Open AccessAn α‐Amanitin‐Resistant DNA‐Dependent RNA Polymerise II from the Fungus Aspergillus nidulans
Author(s) -
STUNNENBERG Hendrik G.,
WENNEKES Lambertus M. J.,
SPIERINGS Titus,
BROEK Hendrikus W. J.
Publication year - 1981
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1981.tb06310.x
Subject(s) - gel electrophoresis , polymerase , rna polymerase , microbiology and biotechnology , biochemistry , biology , aspergillus nidulans , enzyme , rna , polyacrylamide gel electrophoresis , rna polymerase ii , protein subunit , chemistry , chromatography , promoter , gene expression , gene , mutant
An α‐amanitin‐resistant DNA‐dependent RNA polymerise 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 polymerise II can be purified over 6000‐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 170000, 150000, 33000, 27000, 24000, 19000, 18000 and 16000. Only one form of RNA polymerise II could be resolved by electrophoresis. The chromatographic and catalytic properties and the subunit composition of the purified RNA polymerise II are clearly different from RNA polymerise 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 α‐amanitin, even at concentrations up to 400 μg/ml, and appears to be unable to bind O ‐[ 14 C]methyl‐γ‐amanitin at a concentration of 10 μg/ml of the toxin. We conclude that the purified RNA polymerise from A. nidulans is a real, but exceptional, type of the class II RNA polymerises.