Isolation and characterization of two 5-fluorouracil-substituted Escherichia coli initiator methionine transfer ribonucleic acids.
Escherichia coli initiator methionine tRNA labeled in vivo with 5-fluorouracil (FUra) has been isolated and characterized. The tRNA, with essentially all its uracil and uracil-derived minor bases replaced by FUra, was purified by sequential chromatography, first on diethylaminoethylcellulose (DEAE-cellulose), at pH 8.9, followed by chromatography on Sepharose 4B, using a reverse salt gradient, then on DEAE-Sephadex A-50, and finally on benzoylated DEAE-cellulose. The last step resolved two FUra-substituted tRNAfMet-iso-accepting species, each with a specific activity over 1500 pmol/A260. Kinetic analysis shows both are aminoacylated at the same rate; apparent KmS for the two are 0.92 and 0.94 microM, compared with 1.7 microM for normal tRNAfMet. Chromatographic differences between the two forms of fluorinated tRNAfMet persist after aminoacylation, and the two tRNAs are not interconverted by denaturation and renaturation. The isoacceptors have nearly identical nucleoside composition, and both contain 7-methylguanosine and 2'-O-methylcytidine as the only modified nucleosides. Analysis of complete RNase T1 digests of the two methionine tRNAs shows that they differ in only one oligonucleotide. The sequence 20FpApGp, derived from the dihydrouridine loop and stem region, which is found in one of the isoaccepting forms of the tRNA, is replaced by an oligonucleotide containing adenine and guanine, but no FUra in the other. A modified FUra, with the properties of a 5-fluoro-5,6-dihydrouracil derivative, is detected in this tRNA. 19F NMR spectra of the two species of FUra-substituted initiator tRNA show 9-10 resolved resonances for the 12 FUra residues incorporated. The spectra differ primarily in the shift of one peak in the form lacking the sequence 20FpApGp, from 4.8 ppm downfield from free FUra (= 0 ppm) to 14.9 ppm upfield from the standard.
D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000235:genetics*; D005472:Fluorouracil / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D009705:Nucleosides / Q000032:analysis; D012329:RNA, Bacterial / Q000302:isolation & purification*; D012343:RNA, Transfer / Q000302:isolation & purification* / Q000378:metabolism; D012346:RNA, Transfer, Amino Acyl / Q000302:isolation & purification; D012358:RNA, Transfer, Met; D006163:Ribonuclease T1 / Q000378:metabolism
D009705:Nucleosides; D012329:RNA, Bacterial; D012346:RNA, Transfer, Amino Acyl; D012358:RNA, Transfer, Met; C024988:tRNA, formylmethionine-; D012343:RNA, Transfer; D006163:Ribonuclease T1; D005472:Fluorouracil