依赖于DNA的RNA聚合酶与体外转录的研究 Ⅰ.酵母变异株20B-12 RNA聚合酶A和C的纯化及鉴定

从酵母变异株20B-12经过超声波处理、硫酸铵沉淀、DEAE纤维素和磷酸纤维素层析等步骤,纯化依赖于DNA的RNA聚合酶A和C,得到聚丙烯酰胺凝胶电泳均一的条带。其中不含DNase、RNase 和蛋白酶活力,无内源DNA。测定了 RNA 聚合酶A和C对α-鹅膏荤碱的敏感性。酶A在α-鹅膏荤碱为400μg/ml时,活性受到抑制,而酶C在该浓度时,几乎不受抑制。(NH_4)_2SO_4对酶A的最适浓度为20mM,对酶C有二个最适浓度,分别为40mM和240mM。无二价金属离子Mn~(2+)或Mg~(2+),酶A和C几乎无活力。两种酶最适Mn~(2+)浓度均为2.5mM,Mg~(2+)浓度均为5mM。两种酶以热变性小牛胸腺DNA为模板,测活性均较天然小牛胸腺DNA为模板时高。

STUDIES ON DNA-DEPENDENT RNA POLYMERASE AND TRANSCRIPTION IN VITRO I. PURIFICATION AND CHARACTERIZATION OF RNA POLYMERASE A AND C FROM MUTANT 20B-12 OF SACCHAROMYCES CEREVISIAE

DNA-dependent RNA polymerase A and C were purified by means of sonication, ammonium sulfate precipitation, chromatography on DE 52 cellulose and P 11 cellulose. The obtained polymerase A and C were homogenous as analyzed on poly- acrylamide gel electrophoresis and devoid of DNase, RNase, proteinase(s) and endo- genous DNA. Polymerase A and C were characterized based on their chromatography behaviours. sensitivities to α-amanitin and ammonium sulfate requirements. Enzyme C was eluted from phosphocellulose with lower concentration of ammonium sulfate followed by enzy- me A. Enzyme C resistant to α-amanitin of 400 μg/ml which inhibited enzyme A. The optimal concentration of ammonium sulfate for enzyme A and C activity was uniphasic (20 mM) and biphasic (40 mM and 240 mM) respectively. Both enzymes required 2.5 mM Mn~(2+) or 5mM Mg~(2+) for maximal activities and transcribed denatured calf thymus DNA more efficiently than native DNA.