RNA Polymerase from the Fungus Aspergillus nidulans

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Abstract
The DNA‐dependent RNA polymerase I (or A) from the lower eukaryote Aspergillus nidulans has been purified on a large scale to apparent homogeneity by homogenizing the fungal hyphae in liquid nitrogen, extraction of the enzyme at high salt concentration, precipitation of RNA polymerase activity with polymin P (a polyethylene imine), elution of the RNA polymerase from the polymin P precipitate, ammonium sulphate precipitation, molecular sieving on Bio‐Gel A‐1.5m, binding to ion‐exchangers and DNA‐cellulose affinity chromatography. By this procedure 1.6 mg of RNA polymerase I can be purified over 2000‐fold from 500 g wet weight of starting material with a yield of 30–35%. The isolated RNA polymerase I is stable for several months at −20°C.The subunit composition has been resolved by polyacrylamide gel electrophoresis on two‐dimensional gels, using either non‐denaturing or 8 M urea (pH 8.7) cylindrical gels in the first dimension and sodium dodecyl sulphate slab gels in the second dimension. The putative subunits have molecular weights of 190000, 135000, 63000, 62000, 43000, 29000, 29000, (28000), 16000 and probably 13000 and 12000.Two distinct forms of RNA polymerase I (Ia and Ib) have been resolved by DEAE‐Sephadex A‐25 chromatography showing ample differences in enzymatic properties and subunit pattern.Additional information is given on RNA polymerase II (or B) which appears to be highly insensitive to α‐amanitin at concentrations up to 400 μg/ml.