The overall objective of this proposed research is to elucidate the intermediate steps in the initiation of protein synthesis in eukaryotic cells. The major objectives are to elucidate the mechanism of action of the eukaryotic protein synthesis initiation factors (eIF)-3, 4F, 4A, and 4B in the binding of mRNA to the ribosomes and to identify primary, secondary or tertiary structures in the mRNA that affect the interaction of the mRNA with these factors and determine the efficiency with which the mRNA is translated. The specific aims are: 1) to make specific changes in the 5' ends of mRNAs and determine what effect(s) these changes have on the amounts of eIF-3, 4F, 4A, and 4B required to initiate translation; 2) to identify those structures in the mRNA that determine whether initiation of translation is dependent on a M7G cap structure at the 5' end of the mRNA; 3) to identify the recognition and/or binding sites for eIF-3, 4F, 4A, and 4B on the mRNA; 4) to determine when the initiation process eIF-4F, 4A, and 4B are released from the mRNA; 5) to identify the factors required to initiate translation of uncapped mRNAs, such as poliovirus mRNA, at an internal AUG initiation codon; and 6) to determine whether eIF-4B or the cap binding subunits of eIF-4F or (iso)4F are phosphorylated and if so, what effect phosphorylation has on the activities of these factors. These studies will be carried out in the wheat germ system with highly purified preparations of eIF-3, 4A, 4B, 4C, 4F and (iso)4F and with unmodified and modified form of several mRNAs. The mRNAs will be prepared by in vitro transcription of plasmids containing the cDNAs for .- and -globin mRNA, satellite tobacco necrosis virus (STNV) RNA, alfalfa mosaic virus (AMV) RNA 4, barley .-amylase mRNA, poliovirus RNA (Lansing strain, truncated), and Kozak hp7 CAT mRNA. Primer extension and nuclease and/or chemical protection analysis will be used to identify the binding sites for eIF-4F, 4A and 4B or mRNA and antibodies will be used to determine when the factors are released from the mRNA. The results of this investigation will provide additional information will regard to intermediate steps in the initiation of protein synthesis in plants as well as other eukaryotic cells. The results obtained will provide additional insights into the regulation of gene expression at the level of translation.
