Molecules of tRNA translate codons in mRNA into the amino acid sequences of protein. An essential feature of this process is the enzymatic aminoacylation of tRNA. Our long term interest is to determine which aspects of tRNA structure determine aminoacylation specificity. Our general approach is to use computer analysis of tRNA sequences to predict likely determinants, and to test those predictions with mutant tRNAs expressed in Escherichia coli. The aminoacylation specificity of tRNA is ascertained by determining the amino acid inserted in a protein corresponding to the codon read by the mutant tRNA. Two considerations have led us to adopt this approach. First, computer analysis of tRNA sequences reduces the very large number of possible specificity determinants to a testable number. Second, in vivo analysis of tRNA reflects the outcome of cognate and noncognate aminoacylating enzymes competing for the tRNA. The power of this approach has been demonstrated by our successes in elucidating the determinants for several tRNAs. Furthermore, because determinants of tRNA aminoacylation are often conserved, our structure-function analysis, which is most feasible in E. coli, should have broad applicability. During the proposed granting period, we will: 1. Define the aminoacylation determinants of several E. coli tRNAs using nonsense suppressor tRNAs. 2. Develop and implement a system to study missense suppressor tRNAs in E. coli. Having realized that a given tRNA often contains determinants in its anticodon and acceptor end, we will develop a missense tRNA system capable of analyzing determinants in both locations simultaneously; this is not possible with nonsense suppressor tRNAs. 3. Genetically characterize the mode of action of determinants distant from the site of catalysis in the acceptor end of tRNA. Since remote (less than 60 Angstroms) determinants affect catalysis, an allosteric change in tRNA and/or the enzyme structure is implied. We will use a genetic approach to investigate this structure-function relationship in tRNA by selecting secondary mutants that compensate for defects in remote determinants.
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