An α‐Amanitin‐Resistant DNA‐Dependent RNA Polymerise II from the Fungus Aspergillus nidulans

Abstract

An a-amanitin-resistant DNA-dependent RNA polymerase I1 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 I1 can be purified over 6000-fold from 500 g (wet weight) of starting material with a yield of 25% and a specific activity of 550units/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 170000, 150000, 33000, 27000, 24000, 19000, 18000 and 26000. Only one form of RNA polymerase11 could be resolved by electrophoresis. The chromatographic and catalytic properties and the subunit composition of the purified RNA polymerase I1 are clearly different from RNA polymerase 1 from A. nidulans but throughout comparable with other class 11 enzymes. It differs from all other class I1 enzymes by its insensitivity towards the toxin a-amanitin, even at concentrations up to 400 pg/ml, and appears to be unable to bind O-['4C]methyl-y-amanitin at a concentration of 10 pg/ml of the toxin.We conclude that the purified RNA polymerase from A . nidulans is a real, but exceptional, type of the class I1 RNA polymerases.DNA-dependent RNA polymerases have been purified and characterized from a variety of organisms (cf. Roeder [l]), belonging to the higher [2 -51 as well as the lower eukaryotes [6,7]. It appeared to be more difficult to purify RNA polymerases from the lower eukaryotes, like fungi, than higher eukaryotes, due to the difficulties in disintegrating the cell wall. Up to now only a few useful procedures for lower eukaryotes other than yeast are known [7,8]. We were able to disrupt the cell wall of Aspergillus nidulans reproducibly by blending the hyphal mass in liquid nitrogen and to prepare a homogenate suitable for the isolation of RNA polymerase1 [9]. We now present the purification and characterization of RNA polymerase11 from A. niduluns, using the same method of homogenization.Sensitivity towards the toxin a-amanitin is often used as a classification criterion for eukaryotic RNA polymerases [I 01, although large differences in the sensitivity of especially RNA polymerase I1 from various organisms are observed. The SO inhibition level of a-amanitin for mammalian RNA polymerase I1 is as low as 10 -25 ng/ml, while the enzyme from the lower eukaryotes is far less sensitive, e.g. RNA polymerase I1 from yeast is 50% inhibited at 1 pg/ml [ l l ] and the enzyme from the mushroom Agaricus bisporus at 6.8 pg/ml [12].The isolated RNA polymerase from A . nidulans was classified as a class11 enzyme, although it appeared to be insensitive through all stages of its purification towards the toxin a-amanitin, even at high concentr...