Purification of RNA polymerase from actinomycin producing and nonproducing cells of Streptomyces antibioticus

Abstract

DNA-dependent RNA polymerase has been purified approximately 700-fold from 12-h-old cells of Streptomyces antibioticus and 400-fold from 48-h cells. Both enzymes appear nearly homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both enzymes possess subunits corresponding to the [beta], [beta]', and [alpha] subunits of Escherichia coli RNA polymerase but no band corresponding to the [sigma] subunit was observed on polyacrylamide gels. Moreover, neither enzyme appears to have [sigma] activity as judged by the rifampicin and heparin challenge assays using T4 DNA as template. In addition to the [beta], [beta]', and [alpha] subunits, electrophoresis of the polymerase from 12-h cells reveals a 45,000 Mr protein which is present at a level of 0.40 mol/mol of [beta] + [beta]'. The polymerases from 12- and 48-h S. antibioticus cells differ slightly in their template specificity, with the 48-h polymerase showing a slightly greater preference for calf thymus DNA as compared with several other native DNAs which were tested. Further, the polymerase from 48-h cells was slightly more active with poly (dA-dT) (relative to calf thymus DNA) than was the polymerase from 12-h cells. Neither polymerase was capable of catalyzing actinomycin-resistant transcription.