STRUCTURE AND FUNCTION OF EUKARYOTIC INITIATOR TRNA

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The long range objective of this proposal is to understand the relationship between structure and function of eukaryotic initiator tRNAS, including the molecular mechanisms underlying their specific recognition by components of the protein synthesis machinery. Because of their unique role in initiation of protein biosynthesis, initiator tRNAS from prokaryotic and eukaryotic sources possess biochemical properties which are distinct from those of non-initiator tRNAS. These include (i) specific binding to the initiation factor eIF-2, (ii) direct binding to the P site on the ribosome and (iii) exclusion from binding to the A site on the ribosome. Along with these specific properties, initiator tRNAS possess unique sequence and/or structural features which are not found in non-initiator tRNAS. The immediate objective of this proposal is to understand the relationship between these features and the above properties of eukaryotic initiator tRNAS. The approach to be used involves isolation and analysis of properties of mutant human tRNAS. Site specific mutations will be used to generate different classes of mutant tRNAs: (a) those derived from initiator tRNAs which have lost one, two or all three of the features unique to initiator tRNAs and (b) those derived from the non-initiator methionine tRNA which now possess one, two or all three of the structural features unique to initiator tRNA. The function and detailed properties of these and other mutant tRNAs will be studied in vivo, in the yeast S. cerevisiae and in mammalian cells, and in vitro.