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Purification and Characterization of DNA-dependent RNA Polymerases from Cauliflower Nuclei ¹

^a Department of Botany, University of Georgia, Athens, Georgia 30602

DNA-dependent RNA polymerases were solubilized from nuclei of cauliflower inflorescences and purified by agarose A-1.5m, DEAE-cellulose, DEAE-Sephadex, and phosphocellulose chromatography and sucrose density gradient centrifugation. RNA polymerases I + III were separated from II by DEAE-cellulose chromatography. Subsequent chromatography on DEAE-Sephadex resolved RNA polymerase I from III. RNA polymerases I and II were further purified to high specific activity by phosphocellulose chromatography and sucrose density gradient centrifugation. RNA polymerase I was refractory to -amanitin at 2 mg/ml. RNA polymerase II was 50% inhibited at 0.05 µg/ml, and RNA polymerase III was 50% inhibited at 1 to 2 mg/ml of -amanitin. The enzymes were characterized with respect to divalent cation optima, ionic strength optima, and abilities to transcribe cauliflower, synthetic, and cauliflower mosaic virus DNA templates.

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