Purification and characterization of a DNA-dependent RNA polymerase from vaccinia virions

Abstract

A DNA-dependent RNA polymerase has been extracted from vaccinia virions and purified to near homogeneity as judged by glycerol gradient sedimentation and polyacrylamide gel electrophoresis. The native enzyme has a molecular weight of approximately 500,000 and can be dissociated into putative subunits of 140,000, 137,000, 37,000, 35,000, 31,000, 22,000, and 17,000 daltons. Activity was dependent on all four ribonucleoside triphosphates, Mn2+, and a DNA template. Optimal activity occurred at pH 7.9 in the presence of 90 mM KCl or 40 mM (NH4)2SO4. All single-stranded DNAs tested served as templates. By contrast, linear double-stranded DNAs were not effectively transcribed and very low activity was obtained with circular supercoiled DNAs which contain small single-stranded regions. The synthetic alternating copolymer poly(dA-dT), which forms a completely base-paired structure, also was not transcribed, whereas poly(dA,dT) and other random copolymers served as templates. Of the four homopolydeoxribonucleotides, only poly(dC) and poly(dT) were transcribed, suggesting that initiation specifically occurs with purine ribonucleotides. In support of this, higher Km values were obtained for GTP and ATP (333 and 80 micro M, respectively) than for UTP and CTP (22 and 12 micro M, respectively) using a DNA template. The RNA polymerase was inhibited by polyanions but was resistant to rifampicin and alpha-amanitin.