I5N LABELED TRNA--SYNTHESIS AND STRUCTURE

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tRNAs are ubiquitous small stable molecules that perform several important roles related to the synthesis of proteins, among which are translation of the genetic code, transport of amino acids, regulation of amino acid biosynthesis, inhibition of protein biosynthesis in virally infected animal cells, control of protein synthesis during carcinogenic states, priming for reverse transcriptases from RNA tumor viruses, and aminoacyl transfer during cell wall biosynthesis. The topologies of tRNAs and the factors which dictate topology are clearly critical to all of these functions. The goal of this project is to relate structure and function in tRNAs. E. coli tRNAs regiospecifically labeled with 13C and 15N will be produced in multimilligram quantities using expression vectors which overproduce the molecules in auxotrophic bacterial strains. Undermodified tRNAs will be prepared in a similar manner in auxotrophic strains which also lack activity for pseudouridilate synthase I (hisT), thiolation factor A for s4U (nuvA), dimethylallyl pyrophosphate:tRNA transferase (miaA), and tRNA-(m5U)methyltransferase (trmA). The native and undermodified tRNAs will be studied by nuclear magnetic resonance spectroscopy at 500 MHz using heteronuclear 2-dimensional chemical shift correlation, nuclear Overhauser effects, and relaxation measurements to determine structural and kinetic parameters. Biochemical comparisons of tRNAs will be made for aminoacylation and in vitro transcription. NMR studies of tRNA-protein complexes will be conducted with undermodified E. coli tRNAPhe and the modification enzymes pseudouridylate synthase I and tRNA(m5U)methyltransferase.