The broad goal of the project is to elucidate the molecular mechanisms of initiation and control of protein synthesis in eukaryotic cells. Special focus is on the structure/function of mammalian and yeast initiation factors involved in Met-tRNAi or mRNA binding to ribosomes, on the phosphorylation of initiation factors as a general method to regulate translation rates, and on the mobilization or masking of mRNPs. Specifically, human eIF2 assembly and GTP binding will be elucidated by site-directed mutagenesis of the three subunit's cDNAs already cloned in the laboratory. Chemical crosslinking and cDNA cloning will be directed to elucidating the structure of the large multi-subunit protein complex, eIF3. The yeast Prt1 protein will be purified by stimulating a Prtl-defective lysate,and the putative initiation factor will be characterized biochemically and genetically. A detailed study of mammalian eIF4F, eIF4A, eIF4B and eIF3 binding to mRNAs will employ methods to locate the positions of bound proteins on the mRNA. mRNA derivatives with fluorescent reporter groups at specific positions along the RNA will be used to measure the kinetics of 40S ribosome or initiation factor movement during the mRNA scanning process. Phosphorylation of initiation factors is implicated in malignant transformation and cell growth control. The multiple sites of phosphorylation of eIF4B and eIF3 will be identified and altered to Ala by site-directed mutagenesis of the cDNAs to prevent phosphorylation, and the effects of the mutant forms will be assessed by transfection studies. Furthermore, the specific kinases responsible for eIF4B and eIF3 phosphorylation will be identified, since enhanced phosphorylation of these proteins correlates with mitogenic activation of mammalian cells. Lastly, the mechanism whereby mRNPs are functionally masked or repressed will be addressed. The 50 kDa mRNP protein that inhibits the translation of beta-globin mRNA in rabbit reticulocytes will be characterized. Its cDNA will be cloned on the basis of partial amino acid sequence information, and the detailed mechanism of its inhibitory activity will be studied in transfected cells and in highly purified translation assay systems. Thus by a combination of in vitro studies with purified components and in vivo approaches utilizing recombinant DNA techniques, detailed mechanisms of initiation factor action and translational control will be elucidated.
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