RNA polymerase III from Drosophila hydei pupae. Purification and partial characterization.

Abstract

DNA-dependent RNA polymerase III, which is usually highly resistant to alpha-amanitin, has been purified from Drosophila hydei pupae. The enzyme is insensitive to alpha-amanitin concentrations up to 0.1 microM; at 100 microM alpha-amanitin the enzyme activity is inhibited by approximately 10%. The enzyme was extracted at low ionic strength and purified to homogeneity by six purification steps; 0.1--0.2 mg enzyme/kg pupae could be obtained. The subunit composition of the enzyme was determined after sucrose-gradient centrifugation by sodium dodecyl sulphate electrophoresis in polyacrylamide gels. The enzyme was resolved into putative subunits of molecular weight 154 000, 135 000, 62 000, 58 000, 38 000, 32 000, 31 000, 27 200, 26 500, 21 500 and 17 500. This subunit composition is in general accord with that of eucaryotic class III enzymes. The catalytic properties (salt-activation profile, ratio of activity with denatured DNA to that with native DNA) and the order of elution of the enzyme from DEAE-cellulose with respect to RNA polymerase II agree with the classification of the isolated enzyme as an RAN polymerase III.