Baroudy B M, Moss B
J Biol Chem. 1980 May 10;255(9):4372-80.
A DNA-dependent RNA polymerase has been extracted from vaccinia virions and purified to near homogeneity as judged by glycerol gradient sedimentation and polyacrylamide gel electrophoresis. The native enzyme has a molecular weight of approximately 500,000 and can be dissociated into putative subunits of 140,000, 137,000, 37,000, 35,000, 31,000, 22,000, and 17,000 daltons. Activity was dependent on all four ribonucleoside triphosphates, Mn2+, and a DNA template. Optimal activity occurred at pH 7.9 in the presence of 90 mM KCl or 40 mM (NH4)2SO4. All single-stranded DNAs tested served as templates. By contrast, linear double-stranded DNAs were not effectively transcribed and very low activity was obtained with circular supercoiled DNAs which contain small single-stranded regions. The synthetic alternating copolymer poly(dA-dT), which forms a completely base-paired structure, also was not transcribed, whereas poly(dA,dT) and other random copolymers served as templates. Of the four homopolydeoxribonucleotides, only poly(dC) and poly(dT) were transcribed, suggesting that initiation specifically occurs with purine ribonucleotides. In support of this, higher Km values were obtained for GTP and ATP (333 and 80 micro M, respectively) than for UTP and CTP (22 and 12 micro M, respectively) using a DNA template. The RNA polymerase was inhibited by polyanions but was resistant to rifampicin and alpha-amanitin.
已从痘苗病毒颗粒中提取出一种依赖DNA的RNA聚合酶，并通过甘油梯度沉降和聚丙烯酰胺凝胶电泳判断其已纯化至接近均一状态。天然酶的分子量约为500,000，可解离为推定的分子量分别为140,000、137,000、37,000、35,000、31,000、22,000和17,000道尔顿的亚基。活性依赖于所有四种核糖核苷三磷酸、Mn2+和DNA模板。在90 mM KCl或40 mM (NH4)2SO4存在下，pH 7.9时活性最佳。所有测试的单链DNA均可作为模板。相比之下，线性双链DNA不能有效转录，含有小单链区域的环状超螺旋DNA转录活性非常低。形成完全碱基配对结构的合成交替共聚物聚（dA-dT）也不能被转录，而聚（dA,dT）和其他随机共聚物可作为模板。在四种同聚脱氧核糖核苷酸中，只有聚（dC）和聚（dT）能被转录，这表明起始特异性地发生于嘌呤核糖核苷酸。为此提供支持的是，使用DNA模板时，GTP和ATP的Km值（分别为333和80 μM）高于UTP和CTP的Km值（分别为22和12 μM）。该RNA聚合酶受到聚阴离子的抑制，但对利福平及α-鹅膏蕈碱具有抗性。
