Purification and properties of vaccinia virus DNA-dependent RNA polymerase.

Abstract

Vaccinia virus DNA-dependent RNA polymerase has been solubilized from purified virions and purified over 2500-fold by chromatography on DEAE-cellulose and phosphocellulose with a 64% yield. RNA synthesis catalyzed by the purified enzyme requires the presence of a DNA template, a divalent cation (Mn2’), and the four ribonucleoside triphosphates. The polymerase activity is inhibited by actinomycin, by monovalent salt concentrations in excess of 0.1 M, by N-ethylmaleimide, and by pyrophosphate, yet is insensitive to rifampicin and to a-amanitin. Single-stranded DNAs are the most effective templates for the vaccinia RNA polymerase. The enzyme is inactive with duplex DNAs, including vaccinia DNA. Superhelical DNAs do serve as template for vaccinia polymerase, yet their template activity is abolished by relaxation of the DNA by DNA topoisomerase. The binding of RNA polymerase to DNA is also most effective with single-stranded DNAs; duplex DNAs are inert. The binding of polymerase to superhelical DNAs may be eliminated by nicking-closing enzyme. The RNA product synthesized in the presence of single-stranded DNA is of large size (23 S), while RNA made in the presence of superhelical DNAs is very small (<4 S), suggesting that the polymerase binds to and transcribes short single-stranded regions present in superhelical DNAs. Although seemingly incapable of displacing and reading through DNA duplex structures, the vaccinia polymerase is capable of transcribing through an RNA:DNA hybrid with concomitant displacement of the RNA strand.