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。RNA聚合酶与各种反式作用蛋白和核苷酸因子相互作用后，通过调节启动子选择性来改变转录的全局模式。为了了解RNA聚合酶启动子选择性控制的分子机制，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、插入或氨基酸替代突变亚基组成的突变型大肠杆菌RNA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聚合酶与转录因子之间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；（ii）接触位点聚集在每个亚基的短片段上，即ⅰ类因子在α亚基的c端域上，ⅱ类因子在sigma亚基的c端域上；（iii）通过核磁共振分析，蛋白质因子和DNA增强子信号与RNA聚合酶亚基的相同蛋白质表面相互作用。在裂变酵母S.pombe的RNA聚合酶II和流感病毒的RNA聚合酶的蛋白质-蛋白质接触位点的定位方面，也开始了同样的研究。

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.