An alpha-amanitin-resistant DNA-dependent RNA polymerase II from the fungus Aspergillus nidulans.

An alpha-amanitin-resistant DNA-dependent RNA polymerase II has been purified from the lower eukaryote Aspergillus nidulans to apparent homogeneity by extraction of the enzyme at low salt concentration, polymin P (polyethylene imine) fractionation, binding to ion-exchangers and density gradient centrifugation. By this procedure 0.4 mg of RNA polymerase II can be purified over 6,000-fold from 500 g (wet weight) of starting material with a yield of 25% and a specific activity of 550 units/mg. The subunit composition has been resolved by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulphate and by two-dimensional gel electrophoresis using a non-denaturing gel in the first dimension and a dodecylsulphate slab gel in the second dimension. The putative subunits have molecular weights of 170,000, 150,000, 33,000, 27,000, 24,000, 19,000, 18,000 and 16,000. Only one form of RNA polymerase II could be resolved by electrophoresis. The chromatographic and catalytic properties and the subunit composition of the purified RNA polymerase II are clearly different from RNA polymerase I from A. nidulans but throughout comparable with other class II enzymes. It differs from all other class II enzymes by its insensitivity towards the toxin alpha-amanitin, even at concentrations up to 400 micrograms/ml, and appears to be unable to bind O-[14C]methyl-gamma-amanitin at a concentration of 10 micrograms/ml of the toxin. We conclude that the purified RNA polymerase from A. nidulans is a real, but exceptional, type of the class II RNA polymerases.