The major objectives of this proposal are: (1) to understand the relationship between the unique structural features and the highly specific properties of eukaryotic and prokaryotic initiator methionine tRNAs and (2) to use mutagenesis of tRNA genes to isolate a variety of stable animal cell lines which carry nonsense suppressors. Our approach is to use site-specific mutagenesis to generate different classes of mutant tRNAs: (i) those derived from eukaryotic and prokaryotic initiator tRNAs which have lost one, two or all three of their unique features and (ii) those derived from elongator methionine tRNA which have acquired one or more features unique to initiator tRNA? The structure and properties of these and other mutant tRNAs will be studied. Specific questions are: Will the initiator tRNA mutants still act in initiation? Will some of them now act as elongators? Will some of the mutants derived from elongator tRNA acquire one or more of the properties special to initiator tRNAs? Toward this objective nine different mutants of human initiator tRNA lacking one, two or all three unique features have been isolated and are being currently analyzed. With E. coli initiator tRNA, one of the unique features has been shown to be important in initiation whereas the other is important in preventing the tRNA from working as an elongator. Along with these interesting functional changes there are structural changes in the mutant tRNAs. We propose to determine the nature of these structural changes and the relationship of these to the change in function. Use of mutagenesis approach for studying the highly specific recognition of E. coli initiator tRNA by met-tRNA formylase has indicated that the recognition site for the enzyme on the tRNA may be localized at the end of the acceptor stem. We shall investigate this by further mutagenesis of both the initiator and elongator species of E. coli methionine tRNA. The enzyme will be purified and used for studies on the topology of enzyme-met-tRNA interactions. Attempts will also be made to clone the gene for the enzyme and to crystallize the enzyme. Finally, we propose to extend our recent work on the generation of stable monkey kidney cell lines carrying an inducible amber suppressor tRNA gene to include ocher and opal suppressors and to also obtain mouse cell lines carrying such suppressors.
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