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Integrative structure and function study of the bacterial and human protein synthesis machinery

Final Report Summary - TRANSLATIONMACHINERY (Integrative structure and function study of the bacterial and human protein synthesis machinery.)

The translation regulation of the genome and the associated protein synthesis are fundamental cellular mechanisms of major interest to public health and clinical research. The project TRANSLATIONMACHINERY aimed at providing new-level, comprehensive insights into the structure-function relationship of the ribosome machineries both in bacteria (the targets for antibiotics) and in human. In the cell, this involves a fine-tuned molecular regulation through proteins and ARNs that bind transiently to the ribosome. The key questions to be addressed in this project were:

(i) how does the mRNA structure regulate translation initiation?
(ii) what is the molecular mechanism of the association of the ribosomal subunits during translation initiation and what is the role of the initiation factors in this process?
(iii) what is the specificity of the human ribosome in comparison with bacterial ribosomes?
(iv) how do ribosomes cooperate when bound to the mRNA chain and forming larger assemblies?

For each of these questions we defined clear objectives to work on, and we obtained important results for each of the four objectives, some of which can be considered major breakthroughs in the field. (I) In order to address the structural role of regulatory mRNA’s during the initiation step of protein synthesis we have analysed the role of a ribosomal protein (S1) in the unfolding of the mRNA. We have obtained first 3D reconstruction of bacterial S1-ribosomes which show the binding site of this protein, as well as mutant data that show which domain of protein S1 is contacting the small ribosomal subunit or the mRNA. We also analysed an mRNA ribosome complex from eukaryotes, revealing that a nucleotide sequence exists on the 40S subunit for binding histone mRNAs during translation initiation. Because these interactions complement interactions of the 40S subunit with the generally known Kozak consensus sequence this is of general interest as ribosome-binding sites are widely used for cloning strategies. As to topic (II), we have been able to addresses the long-standing question of the structure and function relationship of the bacterial translation initiation factor IF2, a key GTPase involved in the early steps of bacterial protein synthesis when the initiator tRNA is recruited onto the 30S ribosomal subunit. We obtained crystals of IF2, in the free form and bound with either GDP or GTP, and determined the structure which gives unprecedented insights into the molecular mechanism of nucleotide binding and GTP hydrolysis, revealing notably the molecular basis for the enzymatic mechanism of this GTPase. The IF2 structure highlights for the first time the important role of amino acids in nucleotide binding, and we have extended this study by a detailed functional analysis with regards to the mechanism of ribosome subunit joining, a key event that as a key regulatory step could be a good candidate for new antibiotic developments. In order to address key questions regarding antibiotic specificity and side-effects, we have studied the human ribosome (topic III). This project represents a major challenge in the field, technically and scientifically. We have been able to establish the preparation of human ribosomes as a routine procedure in the research group, obtained first crystals illustrating the high homogeneity of the sample, and determined the atomic structure to ~3 Å resolution using in-house high-end electron microscopes and new high-sensitive cameras. The structure gives unprecedented structural insights into rRNA entities and amino acid side-chains and paves the way for analyzing antibiotic complexes and diseases associated with deregulated protein synthesis. As the first high-resolution structure of the human ribosome this represents a major achievement in the field. Finally, on topic IV, we have obtained 3D reconstructions using electron tomography of the ribosomes actively synthesizing proteins within a larger assembly called polysomes which has allowed a full, molecular description of this huge macromolecular complex (~100 MDa). As an illustration of the scientific output, 17 publications have been made in high-impact journals (among which 2 PNAS, 1 Nature Comm, 1 PLOS Biol., 3 Nucleic Acids Res., 2 Acta Cryst D., 1 TIBS, 1 EMBO J.), one on the high-resolution human 80S ribosome is under review, two more are under preparation, and 39 conference contributions (posters or oral presentations including a series of invited speaker presentations) have been presented by the team members at several international conferences.