The proposed research is concerned with the molecular mechanisms of translation-elongation in the model eukaryote, Saccharomyces cerevisiae. In particular, the mechanism of action of the yeast elongation factor 3 (EF-3) will be studied using purified factors isolated from the wild-type and temperature-sensitive yeast strains. EF-3 stimulates EF-lalpha-dependent binding of aa-tRNA to the ribosomal 'A' site. The stimulatory effect of EF-3 requires nucleotide hydrolysis. Mechanistic studies of EF3 will be conducted using radio-iodinated factors. Ribosome-bound complexes will be isolated by FPLC chromatography. EF-3-dependent increased association of EF-lalpha or release of EF-lalpha:CDP complex will be monitored by following the association of the radioactively labeled factors with the ribosomal fractions. An alternate model for the EF-3 effect on the ribosomal 'A' site binding will be followed by monitoring dipeptide bond formation. Photoaffinity labeled nucleotide will be used for the isolation of the active site peptide of EF-3. Attempts will be made to define the site of interaction of EF-3 on the ribosome by using heterobifunctional cross-linking agents. A possible role of EF-3 in natural message translation will be investigated by using polysomes isolated from a yeast mutant temperature sensitive for EF-3. The gene for EF-3 has been cloned. The cloned gene will be exploited for large-scale isolation of the protein for physical and biochemical studies. In vivo analysis with the cloned gene indicates a function of EF-3 in translational fidelity. Based on these studies, experiments will be designed to investigate the effect of mutational alteration and over-expression of EF-3 on transitional accuracy. Since EF-lalpha is functionally dependent on EF-3, a possible compensatory effect of EF-3 will be analyzed in a strain containing mutant EF-lalpha or EF-2. A physical analog of EF-3 has not been identified in higher eukaryotes. We will search for a functional analog of EF-3 by Southern blot analysis with the cloned EF-3 gene and by Western blot analysis with antibody probe. A comparative analysis of the structure and function of EF-3 with that of other eukaryotic translational factors and ribosomal proteins is expected to define the function of this unique yeast protein in translation. A better understanding if the structure and function of EF-3 may lead to the identification of potential antifungal agents.
