Saccharomyces cerevisiae DNA-dependent RNA polymerase III: a zinc metalloenzyme.
Yeast nuclear RNA polymerase III was purified by batch adsorption to phosphocellulose, followed by ion-exchange chromatography on DEAE-Sephadex and affinity chromatography on DNA-Sepharose. Polyacrylamide gel electrophoresis of the purified enzyme showed a single protein band which contained polymerase activity. The molecular weight estimated by sedimentation velocity centrifugation in a glycerol gradient was 380 000. Enzyme activity was inhibited 50% at 0.1 mM 1,10-phenanthroline and 100% of 1.0 mM, but was restored when 1,10-phenanthroline was removed by dialysis. Enzyme activity was not inhibited by 7,8-benzoquinoline, a nonchelating structural analogue of 1,10-phenanthroline. These results strongly suggest that inhibition of enzyme activity occurs by the formation of a reversible enzyme-zinc-phenanthroline ternary complex. The zinc content, measured by atomic absorption spectroscopy, was 2 g-atoms per mol of enzyme. Zinc was not removed from the enzyme by gel filtration on Sephadex G-25, by passage through Chelex-100 resin, or by dialysis against buffer containing 1,10-phenanthroline. Enzyme-bound zinc was removed by dialysis after denaturation of the enzyme with heat and sodium dodecyl sulfate. Enzyme-bound zinc did not exchange with free zinc. These results establish yeast nuclear RNA polymerase III as a zinc metalloenzyme.
D002467:Cell Nucleus / Q000201:enzymology; D012321:DNA-Directed RNA Polymerases / Q000302:isolation & purification; D007700:Kinetics; D008970:Molecular Weight; D012320:RNA Polymerase III / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D015032:Zinc / Q000494:pharmacology
D012321:DNA-Directed RNA Polymerases; D012320:RNA Polymerase III; D015032:Zinc