STRUCTURAL BASIS OF TRANSFER RIBONUCLEIC ACID FUNCTION

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High-resolution 19F nuclear magnetic resonance (NMR) will be used to probe the solution structure of 5-fluorouracil (FUra)-substituted Escherichia coli tRNA. 5-Fluorouridine is incorporated biosynthetically into tRNA, replacing uridine and uridine-derived minor bases, yielding fully functional molecules containing highly sensitive local probes. These are distributed throughout the helical and loop regions of the tRNA and can serve as reporters of structural changes in all parts of the molecule. Three fluorinated tRNAs have been purified and the 19F NMR spectra obtained at 282 MHz resolve virtually all incorporated FUra residues. Assignment of the 19F resonances will permit the effects of aminoacylation, and the interaction of tRNA with cations, proteins, etc. to be studied and interpreted. A start has been made on peak assignments and this work will continue, making use of several approaches including studies with molecular fragments, chemical modification, enzymatic replacement of specific regions of the tRNA, oligonucleotide binding etc. To aid in the interpretation of 19F NMR spectra, synthetic oligo- and polynucleotides containing FUra in different sequence contexts will be prepared. The chemical shifts and relaxation of FUra residues in helical structures and in random coils will be examined to determine the effects of secondary structure, neighboring FUra residues, fraying of helices, and the nature of neighboring bases. Specific interaction of cognate and non-cognate aminoacyl-tRNA synthetases with tRNA will also be examined by 19F NMR to test the proposed formation of a covalent adduct between the enzyme and the uracil or modified uracil at position 8 in the tRNA. Because uridine-derived minor bases are replaced by FUra, the role of modified bases in the initiation of protein synthesis and codon recognition will also be investigated. FUra-substituted E. coli tRNALys, unlike most other tRNAs, is not very active in protein synthesis. This tRNA will purified and the structural basis for the inactivity will be investigated. Recent evidence suggests that the antitumor effects of FUra and its cytotoxicity may in part be use to FUra incorporation into cellular RNA. A study of the consequences of FUra incorporation into the RNA of bacteria, in which incorporation occurs more readily than in mammalian cells, should provide a model for understanding the action of fluoropyrimidines.