Demonstration of RNA polymerase multiplicity in Trypanosoma brucei. Characterization and purification of alpha-amanitin-resistant and -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 micrograms of the drug/ml. The enzymes had different salt optima, but both were of high Mr (greater than 480,000) and demonstrated the template preference: poly[d(A-T)] greater than denatured DNA greater than 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.
D000546:Amanitins / Q000494:pharmacology*; D000818:Animals; D002852:Chromatography, Ion Exchange; D012321:DNA-Directed RNA Polymerases / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D014346:Trypanosoma brucei brucei / Q000201:enzymology*
D000546:Amanitins; D007527:Isoenzymes; D012321:DNA-Directed RNA Polymerases