REGULATION OF EUKARYOTIC PROTEIN SYNTHESIS INITIATION

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Protein synthesis in eukaryotes represents a major control point for the expression of genetic information and accounts for a substantial fraction of cellular energy expenditure. An understanding of protein synthesis and its regulation is central to an understanding of cell growth as well as the action of and cellular defenses against viruses. The long-term objectives of this research project are to understand the mechanism by which initiation of protein synthesis occurs, focussing on the rate- limiting step wherein mRNA becomes bound to the ribosome, and the regulation of this process. The specific aims to be pursued during the current grant period involve the initiation factors of the eIF-4 group which catalyze this step. The first aim deals with the structure and function of eIF-4E, the mRNA cap-binding protein, the complexes it forms with other eIF-4 factors, its binding and release from the 48S initiation complex, and the nature of internal initiation. The second aim is to understand the mechanism by which phosphorylation of eIF-4E at Ser-53 stimulates protein synthesis. The third aim is to determine the structure of eIF-4F, which will entail completing the primary structure of the p220 subunit and examining the interaction of p220 with other eIF- 4 polypeptides. The fourth aim is to complete the cloning and sequencing of the eIF-4E gene and to characterize its upstream regulatory elements. These studies will make use of traditional biochemical and molecular biological methodology as well as a variety of tools developed during the previous grant period, including in vitro transcription/translation vectors, recombinant phage containing portions of the p220 cDNA and eIF- 4E gene, mammalian cell expression vectors to express variant proteins as well as antisense RNA, the ability to arrest initiation of protein synthesis at specific stages and the measurement of association constants by fluorescence quenching.