【摘要】 为研究S .pombeRNApolⅡ各亚基间体内装配成复合体的机制 ,本文首次用酵母双杂交系统鉴定了Rpb2和Rpb3两亚基间体内相互作用的位点。首先将Rpb2的 4个片段克隆至Gal4BD表达载体pAS2上 ,构建BD Rpb2片段融合蛋白重组质粒 ;同时将Rpb3克隆至Gal4AD表达载体pGADGH上 ,构建AD Rpb3融合蛋白重组质粒。其次 ,将pGADGHRpb3分别与pAS2Rpb2各片段重组质粒共转化到受体酵母菌Y1 90感受态细胞内 ,筛选并鉴定β gal活性阳性 (β gal+)的共转化子。最后 ,将β gal+共转化子中的Rpb2片段进行序列分析并进行同源序列比较确定其在Rpb2中的位置。结果表明 ,Rpb2与Rpb3相互作用的位点位于Rpb2的 90 2～ 989aa肽段内更多还原

【Abstract】 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 β\|gal activity assay.The β\|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.更多还原