MODIFIED NUCLEOSIDE STRUCTURE FUNCTION RELATIONS--TRNA

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Modified nucleosides impart unique site-specific properties to nucleic acids, yet determining their significance to structure-function relationships has proven difficult. Presumably the evolution of modified nucleosides is connected with their altering of RNA chemistry and structure, and the potential to accomplish this is as varied as are the chemistries and structures of the modifications. An understanding of the unique chemistry, structure and dynamics that the more than 80 naturally occurring modified nucleosides contribute to nucleic acids would itself provide investigators with exciting possibilities for nucleic acid research, as well as basic knowledge of nucleic acid evolution and function. Modified nucleosides are used as anti-viral agents, and have been used to demonstrate new potentials in transcription and the introduction of unusual amino acids in translation. Our long-term objective is to understand the relationship of modified nucleoside chemistry, structure and dynamics to function, particularly with regard to tRNA function in translation. With more than sixty differently modified nucleosides, tRNA is one of the most heterogeneous bio-polymers known. In this study the unique contributions of modified nucleosides to different local structures in yeast tRNA(Phe) and their specific importance to aminoacylation and anticodon-codon recognition will be determined. The importance of modification in relation to the ribose 2'- OH in secondary interactions and metal ion binding features of tRNA structure will be elucidated. Stable isotope enrichment techniques will be combined with modified nucleoside and RNA chemical syntheses. Modified nucleoside-containing RNA sequences, DNA analogues, and tRNA domains of yeast tRNA(Phe) will be produced. Their structures will be determined by NMR and CD spectrometry. We will elucidate the biological importance of specific modified nucleosides to yeast tRNA(Phe) recognition by yeast phenylalanyl-tRNA synthetase (FRS) and tRNA(Phe) interaction with codon. The importance of modified nucleosides to FRS recognition of tRNA(Phe) will be assayed by determining the kinetics of aminoacylation of the mature tRNA in the presence of competing unmodified and selectively modified tRNA stem-loop domains. Biochemical determinations of codon-dependent binding of tRNA(Phe) to ribosomes will be assayed in the presence and absence of competing anticodon stem-loop sequences with and without selected modified nucleosides and deoxyribose substitutions. The structure and dynamics of anticodon-codon interaction will be determined by CD and NMR spectrometry.