Affinity labeling of Escherichia coli DNA-dependent RNA polymerase with 5-formyl-l-(alpha-D-ribofuranosyl)uracil 5'-triphosphate.
5-Formyl-1-(alpha-D-ribofuranosyl)uracil 5'=triphosphate has been used to affinity label E. coli DNA-dependent RNA polymerase. It is a noncompetitive inhibitor of the enzyme with Ki=0.54 mM. A short preincubation of the enzyme and alpha-fo5UTP is required to achieve maximum inhibition, and the entent of the inhibition is dependent upon the alpha-fo5UTP concentration. When a preincubation mixture of alpha-fo5UTP/enzyme is diluted, the enzyme regains activity with time showing that the inhibition is reversible, presumably occurring by Schiff base formation between an amino group on the enzyme and the formyl group. Upon sodium borohydride reduction of an enzyme/alpha-fo5UTP preincubation mixture the enzyme is irreversibly inhibited. alpha-fo5UTP is more effective in inhibiting the enzyme than alpha-fo5U, and the inhibition is decreased by the presence of ATP, UTP, or GTP in the preincubation mixture, suggesting that inhibition is occurring at a triphosphate binding site. The stoichiometry of binding of alpha-fo5UTP to the enzyme was determined using the gamma-32P-labeled derivative. After a 20-s preincubation of enzyme/alpha-fo5UTP followed by NaBH4 reduction the stoichiometry of binding was 1.1:1 (alpha-fo5UTP bound: inactivated enzyme), and this rose to 2.42:1 after a 10-min preincubation. After a 20-s preincubation the [gamma-32P]-alpha-fo5UTP was shown to be located on the beta subunit of RNA polymerase by cellulose acetate electrophoresis in 6 M urea.
D000345:Affinity Labels; D001665:Binding Sites; D001894:Borohydrides; D004247:DNA; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D046911:Macromolecular Substances; D008274:Magnesium / Q000494:pharmacology; D011485:Protein Binding; D012293:Rifampin / Q000494:pharmacology; D014500:Uracil Nucleotides
D000345:Affinity Labels; D001894:Borohydrides; D046911:Macromolecular Substances; D014500:Uracil Nucleotides; D004247:DNA; D012321:DNA-Directed RNA Polymerases; D008274:Magnesium; D012293:Rifampin