Purification and characterization of a mutant tRNA nucleotidyltransferase.
tRNA nucleotidyltransferase has been extensively purified from a mutant strain of Escherichia coli which displays greatly decreased AMP incorporation, but normal CMP incorporation. The defect in AMP incorporation is retained throughout the purification of the mutant protein. The mutant protein behaves identically to the wild-type protein with regard to elution position on various chromatographic columns, and both have similar molecular weights of about 50000. The defect in the mutant protein is accentuated by the use of yeast tRNA rather than E. coli tRNA-C--C as substrate, by decreased pH, by increased ionic strength and by decreased divalent cation concentration. Substitution of MN2+ for Mg2+ greatly increases the relative activity of the mutant enzyme. In all these cases, CMP incorporation by the mutant enzyme remains the same as the wild-type enzyme. The Km values of the mutant enzyme for its tRNA and triphosphate substrates are unchanged, and the mutant protein is as stable as the wild type with respect to temperature inactivation. These results strongly suggest that the mutation is in the structural gene for tRNA nucleotidyltransferase, and that the mutation probably does not affect the overall structure of the mutant protein, but only a localized region near the AMP-incorporating site.
D000249:Adenosine Monophosphate / Q000378:metabolism; D000818:Animals; D002413:Cations, Divalent / Q000494:pharmacology; D003568:Cytidine Monophosphate / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver; D009154:Mutation; D012316:RNA Nucleotidyltransferases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D012329:RNA, Bacterial / Q000378:metabolism; D012343:RNA, Transfer / Q000378:metabolism; D011817:Rabbits; D012441:Saccharomyces cerevisiae; D013379:Substrate Specificity; D013696:Temperature
D002413:Cations, Divalent; D012329:RNA, Bacterial; D000249:Adenosine Monophosphate; D012343:RNA, Transfer; D012316:RNA Nucleotidyltransferases; C023198:tRNA nucleotidyltransferase; D003568:Cytidine Monophosphate