Eukaryotic translation termination is triggered by peptide release factors eRF1 and eRF3. Whereas eRF1 recognizes the stop codon and induces hydrolysis of peptidyl-tRNA, eRF3's function has for long been obscure. We recently reconstituted all steps of eukaryotic translation in vitro using purified ribosomal subunits, 9 initiation, 2 elongation and 2 termination factors and aminoacyl-tRNAs on mRNA encoding a tetrapeptide followed by a stop codon. This allowed us to propose a model for eukaryotic termination that accounts for the cooperative action of eRF1 and eRF3 in ensuring fast release of nascent polypeptide. According to this model, binding of eRF1, eRF3 and GTP to pre-termination complexes first induces a structural rearrangement, which is manifested as a two-nucleotide forward shift of the toeprint attributed to pre-termination complexes, that leads to GTP hydrolysis followed by rapid hydrolysis of peptidyl-tRNA. Cooperativity between eRF1 and eRF3 requires their direct binding through their C-terminal domains. The overall objective of this proposal is to further characterize the mechanism of eukaryotic translation termination and to investigate the completely unknown mechanism of the next, final stage of eukaryotic protein synthesis, ribosomal recycling. Fast kinetics techniques (quench-flow and stopped-flow) will be applied to determine rate constants for GTP hydrolysis and peptide release, to identify intermediate steps (e.g. conformational changes in termination complexes) and to define their kinetics in order to establish a complete kinetic frame-work of termination. The interaction between eRF1 and eRF3 will be studied by small-angle X-ray and neutron scattering. Sharply focused mutagenesis combined with detailed functional assays will be employed to determine the mechanism, by which interaction of eRF3 with eRF1 stimulates eRF3 s GTPase activity and its binding to GTP. To obtain a comprehensive structural overview of termination, the positions of tRNA, mRNA and both release factors, and conformational states of SOS ribosomes in pre-termination, post-termination and various termination complexes will be determined using a combination of directed UV cross-linking and cryo-electron microscopy. Our success in reconstituting in vitro initiation, elongation and termination will now allow us to investigate the mechanism of the last stage of protein synthesis, ribosomal recycling. To delineate the mechanism of eukaryotic post-termination ribosomal recycling and to establish the order of events, the factor requirements for all steps in this process (release of deacylated tRNA and mRNA, and dissociation of 808 ribosomes into subunits) will be determined. This approach will yield the first model of eukaryotic ribosomal recycling. Translation termination at premature stop codon (PSC) is a frequent cause of genetic disease. Detailed understanding of the mechanism of termination will facilitate rational development of more efficacious agents for PSC suppression therapy. ? ? ?
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