Characterization of phage-Xp10-coded RNA polymerase.
A bacteriophage-coded RNA polymerase was isolated from bacteriophage-Xp10-infected Xanthomonas campestris pv. oryzae. The enzyme was purified to homogeneity through precipitation by poly(ethylene glycol) and chromatography on DEAE-cellulose, heparin--Sepharose 4B and blue-dextran--Sepharose 4B. It is composed of a single polypeptide of Mr96,000. The enzyme preferred denatured Xp10 DNA, calf thymus DNA, host bacterium DNA and poly[d(A-T)] as templates. The optimal concentration of MgCl2 is 16 mM. The optimal temperature and pH are 37 degrees C and 8.0, respectively. The Km of ATP is 26 microM. DNA, MgCl2 and four ribonucleotides were required for enzyme activity. If ATP alone was present, half of the Xp10 RNA polymerase activity was retained. The enzyme activity was inhibited by KCl, spermidine, actinomycin D, heparin, blue dextran and ethidium bromide; it was resistant to rifampicin and streptovaricin. N-Ethylmaleimide did not affect the enzyme activity. The transcription site and product of Xp10 RNA polymerase upon Xp10 DNA were analyzed by DNA/RNA hybridization and polyacrylamide-agarose composite gel electrophoresis. The enzyme could specifically transcribe the late region of Xp10 genome and produce two RNA bands.
D000255:Adenosine Triphosphate / Q000494:pharmacology; D001435:Bacteriophages / Q000201:enzymology*; D002412:Cations; D002845:Chromatography; D004247:DNA / Q000378:metabolism; D004269:DNA, Bacterial / Q000378:metabolism; D004279:DNA, Viral / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D013095:Spermidine / Q000494:pharmacology; D013379:Substrate Specificity; D013698:Templates, Genetic; D014158:Transcription, Genetic; D014974:Xanthomonas
D002412:Cations; D004269:DNA, Bacterial; D004279:DNA, Viral; D000255:Adenosine Triphosphate; D004247:DNA; D012321:DNA-Directed RNA Polymerases; D013095:Spermidine