CRYOTRANSLATION

High Resolution cryo-EM Analysis of Ribosome-associated Functions

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 Obiettivo del progetto (Objective)

'Translation of the genetically encoded information into polypeptides, protein biosynthesis, is a central function executed by ribosomes in all cells. In the case of membrane protein synthesis, integration into the membrane usually occurs co-translationally and requires a ribosome-associated translocon (SecYEG/Sec61). This highly coordinated process is poorly understood, since high-resolution structural information is lacking. Although single particle cryo-electron microscopy (cryo-EM) has given invaluable structural insights for such dynamic ribosomal complexes, the resolution is so far limited to 5-10 Å for asymmetrical particles. Thus, the mechanistic depth and reliability of interpretation has accordingly been limited. Here, I propose to use single particle cryo-EM at improved, molecular resolution of 3-4 Å to study two fundamental ribosome-associated processes: (i) co-translational integration of polytopic membrane proteins and (ii) recycling of the eukaryotic ribosome. First, we will visualize nascent polytopic membrane proteins inserting into the lipid bilayer via the bacterial ribosome-bound SecYEG translocon. Notably, the translocon will be embedded in a lipid environment provided by so-called nanodiscs. Second, we will visualize in a similar approach membrane protein insertion via the YidC insertase, the main alternative translocon. Third, as a novel research direction, we will determine the structure and function of eukaryotic ribosome recycling complexes involving the ABC-ATPase RLI. The results will allow, together with functional biochemical data, an in-depth molecular structure-function analysis of these fundamental ribosome-associated processes. Moreover, reaching molecular resolution for asymmetrical particles by single particle cryo-EM will lift this technology to a level of analytical power approaching X-ray and NMR methods. ERC funding would allow for this highly challenging research to be conducted in an internationally competitive way in Europe.'