A multi-resolution approach for the structure of translation initiation super-complexes – MASTIC

1-Scientific background and objectives : Protein synthesis is a highly regulated process, in particular at the level of initiation, the limiting step in translation. Such regulations play crucial roles in many cellular processes, rendering initiation of translation an attractive target for new drugs. For initiation to succeed, a series of macromolecular complexes are formed, involving the small ribosomal subunit, the initiator tRNA, the mRNA and several peripheral proteins called initiation factors. The aim of this project is to describe the structures of some of these complexes in eukaryotes, using approaches giving information at various resolutions. This project is particularly attractive since to date, very few structural images of the initiation steps are available, even though many results obtained for elongation and termination of translation have shown that structural studies of such large assemblies are feasible. Moreover, to help the study of eukaryotic complexes, archaeal systems will be used at the beginning of the project as simplified models representative of some sub-complexes involved in eukaryotic initiation of translation. 2-Description of the project, methodology : The final goal of this project is to describe the 43S initiation complex from Saccharomyces cerevisiae, containing the small ribosomal subunit, the initiator tRNA and the eIF1, eIF1A, eIF2, eIF3 and eIF5 factors. The strategy will use a combination of approaches, including X-ray crystallography, mass-spectrometry and cryo-electron microscopy. These approaches, applied to the full complex or to various sub-complexes, will be used iteratively to produce images at the highest possible resolution. For the preparation of complexes, each component will be produced, individually or using co-expression plasmids, in E. coli using recombinant DNA techniques (except the ribosomal subunits which will be prepared from the desired archaeal or eukaryotic cells, Pyrococcus abyssi, Sulfolobus solfataricus and S. cerevisiae). The complexes of interest will be reconstituted in vitro from purified components and crystallization will systematically be attempted. In addition, low resolution strategies will be used. Isolated domains of the factors will be produced and their interactions studied, in order to define the topology of the complexes. The precise regions of contacts between polypeptides will be determined using an original mass-spectrometry-based approach, to be developed in the course of this project, which should be generally applicable for the study of any macromolecular assembly. Obtained data will be used at two levels: (i) in order to define simplified versions of the complexes having a better chance to crystallize and (ii) together with high resolution X-ray structures of individual components in order to allow the three-dimensional (3D) reconstruction of structures from cryo-electron microscopy (cryo-EM) images. Such cryo-electron microscopy structures will allow a first description of the assemblies, but will also in turn be used to guide further crystallization attempts, aimed at obtaining higher resolution structures. 3-Expected results : This project has the ambition to describe complexes of unknown structure involved in the eukaryotic translation initiation process. Among them, the multisubunit factor eIF3 is an important objective, and a high resolution structure is more and more required to help understanding its function. Moreover, an image of the assembly involving eIF2-initiator tRNA, eIF3, eIF1 and eIF5, called the multi-factor complex (MFC), becomes necessary to understand how all these proteic factors contribute to the channeling of the initiator tRNA towards the ribosome. Finally, obtention of images of the 43S complex (MFC+small ribosomal subunit) is a key step towards functional and dynamic studies of the translation initiation process in eukaryotes.