Miyao T, Yasui K, Sakurai H, Yamagishi M, Ishihama A
National Institute of Genetics, Department of Molecular Genetics, Mishima, Shizuoka, Japan.
Genes Cells. 1996 Sep;1(9):843-54. doi: 10.1046/j.1365-2443.1996.730274.x.
Eukaryotic RNA polymerase II is composed of more than 10 polypeptide chains. The minimum and essential subunits for RNA synthesis have not yet been identified. Toward this ultimate goal, we analysed the topological arrangement of the putative subunits. Here we report a subunit-subunit contact network involving subunit 5 of the fission yeast Schizosaccharomyces pombe RNA polymerase II.
The rpb5+ gene encoding subunit 5 of RNA polymerase II was cloned from the fission yeast Schizosaccharomyces pombe. The polypeptide predicted from DNA sequence of the rpb5+ gene consists of 210 amino acids with a calculated molecular weight of 23914. The homology of the amino acid sequence is 55% and 43% with Saccharomyces cerevisiae RPB5 and human hRPB25, respectively. Far-Western blot analysis of S. pombe RNA polymerase II using 32P-labelled recombinant Rpb5 fused to glutathione S-transferase (GST) as a probe, indicated that Rpb5 binds strongly to membrane-immobilized Rpb1, Rpb2 and Rpb3 and weakly to Rpb5 and a 15-kDa subunit (Rpb8 or Rpb11). In agreement with this result, the 32P-labelled Rpb3 probe showed a strong binding signal against Rpb5 in addition to Rpb1 and Rpb2. The existence of Rpb5-Rpb3 contact was supported by detection of complexes formed between these two proteins synthesized in vitro using protein-immobilized beads.
Rpb3 and Rpb5, the putative subunits of RNA polymerase II, associate each other to form binary complexes. These two subunits also bind to the two large subunits, Rpb1 and Rpb2, independently.
真核生物RNA聚合酶II由10多条多肽链组成。RNA合成所需的最小且必需的亚基尚未确定。为了实现这一最终目标，我们分析了假定亚基的拓扑排列。在此，我们报道了一个涉及裂殖酵母粟酒裂殖酵母RNA聚合酶II亚基5的亚基-亚基接触网络。
从裂殖酵母粟酒裂殖酵母中克隆了编码RNA聚合酶II亚基5的rpb5+基因。根据rpb5+基因的DNA序列预测的多肽由210个氨基酸组成，计算分子量为23914。其氨基酸序列与酿酒酵母RPB5和人hRPB25的同源性分别为55%和43%。用与谷胱甘肽S-转移酶（GST）融合的32P标记重组Rpb5作为探针，对粟酒裂殖酵母RNA聚合酶II进行远缘Western印迹分析，结果表明Rpb5与膜固定的Rpb1、Rpb2和Rpb3强烈结合，与Rpb5和一个15 kDa亚基（Rpb8或Rpb11）弱结合。与此结果一致，32P标记的Rpb3探针除了与Rpb1和Rpb2结合外，还显示出与Rpb5的强结合信号。通过检测使用蛋白质固定珠体外合成的这两种蛋白质之间形成的复合物，证实了Rpb5-Rpb3接触的存在。
RNA聚合酶II的假定亚基Rpb3和Rpb5相互结合形成二元复合物。这两个亚基也分别与两个大亚基Rpb1和Rpb2结合。
