We propose to study the following areas: (1) Examination of rate-limiting elongation in Escherichia coli as a means of controlling protein synthesis, including looking at the mechanism, asking if a postulated mathematical description is general for other messages and codons, varying the total tRNA concentration in cells to see what effects this has and if uncharged tRNA plays a role in regulation, testing mutant tRNAS for translational efficiency, determining what amino acid is incorporated when rate-limiting an elongation step, seeing if methods of perturbing fidelity affect rate-limiting elongation, analyzing the partial growth defect of phage MS2 on Su6+ cells, characterizing a cloned gene from Su6+ cells which led to a bizarre phenotype, completing analysis of our new method for measuring aminoacylated tRNA levels in vivo, and examining a new feedback loop mechanism from the rate of protein synthesis to tRNA aminoacylation levels. (2) Determination of codon recognition by E. coli tRNA isoacceptors using protein synthesis in vitro directed by sequenced mRNA or DNA (in a coupled system). Site-specific incorporation of a radioactive amino acid into a known position in a protein encoded by a known codon will permit a quantitative assessment of specific tRNA function, including tRNA-Ser and tRNA-Leu species, possible effects of mRNA superstructure, reading context, and preferences for one isoacceptor over another. (3) Effect of ribosomal mutations on tRNA coding properties, including ribosomal """"""""flexibility"""""""" mutants, strains which fail to grow tRNA mutants of phage T4, and relaxed mutants; these experiments will also test our model of """"""""translational hierarchy"""""""" of tRNAs. (4) Codon recognition properties of mutant tRNAs and nutritionally obtained undermodified tRNAs, which will help assess the contribution of various modifications to function. (5) Exploration of misreading in vitro under tRNA dependent conditions, including determining substituted amino acids, testing amino acid starvation in vitro, and mixing extracts of ribosomal and tRNA-synthetase deficient mutants. The experiments proposed in this application should further our understanding of tRNA structure and function, and of the mechanism and regulation of protein synthesis, with possible implications for our understanding of aging and/or cancer.
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