Qu Z, Zheng S, Gu H, Shi B
Cancer Institute, Zhejiang University, Hangzhou 310009, China.
Wei Sheng Wu Xue Bao. 2001 Oct;41(5):592-7.
To map the interacting site of subunit Rpb2 to subunit Rpb3 of RNA polymerase II in fission yeast Schizosaccharomyces pombe, the yeast two-hybrid system was employed in this paper to screen the interacting clones between Rpb2 and Rpb3.4 fragments of Rpb2 cDNA were cloned into the Ga14 BD vector pAS2. The 4 clones were named as pAS2 Rpb2-1, 2-2, 2-3 and 2-4, respectively. The complete cDNA of Rpb3 was cloned into the Gal 4 AD vector pGADGH. The clone was named as pGADGH Rpb3. The two-hybrid plasmids pGADGH Rpb3 and pAS2Rpb2-1, 2-2, 2-3 or 2-4 respectively were cotransformed into host cell yeast Y190. The interaction positive cotransformants were identified by beta-gal activity assay. The beta-gal positive cotransformants were selected from pGADGH Rpb3 and pAS2Rpb2-4 two-hybrid system. DNA sequencing and alignment results showed that the interacting site of Rpb2 to Rpb3 located within the fragment from base 2701 to 2966 of Rpb2 cDNA, or within the C-termini polypeptide from amino acid 902 to 989 of Rpb2 protein.
为了定位裂殖酵母粟酒裂殖酵母中RNA聚合酶II的Rpb2亚基与Rpb3亚基的相互作用位点，本文采用酵母双杂交系统筛选Rpb2和Rpb3之间的相互作用克隆。将Rpb2 cDNA的4个片段克隆到Gal4 BD载体pAS2中。这4个克隆分别命名为pAS2 Rpb2-1、2-2、2-3和2-4。将Rpb3的完整cDNA克隆到Gal4 AD载体pGADGH中。该克隆命名为pGADGH Rpb3。将双杂交质粒pGADGH Rpb3分别与pAS2Rpb2-1、2-2、2-3或2-4共转化到宿主细胞酵母Y190中。通过β-半乳糖苷酶活性测定鉴定相互作用阳性共转化体。从pGADGH Rpb3和pAS2Rpb2-4双杂交系统中筛选出β-半乳糖苷酶阳性共转化体。DNA测序和比对结果表明，Rpb2与Rpb3的相互作用位点位于Rpb2 cDNA第2701至2966位碱基的片段内，或位于Rpb2蛋白第902至989位氨基酸的C末端多肽内。
