Characterization of phage-Xp10-coded RNA polymerase.

A bacteriophage-coded RNA polymerase was isolated from bacteriophage-Xpl0-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 M, 96000. 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°C and 8.0, respectively. The Km of ATP is 26 μM. DNA, MgCl2 and four ribonucleotides were required for enzyme activity. If ATP alone was present, half of the Xpl0 RNA polymerase activity was retained. The enzyme activity was inhibited by KC1, 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 Xpl0 RNA polymerase upon Xpl0 DNA were analyzed by DNA/RNA hybridization and polyacrylamide-agarose composite gel electrophoresis. The enzyme could specifically transcribe the late region of Xpl0 genome and produce two RNA bands. [ABSTRACT FROM AUTHOR]