The overall objectives of this research are to elucidate the intermediate steps in the initiation of polypeptide synthesis on cytoplasmic ribosomes of eukaryotic cells. The major objectives are to elucidate the mechanism of action of ATP and eukaryotic initiation factors, eIF-3, eIF-4A, eIF-4B, and eIF-4F in the initiation process and to determine what aspects of the primary, secondary or tertiary structure of mRNA affect the efficiency of translation.
The specific aims are to: 1) learn more about the physical and functional properties of eIF-3, how the 10 subunits of eIF-3 are arranged, which of the subunits are required for assembly and/or activity, and what the functions of the various subunits are; 2) determine whether there is a physical and/or functional relationship between eIF-4B and eIF-4F; 3) determine whether there are differences in the amounts of eIF-3, 4A, 4B, or 4F required for the initiation of translation of various mRNAs and, if so, how these differences correlate with the primary or postulated secondary or tertiary structures of the mRNAs; 4) determine the role that ATP plays in the interaction of eIF-4A, 4B and 4F with mRNA and in the subsequent binding of mRNA to the small ribosomal subunit; 5) determine the recognition and/or binding site(s) for eIF-4A, 4B and 4F on mRNA. These studies will be carried out with highly purified preparations of eIF-2, 3, 4A, 4B and 4F from wheat germ, and a variety of naturally occurring mRNAs (Alpha and Beta globin, satellite tobacco necrosis virus (STNV), large and small turnip yellow mosaic virus (TYMV), large and small tobacco mosaic virus, cow pea strain (CcTMV)). In addition, truncated or modified forms of these mRNAs will be utilized. Crosslinking reagents and monoclonal antibodies will be used to determine how the subunits of eIF-3 are arranged and what the functions of the subunits are. Polyclonal and monoclonal antibodies to eIF-4B and eIF-4F will be used to determine the structural and functional relationship between these two factors. Deoxyoligonucleotides complementary to sequences in the mRNAs will be used to determine what regions of the mRNA are involved in the recognition and/or binding of the initiation factors and in the hydrolysis of ATP.
