Molecular Anatomy of RNA Polymerase : Mapping of Transcription Factor Contact Sites

RNA 聚合酶的分子解剖学：转录因子接触位点的作图

基本信息

批准号：
06454672
负责人：
ISHIHAMA Akira
金额：
$ 4.54万
依托单位：
National Institute of Genetics
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1995
项目状态：
已结题

项目摘要

RNA polymerase with the catalytic activity of RNA synthesis plays a Key role in gene transcription. Swiching of the global pattern of transcription takes place by modulating the promoter selectivity of RNA polymerase after interaction with various trans-acting protein and nucleotide factors. In order to understand the molecular mechaninism of promoter selectivity control of RNA polymerase, we have carried out a systematic mapping of the transcription factor contact sites on RNA polymerase. For this purpose, we reconstituted mutant E.coli RNA polymerases consisting of deletion, insertion or amino acid substitution mutant subunits and examined their responses to various transcription factors. Results indicated that : (i) the molecular contact between RNA polymerase and transcription factors involves narrow regions of about 10 amino acid residues on each component ; (ii) the contact sites are clustered on a short segment of each subunit, i.e., class-I factors on the C-terminal domain of alpha subunit and class-II factors on the C-terminal domain of sigma subunit ; and (iii) both protein factors and DNA enhancer signals interact with the same protein surface of RNA polymerase subunits as analyzed by NMR.The same line of studies has been initiated for mapping of protein-protein contact sites on RNA polymerase II from fission yeast S.pombe and on RNA polymerase from influenza viruses.

与RNA合成的催化活性的RNA聚合酶在基因转录中起关键作用。与各种反式作用蛋白和核苷酸因子相互作用后，通过调节RNA聚合酶的启动子选择性来进行全局转录模式的旋转。为了了解RNA聚合酶的启动子选择性控制的分子机械学，我们对RNA聚合酶上的转录因子接触位点进行了系统的映射。为此，我们重新建立了由缺失，插入或氨基酸取代突变体亚基组成的突变大肠杆菌RNA聚合酶，并检查了它们对各种转录因子的反应。结果表明：（i）RNA聚合酶和转录因子之间的分子接触涉及每个成分上约10个氨基酸残基的狭窄区域；（ii）接触位点聚集在每个亚基的一个短段，即Alpha亚基的C末端域上的I类因子和II类因子上的Sigma亚基C末端域的一个因子；（iii）NMR分析的RNA聚合酶亚基的蛋白质因子和DNA增强子信号与相同的RNA聚合酶亚基相同的蛋白质表面相互作用。已经启动了相同的研究，用于映射来自裂变酵母菌的RNA聚合酶II上的RNA聚合酶II上的蛋白质 - 蛋白质接触位点。