin-5

<p><b>Copyright information:</b></p><p>Taken from "Multiple molecular dynamics simulation of the isoforms of human translation elongation factor 1A reveals reversible fluctuations between "open" and "closed" conformations and suggests specific for eEF1A1 affinity for Ca-calmodulin"</p><p>http://www.biomedcentral.com/1472-6807/8/4</p><p>BMC Structural Biology 2008;8():4-4.</p><p>Published online 25 Jan 2008</p><p>PMCID:PMC2275276.</p><p></p

Multiple molecular dynamics simulation of the isoforms of human translation elongation factor 1A reveals reversible fluctuations between "open" and "closed" conformations and suggests specific for eEF1A1 affinity for Ca-calmodulin-2

<p><b>Copyright information:</b></p><p>Taken from "Multiple molecular dynamics simulation of the isoforms of human translation elongation factor 1A reveals reversible fluctuations between "open" and "closed" conformations and suggests specific for eEF1A1 affinity for Ca-calmodulin"</p><p>http://www.biomedcentral.com/1472-6807/8/4</p><p>BMC Structural Biology 2008;8():4-4.</p><p>Published online 25 Jan 2008</p><p>PMCID:PMC2275276.</p><p></p

Multiple molecular dynamics simulation of the isoforms of human translation elongation factor 1A reveals reversible fluctuations between "open" and "closed" conformations and suggests specific for eEF1A1 affinity for Ca-calmodulin-4

Ctory 9, cyan – trajectory 10, magenta – trajectory 11, yellow – trajectory 12, orange – trajectory 13. Black – average curves.<p><b>Copyright information:</b></p><p>Taken from "Multiple molecular dynamics simulation of the isoforms of human translation elongation factor 1A reveals reversible fluctuations between "open" and "closed" conformations and suggests specific for eEF1A1 affinity for Ca-calmodulin"</p><p>http://www.biomedcentral.com/1472-6807/8/4</p><p>BMC Structural Biology 2008;8():4-4.</p><p>Published online 25 Jan 2008</p><p>PMCID:PMC2275276.</p><p></p