Isolation and characterization of DNA-dependent RNA polymerase III from Leishmania mexicana and inhibition by purine analogs.
A DNA-dependent RNA polymerase has been isolated and characterized from the parasitic flagellated protozoan Leishmania mexicana. The initial stages of purification utilized high-ionic-strength extraction and protamine sulfate treatment. The enzyme was further purified by differential elution by heparin-Sepharose, DEAE-Sephadex, and carboxymethyl-Sephadex chromatography. Analysis of the chromatographically purified RNA polymerase on nondenaturing gels revealed two electrophoretic forms. The enzyme isolated had characteristics of true DNA-dependent RNA polymerase since it required DNA and all four nucleoside triphosphates for synthesis of RNase-sensitive products. Analysis of ammonium sulfate and metal ion optima, as well as relative activities of the enzyme with Mn2+ versus Mg2+, gave results similar to those reported for other RNA polymerase IIIs in eucaryotes. Formycin A triphosphate was found to be a noncompetitive inhibitor of RNA polymerase III, and cordycepin triphosphate was found to be inhibitory, although the exact mode of inhibition was not determined.
Division of Public Health, Morrill Science Center, University of Massachusetts, Amherst 01003-0081.
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Nolan
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Fehr
D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology*; D002845:Chromatography; D012321:DNA-Directed RNA Polymerases / Q000302:isolation & purification*; D003838:Deoxyadenine Nucleotides / Q000494:pharmacology*; D005573:Formycins / Q000494:pharmacology*; D007894:Leishmania mexicana / Q000201:enzymology*; D012320:RNA Polymerase III / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D012265:Ribonucleotides / Q000494:pharmacology*
D000903:Antibiotics, Antineoplastic; D003838:Deoxyadenine Nucleotides; D005573:Formycins; D012265:Ribonucleotides; C032552:formycin triphosphate; C011746:3'-deoxyadenosine 5'-triphosphate; D012321:DNA-Directed RNA Polymerases; D012320:RNA Polymerase III