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

RNA聚合酶具有催化RNA合成的活性，在基因转录中起着关键作用。全球转录模式的改变是通过调节RNA聚合酶与各种反式作用蛋白和核苷酸因子相互作用后的启动子选择性而发生的。为了了解RNA聚合酶启动子选择性调控的分子机制，我们对RNA聚合酶上的转录因子接触位点进行了系统的定位。为此，我们重组了由缺失、插入或氨基酸替换突变亚基组成的突变型E.coliRNA聚合酶，并检测了它们对各种转录因子的反应。结果表明：(1)RNA聚合酶与转录因子的分子接触涉及每个组分上约10个氨基酸残基的狭窄区域，(2)接触位点聚集在每个亚基的一小段上，即α亚基C-末端结构域上的I类因子和Sigma亚单位C-末端结构域上的II类因子；和(Iii)蛋白质因子和DNA增强子信号都与NMR分析的RNA聚合酶亚基的同一蛋白质表面相互作用。同样的研究已经启动，以绘制来自裂解酵母S.pombe的RNA聚合酶II和来自流感病毒的RNA聚合酶的蛋白质-蛋白质接触位点。

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.