Euglena gracilis DNA dependent RNA polymerase II: a zinc metalloenzyme.
Zinc is essential for cellular proliferation. Zinc deficiency of Euglena gracilis results in arrest of cell division and deranges nucleic acid and protein metabolism pointing to a decisive role of zinc in transcription and translation. We have, therefore, investigated the role of zinc in the function of the DNA-dependent RNA polymerases of this organism. Two RNA polymerases from zinc sufficient organisms were purified first by affinity chromatography on a DNA cellulose column and subsequently separated on diethylaminoethyl (DEAE)-Sephadex A-25. The two fractions were characterized as polymerase I and II by their elution pattern from DEAE-Sephadex and sensitivity to alpha-amanitin. RNA polymerase II has a provisional molecular weight of 700 000 and contains an average of 2.2 g=atoms of zinc per mol of enzyme, but not Mn, Cu, or Fe, as measured by microwave emission spectroscopy. Chelating agents, such as 1,10-phenanthroline, 8-hydroxyquinoline, 8-hydroxyquinoline-5-sulfonic acid, and lomofungin, inhibit activity. In contrast, the nonchelating analogues, 1,7-and 4,7-phenanthroline, do not affect activity. Inhibition by 1,10-phenanthroline is instantaneous and fully reversible by dilution. 1,10-Phenanthroline also inhibits RNA polymerase I, suggesting a role of zinc in its function. The demonstration that RNA polymerase II is a zinc enzyme indicates the involvement of zinc in eukaryotic RNA synthesis and serves as a further basis for the definition of the role of this element in eukaryotic cell growth, division, and differentiation.
D000546:Amanitins / Q000494:pharmacology; D000818:Animals; D002614:Chelating Agents / Q000494:pharmacology; D003300:Copper / Q000494:pharmacology; D004257:DNA Polymerase II / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D005056:Euglena gracilis / Q000201:enzymology*; D007501:Iron / Q000494:pharmacology; D007700:Kinetics; D008667:Metalloproteins / Q000378:metabolism; D008970:Molecular Weight; D010618:Phenanthrolines / Q000494:pharmacology; D012319:RNA Polymerase II / Q000302:isolation & purification; D015032:Zinc / Q000494:pharmacology*
D000546:Amanitins; D002614:Chelating Agents; D008667:Metalloproteins; D010618:Phenanthrolines; D003300:Copper; D007501:Iron; D012319:RNA Polymerase II; D004257:DNA Polymerase II; D004259:DNA-Directed DNA Polymerase; D015032:Zinc