DEMONSTRATION OF RNA-POLYMERASE MULTIPLICITY IN TRYPANOSOMA-BRUCEI - CHARACTERIZATION AND PURIFICATION OF ALPHA-AMANITIN-RESISTANT AND ALPHA-AMANITIN-SENSITIVE ENZYMES

We have isolated, characterized and substantially purified two distinct RNA polymerase activities from the flagellate protozoan parasite Trypanosoma brucei. RNA polymerases from this organism were resolved poorly on DEAE-Sephadex, but could be separated with CM-Sephadex. One form was totally resistant to .alpha.-amanitin, whereas the second was 50% inhibited by 10-20 .mu.g of the drug/ml. The enzymes had different salt optima, but both were of high Mr (> 480,000) and demonstrated the template preference: poly[d(A-T)] > denatured DNA > native DNA, and both were more active with Mn2+ than with Mg2+. The amanitin-resistant enzyme, polymerase R, was partially purified by chromatography on CM-Sephadex, DEAE-Sephadex and heparin-Sepharose. This enzyme was very labile, and activity yields were around 9%; after purification, one or two protein bands could be discerned after electrophoresis under non-denaturing conditions, but about 20 polypeptides were resolved on denaturing gels, including a major component (not thought to be part of the enzyme) of Mr 65,000. Polymerase S, sensitive to low .alpha.-amanitin concentrations, was more extensively purified, with an 18% recovery, and yielded a single major band with two minor ones after native gel electrophoresis. Analysis under denaturing conditions permitted a possible subunit structure for this enzyme to be ascribed.