Demonstration of RNA polymerase multiplicity in Trypanosoma brucei. Characterization and purification of alpha-amanitin-resistant and -sensitive enzymes

• Published in: Biochemical journal 1987-02, Vol.241 (3), p.649-655
• Main Authors: Earnshaw, D L, Beebee, T J, Gutteridge, W E
• Format: Article
• Language: English
• Subjects: alpha -amanitin; Amanitins - pharmacology; Animals; Chromatography, Ion Exchange; DNA-directed RNA polymerase; DNA-Directed RNA Polymerases - antagonists & inhibitors; DNA-Directed RNA Polymerases - isolation & purification; Electrophoresis, Polyacrylamide Gel; Isoenzymes - antagonists & inhibitors; Isoenzymes - isolation & purification; RNA biosynthesis; Trypanosoma brucei; Trypanosoma brucei brucei - enzymology
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/bj2410649

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.