Isolation and purification of RNA polymerases from rye embryos

Abstract

A procedure has been developed for the purification of soluble DNA-dependent RNA polymerase (EC 2.7.7.6) from rye embryos. The enzyme solubilized by high salt extraction with sonication and resolved by DEAE-cellulose chromatography yields two activities. Enzyme I eluted at 0.15 M (NH 4) 2SO 4, was insensitive to α-amanitin and was extremely labile. Enzyme II eluted at 0.25 M (NH 4) 2SO 4 was inhibited by α-amanitin. However, DEAE-Sephadex chromatography yields three DNA-dependent RNA polymerases. Enzyme I is resistant to amanitin, while II and III enzymes are inhibited by this poison. Partially purified on DEAE-cellulose, polymerase II was further purified by hydrophobic chromatography on an ω-aminobutyl-Sepharose column. After ω-aminobutyl-Sepharose chromatography, enzyme II was stable and was more active with denatured than with native DNA as template. The activity of purified RNA polymerase II is dependent on the DNA, Mn 2+ and Mg 2+ added and requires ATP, GTP, CTP and UTP for its maximum activity. Transcription is inhibited besides by α-amanitin, by chromomycin A 3, daunomycin, ethidium bromide and actinomycin D. Rifampin and rifamycin SV do not inhibit the enzyme. Synthetic copolymers were also effective as templates.