Fabisz-Kijowska A, Dullin P, Walerych W
Biochim Biophys Acta. 1975 Apr 16;390(1):105-16. doi: 10.1016/0005-2787(75)90013-1.
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 (NN4)2SO4, was insensitive to alpha-amanitin and was extremely labile. Enzyme II eluted at 0.25 M (NH4)2SO4 was inhibited by alpha-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 omega-aminobutyl-Sepharose column. After omega-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 alpha-amanitin, by chromomycin A3, daunomycin, ethidium bromide and actinomycin D. Rifampin and rifamycin SV do not inhibit the enzyme. Synthetic copolymers were also effective as templates.
已开发出一种从黑麦胚胎中纯化可溶性DNA依赖性RNA聚合酶（EC 2.7.7.6）的方法。通过超声处理用高盐提取溶解的酶，经DEAE-纤维素色谱分离产生两种活性。酶I在0.15M硫酸铵中洗脱，对α-鹅膏蕈碱不敏感且极其不稳定。酶II在0.25M硫酸铵中洗脱，被α-鹅膏蕈碱抑制。然而，DEAE-葡聚糖凝胶色谱产生三种DNA依赖性RNA聚合酶。酶I对鹅膏蕈碱有抗性，而酶II和III被这种毒素抑制。在DEAE-纤维素上部分纯化后，聚合酶II通过在ω-氨基丁基-葡聚糖凝胶柱上进行疏水色谱进一步纯化。经过ω-氨基丁基-葡聚糖凝胶色谱后，酶II稳定，以变性DNA为模板比以天然DNA为模板时活性更高。纯化的RNA聚合酶II的活性取决于所添加的DNA、锰离子和镁离子，并且其最大活性需要ATP、GTP、CTP和UTP。转录除了被α-鹅膏蕈碱抑制外，还被放线菌素A3、柔红霉素、溴化乙锭和放线菌素D抑制。利福平和平阳霉素不抑制该酶。合成共聚物也可作为模板有效发挥作用。
