Partial Purification and Characterization of RNA Polymerase from Nuclei of Ehrlich Ascites Tumor Cells

Abstract

DNA-dependent RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase) was partially purified from nuclei of Ehrlich ascites tumor cells by the techniques of sonic disruption in the presence of 0.3 M ammonium sulfate, ultracentrifugation, salt fractionation, and dialysis. The purified enzyme had an absolute requirement for DNA and was inhibited by actinomycin D and other known inhibitors of DNA-dependent RNA polymerase, but not by rifamycin or rifampicin. The four ribonucleoside triphosphates and Mn2+ ions were also required for optimal activity. Optimal concentrations for Mn2+ and Mg2+ ions and (NH4)2SO4 were 2.4 mM, 6.0 mM, and 0.05 M, respectively. All the native and heat-denatured DNA's that were tested served as templates, but the relative activities with the two forms of DNA depended upon the composition of the reaction medium. Of the natural RNA's and 11 synthetic homo- and heteropolyribonucleotides that were tested, the RNA's, poly(A), poly(A,U), and poly(A,C) were slightly active, but less than 5% as efficient as calf thymus native DNA, with 3H-UTP as the labeled substrate.