Purification and characterization of RNA polymerase II resistant to .alpha.-amanitin from the mushroom Agaricus bisporus

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Abstract
The DNA-dependent RNA polymerase II or B (ribonucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) from the mushroom A. bisporus was purified to apparent homogeneity. The purification procedures involved precipitation with polyethylenimine, selective elution of RNA polymerase II from the polyethylenimine precipitate, ammonium sulfate fractionation, DEAE-cellulose chromatography, CM[carboxymethyl]-cellulose chromatography and exclusion chromatography on Bio-Gel A-1.5 M. With this procedure 11 mg of RNA polymerase II was recovered from 1.5 kg of mushroom tissue. RNA polymerase II from A. bisporus had 12 subunits with the following MW: 182,000, 140,000, 89,000, 69,000, 53,000, 41,000, 37,000, 31,000, 29,000, 25,000, 19,000 and 16,500. Purified RNA polymerase II from A. bisporus was half-maximally inhibited by the mushroom toxin a-amanitin at a concentration of 6.5 .mu.g/ml (7 .times. 10-6 M), which is 650-fold more resistant than mammalian RNA polymerases II. The apparent Ki for the .alpha.-amanitin-RNA polymerase complex was 12 .times. 10-6 M. The activity of purified RNA polymerase II from the mushroom was quite typical of other eukaryotic RNA polymerases II with regard to template preference, salt optima and divalent metal cation optima.