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Analysis of the role of the ribosomal mRNA docking site during translation initiation – STIR

Submission summary

The determination of the three-dimensional structure of the prokaryotic ribosome at high resolution has provided vast amounts of information that allows a better understanding of the function of the ribosome. It is now possible to dissect the different functional states of the ribosome and obtain a dynamic view of translation. Translation initiation is a multi-step process that leads to the formation of a complex where the initiator tRNA recognises the initiation codon of the mRNA in the P-site of the ribosome. During initiation, about 30 unpaired nucleotides of the mRNA are wrapped in a groove that encircles the neck of the 30S subunit. The 5'-untranslated regions of prokaryotic mRNAs often carry complex secondary structures that can alter the kinetics for the assembly of the initiation complex. These structures are also associated with multiple functions. They can be recognized by various molecules, ranging from metabolites, to non-coding RNAs and proteins allowing the level of translation to be regulated in response to specific environmental conditions. The goal of this project is to understand how mRNA structures modulate the different steps leading to the assembly of the initiation complex giving a dynamic picture of translation initiation and its regulation. Recent cryo-EM experiments performed by partner 2, in collaboration with partner 1, have shown that the mRNA encoding ribosomal protein S15 of Escherichia coli can bind to the ribosome in two very different ways. S15 is known to negatively regulate the translation of its own mRNA by binding to a pseudoknot structure that straddles the translation initiation site. The cryo-EM experiments show that in the absence of S15, the mRNA occupies the classical position in the groove around the neck of the 30S in a way that allows the initiator tRNA to bind to the initiation codon in the P-site. However, in the presence of S15, the mRNA is found in a different location, in a precise ribosomal environment at the surface of the platform of the 30S. These experiments indicate that S15 negatively regulates the translation of its own mRNA by preventing the unfolding of the pseudoknot required for the RNA to enter the mRNA groove. This study permits visualisation of the key intermediates of the docking and unfolding processes of structured mRNAs for the first time. Furthermore, comparative sequence and structure analysis of the ribosomal components forming the platform-binding center strongly suggest that the docking area on the 30S subunit is a universal site for transiently anchoring structured mRNAs. From this work, it is hypothesized that the formation of the initiation complex with structured mRNAs involves at least three steps (binding, unfolding and accommodation into the ribosome decoding center) in a precise order. We suspect that these steps can be generalized to many other structured RNAs. The objectives of the project are (i) to characterize the functional significance of the ribosomal mRNA docking site and its protein/RNA components, (ii) to study the ribosome-dependent kinetics of mRNA unfolding during translation initiation, and (iii) to study the mRNA docking site using different representative mRNA structures with the purpose of proving that the mechanism of recognition of structured mRNAs is universal. We hope to obtain a dynamic view of the different steps leading to a productive initiation complex involving structured mRNAs. This will be done using the rpsO mRNA as a model system, and will be generalized to other well known structured mRNAs such as those encoding hydroxyethylthiazole kinase (thiamine biosynthesis), ribosomal proteins (L20, S20, S4) or threonyl-tRNA synthetase. We will use Escherichia coli and the pathogenic bacterium Staphylococcus aureus as model organisms. The network involves three teams with different and complementary expertises, allowing a multidisciplinary approach: partner 1 (P. Romby / P. Dumas) will be involved in the crystallography of defined RNA-RNA or RNA-protein complexes, kinetics studies (fluorescence based approaches), biochemistry (footprinting, toeprinting, mutagenesis, proteomics); partner 2 (B. Klaholz) will perform the structural and functional analyses of ribosomal complexes by cryo-EM. He will also be involved in the crystallography of subcomplexes; partner 3 (M. Springer) will perform the in vivo experiments using molecular genetics and transcriptome techniques. This synergy represents a unique opportunity to unite biochemical, genetic and structural skills, in a common effort to take up this challenge of understanding the molecular mechanism of translation initiation regulation by structured mRNAs.

Project coordination

Pascale ROMBY (Organisme de recherche)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

GIE CENTRE EUROPEEN DE RECHERCHE EN BIOLOGIE ET EN MEDECINE

Help of the ANR 462,000 euros
Beginning and duration of the scientific project: - 36 Months

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