Cotranslational processing mechanisms: towards a dynamic 3D model – RIBO-Dyn

We are using interdisciplinary experimental approaches combining in vivo studies with detailed biophysical studies ranging from supramolecular mass spectrometry, single molecule fluorescence spectroscopy to crystallography. For this project we will focus only on the prokaryote organism; this will be important to integrate the results of the various methods and to build the proof of concept transposable in the future to the most complicate eukaryotic system.

Team 1 has set up several conditions to get very good bacterial non-translating ribosomes which have been used by all teams. Moreover, team 1 has spent a majority of the latest 18 months to engineer constructs to use for the overexpression and purification of bacterial and viral NME proteins with and without specific tags which have been used to reveal binding of bacterial METAP and PDF from V. parahaemolyticus to ribosome in the proximity of the exit tunnel. Our data suggest a possible competition mechanism between METAP and VpPDF for ribosome binding at L24/L22 site that seems to be not the case for E.coli PDF and METAP. This latest data has been confirmed by a fluorescence assay set up by team 3. Indeed, team 3 has developed a fluorescence assay able to monitor the interaction of PDF with the ribosome and the possible competition with other RPBs. Concerning the single molecule experiments, team 4 has made important progress in surface chemistry and studied the fluorophore photostability in PURE System. Finally, team 2, after verification of the purity, homogeneity of all constructs prepared by team 1, has determined by MS analysis under non-denaturing conditions the oligomeric state of all RPBs used and technical optimizations were performed to characterize the ribosome by native MS.

We are only beginning to understand the mechanistic aspects of the cotranslational events. The present project will allow to get details about the existence or not of a concerted and temporal dialogue between three cotranslational processes, processing, folding and mRNA translational rate, for an efficient protein synthesis in the cell. Owing the complexity of these processes and the large size of ribosome, our experiments will present formidable challenges, but we are confident that the future in this field will bring many exciting discoveries at both scientific and technological levels. The results concerning the interplay between ribosome and RPBs will be expected to attract pharmaceutical companies involved in the development of new therapeutic drugs.

3 articles under preparation (2 by team 1&3 and 1 by team 4&1).
1 conference proceeding (OptDiag 2012) involving team 2 and 4, to appear by the end of 2012
2 invited talks (international)
1 invited talk (national)
9 poster presentations
Organization together with T. Meinnel and T. Arnesen of a Jacques Monod Conference “The translating ribosome: towards mature proteins” which took place between 2th and 6th June 2012.

The Royal Swedish Academy of Sciences has decided to award the Nobel Prize in Chemistry for 2009 T.A.Steitz, V. Ramakrishnan and A.E. Yonath for having shown what the ribosome, one of the cell’s most complex machineries, looks like at the atomic level. The ribosome is composed of two subunits, small and large. In the small subunit, transfer RNAs (tRNAs) recognize protein-encoding information on messenger RNA (mRNA) transcribed from the genetic code. The large subunit includes the ribosome’s active site, where proteins are actually assembled by one-at-a-time addition of amino acids. The Nobel Prize winners all used X-ray crystallography to map the ribosome’s structure. The determination of high resolution structures of the stable “core” of this machine, which appears to be structurally similar in all living kingdoms, is one of the major breakthroughs of the past years. An understanding of the ribosome's innermost workings is important for a scientific understanding of life. Concomitantly to this major advance, new proteomics methods, high-throughput protein production combined to crystallization approaches have made a marked impact on the relevance of non-ribosomal proteins that permanently or transiently might associate to the ribosome. Among non-ribosomal proteins, ribosome-associated protein biogenesis factors (RPBs) are molecules acting as soon as a nascent polypeptide reaches the tunnel exit of the ribosome. RPBs are involved in the so-called cotranslation events including protein folding, modifications and/or translocation in various compartments. Altogether, RPBs can be considered as a welcoming committee of the nascent chain emerging from the ribosome. It is still a conundrum how RPBs work on the ribosome for the sake of efficient and rapid translation of any nascent polypeptide chains i.e. it is difficult to envisage that the full set of RPBs interacts simultaneously with one ribosome. To take into account the awareness that coordination is needed a new “concerted model” recently proposed simultaneous action of several of these processes on the ribosome. Although fascinating, this model clearly raises more questions than it answers. In 2008, the Giglione’s group has received from CNRS a small starting grant called PEPS (Exploratory Pluridisciplinary Projects) to start and validate the feasibility of the core of the project presented here and aimed to understand the dynamic process of how nascent chains emerging from the ribosome are supported by protein biogenesis factors to ensure both processing and folding mechanisms. This will be the primary goal of this proposal. To succeed in this ANR proposal, collaboration with the Van Dorsselaer’s group has been developed at the beginning of 2009 and preliminary experiments have conducted confirming the feasibility of the approaches proposed. This collaboration will allow investigating the ribosome-RPBs interactions by supramolecular mass spectrometry, an original application of mass spectrometry which enables the direct detection of noncovalent complexes in the gas phase of the mass spectrometer. Finally, thanks to the recent collaboration with the Fourmy’s and Perronet’ groups, the project will take advantage to a supplementary dimension in the dynamics of protein synthesis using single molecules. Together, we will explore how the ribosome moves along the mRNA and how the primary and secondary structures of mRNA could influence ribosomal relationship linking ribosome processivity and RPBs interactions. In this context, another important complementary goal will be to assess how non-uniform elongation rates along a messenger RNA can influence co-translational folding. The obtained results will give details about the existence or not of a concerted and temporal dialogue between the two explored processes for efficient protein synthesis in the cell.