The ultimate aim of the project is to understand the structural rearrangements of the ribosome that underlie the different phases of protein synthesis in bacteria and yeast. To achieve this goal, functional and structural analyses will be fully integrated to maximize synergisms between the different competence areas of the participating groups. In detail, the project aims ? ? (1) To clarify how initiation of protein synthesis takes place with the help of the three initiation factors IF1, IF2 (a G protein) and IF3. ? (2) To study how translocation of tRNAs occurs on the eubacterial ribosome with the help of elongation factor EF-G (a G protein). ? (3) To study the solution structure of eubacterial class-1 release factors to decide if it is similar to the crystal structure of RF2. ? (4) To clarify the role of domain 1 in eubacterial release factors and the role of protein L11 in the large ribosomal subunit in termination of protein synthesis. ? (5) To clarify the mechanism behind ribosomal recycling, catalysed by EF-G and ribosomal recycling factor RRF, back to a new round of initiation. ? (6) To clarify the role of peptide release factor eRF3 (a G protein) in eukaryotic protein synthesis. ? ? All experiments are based on in vitro systems with components of high purity from E. coli and yeast. The approach is to integrate functional assay carried out with biochemical tools, including fast kinetics methods (quench-flow, stopped flow) with structural analysis carried out mainly with cryo-electron microscopy but also with low angle X-ray scattering. Ribosomal complexes will be shipped from Uppsala to Albany and prepared under controlled temperature conditions for cryo-EM studies, including time resolved cryo-EM when applicable. Grids will be imaged with a liquid nitrogen or liquid helium cooled cryo-EM microscope, and the images will be analyzed with the SPIDER image processing system. Between 30,000 and 100,000 images will be collected for 10-12A resolution. The density maps will be analyzed by manual or automated fitting of x-ray structures or by real space refinement to obtain atomic models. ? ?
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