Mechanism of inhibition of RNA polymerase II and poly(adenylic acid) polymerase by the O-n-octyloxime of 3-formylrifamycin SV.
Factors affecting the inhibition of RNA polymerase II from rat liver by the O-n-octyloxime of 3-formylrifamycin SV (AF/013) were investigated. Using either native or denatured calf-thymus DNA as template, almost complete inhibition of RNA polymerase II was observed when AF/013 was added directly to the enzyme. Considerable resistance to AF/013 was observed when RNA polymerase II was preincubated with denatured DNA at either 0 or 37 degrees. However, under similar conditions, no resistance was observed when enzyme was preincubated with native DNA. Only when AF/013 was added to the ongoing reaction using native DNA did a resistance to AF/013 occur. The inhibition of RNA polymerase II by AF/013 was competitive with respect to all four nucleoside triphosphate substrates. The inhibition by AF/013 remaining after enzyme-DNA complex formation also appeared competitive with nucleoside triphosphate levels. The effect of exogenous protein (bovine serum albumin, BSA) on the inhibition of RNA polymerase II was also investigated. BSA reduced the extent of inhibition by AF/013, but did not alter the competitive nature of inhibition. Concurrently, the inhibition of highly purified nuclear poly(A) polymerase from rat liver, a template independent enzyme which incorporates AMP in a chain elongation reaction, was examined. As in the case of RNA polymerase, poly(A) polymerase was inhibited by AF/013 in a manner competitive with the nucleoside triphosphate substrate. The competitive nature of inhibition of RNA polymerase by AF/013 with respect to all four nucleoside triphosphate substrates, before and after enzyme-DNA complex formation, as well as the competitive nature of inhibition of poly(A) polymerase with respect to ATP tend to indicate that the major effect of AF/013 on RNA polymerase II is at the level of the substrate binding as opposed to a specific inhibition of initiation.
D000818:Animals; D002417:Cattle; D002467:Cell Nucleus / Q000201:enzymology*; D002852:Chromatography, Ion Exchange; D004247:DNA; D012321:DNA-Directed RNA Polymerases / Q000037:antagonists & inhibitors*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D009713:Nucleotidyltransferases / Q000037:antagonists & inhibitors*; D011062:Polynucleotide Adenylyltransferase / Q000037:antagonists & inhibitors*; D051381:Rats; D012294:Rifamycins / Q000494:pharmacology*; D012710:Serum Albumin, Bovine / Q000494:pharmacology; D013698:Templates, Genetic; D013997:Time Factors
D012294:Rifamycins; D012710:Serum Albumin, Bovine; D004247:DNA; D009713:Nucleotidyltransferases; D011062:Polynucleotide Adenylyltransferase; D012321:DNA-Directed RNA Polymerases