Animal DNA-dependent RNA polymerases: X. General enzymatic properties of purified calf thymus RNA polymerases AI and B

Abstract

Summary The requirements of pure calf thymus DNA-dependent RNA polymerases AI and B for DNA, divalent cations and ionic strength were analyzed. It was found, using «common calf thymus DNA, that neither the preference for native versus denatured DNA, nor the ratio of stimulation by Mn2+ versus Mg2+, nor the ionic strength optimum could be considered as invariant characteristics of the two types of enzymes. In particular, the ionic strength optimum and the relative stimulating effect of Mn2+ and Mg2+ were highly dependent on the state of the DNA (native or denatured) and on its concentration in the reaction. Studies of incorporation of [γ-32P] nucleoside triphosphates have shown that both AI and B enzymes initiated more RNA chains on denatured DNA than on native «common DNA. ATP and GTP, but not UTP and CTP, were found for both AI and B enzymes at the 5′ end of the RNA chains. However, the ATP/GTP ratio was markedly dependent both on the nature of the divalent cation present in the reaction and on the state of the DNA. The lower template efficiencies of denatured DNA for enzyme AI in the presence of Mn2+ or Mg2+ or for enzymes B in the presence of Mg2+, were related to the synthesis of shorter RNA chains. Unlike the yeast RNA polymerases which have an absolute requirement for single-stranded regions for initiating RNA synthesis, both calf thymus enzymes AI and B can also initiate RNA chains on single-strand nicks, as shown by their ability to transcribe a native «common calf thymus DNA which has been treated with nuclease S1 to remove any single-stranded region. The mechanism of the stimulation of enzymes AI and B brought about by increasing the ionic strength was analyzed. It was shown that the small stimulation observed for enzyme AI, when the ionic strength was increased, was essentially related to a stimulation of the initiation step, while the large stimulation observed for enzymes B was due to a stimulation of both the initiation and elongation steps of RNA synthesis. In addition, our results demonstrate that the initiation of RNA chains is more sensitive to high ionic strength than their elongation. In general, initiation occurs more readily in the presence of Mn2+ than in the presence of Mg2+, and also more readily on denatured than on native «common calf thymus DNA. Km values for the four nucleoside triphosphates, optimum pH, effect of phosphate and pyrophosphate, heat and urea inactivation patterns were also determined for the purified calf thymus RNA polymerases AI and B.