Kimura M, Ishiguro A, Ishihama A
Department of Molecular Genetics, National Institute of Genetics, Mishima, Shizuoka 411, Japan.
J Biol Chem. 1997 Oct 10;272(41):25851-5. doi: 10.1074/jbc.272.41.25851.
RNA polymerase II purified from the fission yeast Schizosaccharomyces pombe consists of 10 species of subunit polypeptide. We introduced a histidine cluster tag sequence into the chromosomal rpb1 and rpb3 genes, which encode subunit 1 (Rpb1) and subunit 3 (Rpb3), respectively, and purified the RNA polymerase by Ni2+ affinity chromatography. After stepwise dissociation of the Rpb1- and Rpb3-tagged RNA polymerases fixed on Ni2+-resin by increasing concentrations of urea or guanidium hydrochloride, Rpb2-Rpb3-Rpb11 or Rpb2-Rpb3-Rpb11-Rpb10 complexes were obtained. Since the complex consisting of Rpb2, Rpb3, and Rpb11 cannot be dissociated even after treatment with 6 M urea buffer, we propose that this complex represents a core subassembly of the RNA polymerase II, analogous to the alpha2beta complex in the assembly of Escherichia coli RNA polymerase. Both the Rpb2-Rpb3-Rpb11 complex and the free Rpb1 protein showed DNA binding activity, although the affinity was weaker compared with the intact RNA polymerase.
从裂殖酵母粟酒裂殖酵母中纯化得到的RNA聚合酶II由10种亚基多肽组成。我们将组氨酸簇标签序列分别引入编码亚基1（Rpb1）和亚基3（Rpb3）的染色体rpb1和rpb3基因中，并通过Ni2+亲和层析纯化RNA聚合酶。通过逐步增加尿素或盐酸胍的浓度，使固定在Ni2+树脂上的带有Rpb1和Rpb3标签的RNA聚合酶解离，得到了Rpb2-Rpb3-Rpb11或Rpb2-Rpb3-Rpb11-Rpb10复合物。由于即使在用6 M尿素缓冲液处理后，由Rpb2、Rpb3和Rpb11组成的复合物也不能解离，我们推测该复合物代表RNA聚合酶II的核心亚组件，类似于大肠杆菌RNA聚合酶组装中的α2β复合物。Rpb2-Rpb3-Rpb11复合物和游离的Rpb1蛋白都显示出DNA结合活性，尽管与完整的RNA聚合酶相比亲和力较弱。
