The goals of this research are to define the sequence and structural elements of tRNA required for its recognition by proteins. Our particular interest is the recognition of E. coli tRNAVal by aminoacyl-tRNA synthetases and by the tRNA modifying enzyme, tRNA (uracil-5- )methyltransferase. Detailed understanding of these interactions will clarify questions concerning the fidelity of protein synthesis, the mechanisms of tRNA modification, and the role of modified nucleosides in tRNA function. The elements of tRNAVal essential for its recognition by valyl-tRNA synthetase (VRS) will be determined by examining the charging kinetics of tRNAVal variants synthesized in vitro by transcription from (cloned) mutant tRNA genes. Minimal requirements for synthetase recognition will be explored with tRNA fragments containing the essential recognition elements of tRNAVal. The structure of wild-type and mutant tRNAVal will be probed by 19F NMR spectroscopy of tRNAs labeled with fluorine by incorporation of 5-fluorouracil (FUra) in place of uracil. Fluorinated tRNAVal is ideally suited for such studies because it remains fully functional and contains highly sensitive 19F probes distributed throughout all the helical stems and loops of the tRNA molecule where they can serve to monitor local structural changes. The 19F NMR spectrum of (FUra)tRNAVal shows a resolved resonance for each of the 14 incorporated FUra residues. Now that each of these has been assigned, we plan to use two-dimensional heteronuclear NOE (HOESY) techniques to examine anticodon loop structure. 19F NMR spectroscopy will also be used to compare the interaction of (FUra)tRNAVal and mutants with cognate and non-cognate synthetases, to help clarify differences in the way these enzymes recognize tRNA. The interaction of tRNAVal with tRNA (uracil-5-)methyltransferase, will also be followed by 19F NMR, to explore conformational changes induced in the tRNA by the enzyme, and to elucidate details of the enzyme mechanisms and of the structures of covalent tRNA-protein reaction intermediates. The results of these investigations will provide basic information on protein-tRNA interactions and on the relation between tRNA structure and function. Study of the consequences of FUra incorporation into tRNA should contribute to a better understanding of the antitumor effects of fluoropyrimidines and their cytotoxicity.
