RNA Polymerase III from Drosophila hydei Pupae

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Abstract
DNA-dependent RNA polymerase III, which is usually highly resistant to .alpha.-amanitin, was purified from D. hydei pupae. The enzyme is insensitive to .alpha.-amanitin concentrations up to 0.1 .mu.M; at 100 .mu.M .alpha.-amanitin the enzyme activity is inhibited by .apprx. 10%. Then enzyme was extracted at low ionic strength and purified to homogeneity by 6 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 sulfate electrophoresis in polyacrylamide gels. The enzyme was resolved into putative subunits of MW 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 eukaryotic 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 RNA polymerase III.