CAREER: Mechanistic Investigation of Nucleoside Hypermodification in tRNA

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9733746 Iwata-Reuyl TECHNICAL Modification of transfer RNA (tRNA) is fundamental to expression of the genetic code, its apparent importance is implied by both the phylogenetic conservation and the rich structural diversity. This study is designed to understand the biosynthesis of queuosine, a modified nucleoside ubiquitous throughout the Bacteria and Eukarya. Queuosine possesses an unusual 7-deazaguanosine core structure, occurs specifically at the wobble position of tRNAıs coding for asparagine, aspartic acid, histidine, and tyrosine, which suggests a role in modulating translational fidelity and/or efficiency. More specifically, the molecular mechanism involving the enzyme S-adenosylmethionine:tRNA ribosyltransferase-isomerase (QueA), which catalyzes the penultimate step of queuosine biosynthesis and the transfer of ribosyl from the co-factor S-adenosylmethionine (AdoMet) to a queuosine-tRNA precursor is studied. The experiments include studies of purified recombinant enzymes for their regio- and stereochemistry and reaction kinetics with the natural substrates and analogs, and do structural studies. The biosynthesis and function of the 7-deazaguanosine class of hypermodified tRNA nucleosides, such as glycosylated derivatives of queuosine and the hypermodified nucleoside archaeosine is also studied. The educational focus is placed on encouraging the participation of undergraduates in biochemical research through an honors research thesis program, which is a pilot program with an attempt to replace and to revitalize the current departmental honors degree requirements. 2. NON-TECHNICAL. Molecular interactions involving transfer RNA (tRNA) are fundamental to expression of the genetic code. It is in the correct attachment of amino acids to tRNA, and subsequent protein synthesis through codon-anticodon pairing of the messenger RNA (mRNA) with the tRNA, that the physical manifestation of genetic information as protein is realized. A characteristic structural feature of tRNA is the presen ce of an astonishing diversity of modified nucleosides, many of which are highly conserved among disparate organisms. Modification can range from relatively minor changes to complex hypermodification, which results in radical structural changes to the nucleoside and requires multiple steps to complete. This study constitutes a broad set of experimental approaches designed to develop a detailed understanding of a critical step in the biosynthesis of the hypermodified nucleoside queuosine, which occurs exclusively at the first position (wobble position) of the anticodon in tRNAıs coding for the amino acids asparagine, aspartic acid, histidine, and tyrosine. Its specific location in the anticodon suggests a role in modulating the fidelity and/or efficiency of protein synthesis, for which further experimental support is needed. The educational component focuses on increasing the participation of undergraduates in biochemical research through the design and implementation of an honors research thesis program, which serves to test the feasibility of instituting a general requirement, in lieu of the current departmental honors degree, for the Chemistry and Biochemistry degrees offered by the department.