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 and mRNA binding to ribosomes and on the phosphorylation of initiation factors as a general method to regulate translation rates. A major aim is to determine the subunit composition and structure of human and yeast eIF3, a complex of 10 or more protein subunits that plays a central role in initiation. Capitalizing on recent cloning of cDNAs and genes encoding these subunits, investigators will express fusion proteins tagged with his6, then purify the eIF3 complex by Ni or glutathione affinity chromatography and analyze the subunit composition. The structure of these complexes will be elucidated by determining subunit-subunit binding interactions by Far-western blotting, the yeast 2-hybrid system, affinity chromatography and/or chemical cross-linking methods. Mutant and suppressor analyses with yeast genes will provide evidence for interactions and help elucidate functional roles. Higher order structures involving eIF3 subunit interactions with other initiation factors, mRNA and/or ribosomes will be probed by similar methods. In addition, tapping mode atomic force microscopy will be applied to elucidate the structures of initiation complexes and a fluorescent-labeled mRNA will be employed to measure the rate of ribosome scanning. Studies of the role of phosphorylation will focus on mammalian eIF4B and eIF3, and on yeast eIF1A. Following the determination of the site of eIF4B phosphorylation by the p70 S6 kinase which is regulated by the rapamycin-sensitive signal transduction pathway, investigators will evaluate the importance of this phosphorylation event for regulating the factor's activity. They also will identify other sites of eIF4B phosphorylation and the kinases involved. eIF3 is known to be phosphorylated on the p116 subunit, although other subunits may be modified as well. They will exploit their knowledge of the primary structures of these proteins to determine sites of phosphorylation and their kinases. Identified sites will be mutated to alanine and aspartate and the mutant forms expressed in transfected cells to elucidate the effects of preventing or mimicking phosphorylation. Identification of the site of eIF1A phosphorylation and expressing appropriate mutant forms as the sole source of this essential protein will be carried out. The studies will extend the understanding of how phosphorylation regulates translation rates.
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