: The overall goal of the project is to elucidate the detailed molecular mechanisms whereby translational controls contribute to the regulation of gene expression in eukaryotic cells. A broad experimental approach is taken that involves biochemical, biophysical, recombinant DNA and genetic techniques to study the structure, function and regulation of specific initiation factors in human and yeast cells. Two general hypotheses will be tested: i) That elF3 plays an essential organizational/structural role in forming multi-initiation factor complexes on the 40S ribosomal subunit; and ii) That the phosphorylation of elF3 and eIF4B enhances the activities of these factors and stimulates initiation of protein synthesis. The principal investigator will exploit the recent finding that essentially all of the individual subunits of human eIF3 can be expressed as stable tagged or untagged proteins in baculovirus-infected insect cells but not in bacteria, and that an active 5-subunit core complex can be formed. He proposes to generate and purify various sub-complexes of eIF3 for functional and high-resolution structural studies. The core subunits that bind to elF1, 4B, 4G and 5 and to the ancillary eIF3 subunits will be identified. The role of the p33 subunit in the function of yeast eIF3 will be elucidated by a mutant suppressor screen and by determining its physiologic RNA binding target by the SELEX procedure. Investigation of eIF3 subunit mRNA levels by kinetic RT-PCR, protein levels by Western immunoblotting and mRNA translation efficiencies by polysome profile analyses will determine which subunit genes are coordinately regulated or which may be overexpressed, thereby possibly serving other functions. The principal investigator's prior work shows that both eIF3 and eIF4B phosphorylation correlates with translation activation. Work to identify phosphorylation sites and responsible kinases will continue for eIF4B, and be extended to the 4 phosphorylated eIF3 subunits, especially p110 which interacts with many other initiation factors. Effects of phosphorylation will be tested for individual subunits by expression of wild type and site-substituted mutant forms in the baculovirus system, followed by measurement of their interaction with other proteins. The experiments will help elucidate the function and regulation of these key initiation factors.
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