TRANSFER RNA STRUCTURE DURING PROTEIN SYNTHESIS

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Response of gene expression to normal yet changing physiological states and to disease states has requisite recognition of nucleic acid structure by a host of proteins and results in the normal and correct or abnormal and incorrect synthesis of proteins. The ability of nucleic acids to interact specifically and successfully in enzymatic reactions would be expected to depend on pliant conformational recognition of the nucleic acid by the protein in three dimensions. Thus, we need to understand not only nucleic acid structure but also structural flexibility and motional capability, particularly internal motion which is important for mechanisms of catalysis or receptor activation. These areas of investigation are significant to the fundamental understanding of the regulation of gene expression, transcription and post transcriptional processing of RNAs, and to codon recognition and fidelity of translation. The native three-dimensional structure, overall and site specific dynamics of nucleic acids in solution are essentially unknown. This research program is the combined effort of two laboratories to investigate the structural dynamics of native transfer RNA in solution alone and during interaction with various proteins. Carbon-13 and proton nuclear magnetic resonance (NMR) spectrometry will be used to ascertain overall and specific site dynamics of the tRNA structure. Transfer RNA will be 13C-enriched in its methyl groups and at various other locations in vivo, and specific tRNA species purified. Carbon-13 NMR studies of transverse and longitudinal relaxation (T1, T2) and NOE will then be accomplished on these tRNAs at 75.5 MHz (13C) under various conditions. The structure and dynamics of amino-acylated and unacylated species will be compared. Interaction of 13C-methyl enriched tRNA species with purified cognate and non-cognate aminoacyl-tRNA synthetases and elongation factor will be studied by 13C-NMR. Analogous unlabeled tRNA species will be studied by 1H-NMR. To aid in making signal assignments of high field proton resonances, 13C-methyl enriched tRNA species will be studied by 13C-coupled and decoupled 1H-NMR. Key advantages of the methodology of 13C-enrichment are the ability to make unequivocal signal assignments and to assess native structure under physiological conditions using the non-destructive, non-perturbing technology of NMR.