Date of Award
Doctor of Philosophy
Biochemistry, Biophysics and Molecular Biology
Initiator tRNA(,f)('Met) was prepared from Escherichia coli treated with the uracil analog 5-fluorouracil (FUra). FUra-substituted tRNAs have essentually all of their uridine and uridine-derived modified nucleosides replaced by fluorouridines and are useful tools for the investigation of the role of modified nucleosides in tRNA function and for ('19)F NMR studies of tRNA structure. In previous studies, FUra-substituted tRNA(,l)('Val) has been isolated and characterized (Horowitz et al., (1974) J. Mol. Bio. 88, 301-312; Ofengand et al., (1974) J. Mol. Biol. 88, 313-325) and ('19)F NMR spectra have been obtained (Horowitz et al. (1977) J. Biol. Chem. 252, 4418-4420);FUra-substituted tRNA(,f)('Met) was purified by a series of chromatographic steps, first on DEAE-cellulose at pH 8.9 followed by Sepharose 4B using a reverse salt gradient, then DEAE-Sephadex A-50, and finally BD-cellulose. The last step resolved two forms of FUra-tRNA(,f)('Met), designated (A) and (B). These had specific activities of 1600 and 1550 pmole methionine acceptance/A(,260) respectively. Both forms were fully active in accepting methionine. Apparent K(,m)s for (A) and (B) were 0.92 (mu)M and 0.94 (mu)M compared to 1.7 (mu)M for normal tRNA(,f)('Met);Chromatographic differences between (A) and (B) persisted after aminoacylation, and the two tRNAs were not interconverted by denaturation and renaturation. Each of the two FUra-tRNA(,f)('Met) species migrated as a single band when electrophoresed on polyacrylamide gels under denaturing conditions. Both (A) and (B) contained Cm and m('7) Guo, and their nucleoside compositions were almost identical. RNase T(,1)-alkaline phosphatase oligonucleotide maps of the two FUra-tRNA(,f)('Met)s revealed that form (B) contained an oligonucleotide that was missing from form (A). This oligonucleotide was identified as FpApG' (DpApG') in normal tRNA(,f)('Met), which is located at positions 20 - 23 in the D arm. The ('19)F NMR spectrum of FUra-tRNA(,f)('Met) form (A) shows 7 well resolved resonances. The spectrum of (B) was very similar except there were two additional resonances at 5.2 and 7.1 ppm downfield from FUra. Recently, Cotten and Horowitz (personal communication, Dept. of Biochemistry, Iowa State Univ.) have tentatively assigned the resonance at 5.2 ppm to the fluorouridine at position 20 of form (B) and observed a far upfield resonance in the spectrum of form (A) that may be due to a modified fluorouridine.
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/
Diane Churchill Hills
Hills, Diane Churchill, "Purification and partial characterization of 5-fluorouracil-substituted methionine tRNA from Escherichia coli " (1982). Retrospective Theses and Dissertations. 8351.