Molecular Anatomy of Transcription Apparatus

大肠杆菌RNA聚合酶的启动子选择性取决于与转录因子分子相互作用的两个步骤：(1)核心酶与不同的Sigma亚基结合，每个亚基识别一组独特的启动子，构成不同形式的全酶；(2)全酶与大约100种辅助转录因子之一的结合。用定量免疫印迹法测定每个sigma亚单位在细胞内的浓度。结果表明，sigma亚单位的浓度及其与核心酶的亲和力取决于细胞生长条件。为了揭示RNA聚合酶特异性的第二步调节，对20多个转录因子在RNA聚合酶上的接触位点进行了测定，结果表明，每个因子与RNA聚合酶在一个由10个氨基酸残基组成的狭窄区域内相互作用，接触位点聚集在每个亚基的调控区域内，对α亚基上的接触位点与I类因子进行了精细的定位，并且发现I类因子在α亚基上的接触面参与了DNA增强子元件的结合。

The promoter selectivity of Escherichia coli RNA polymerase is determined by two steps of molecular interaction with transcription factors : (1) association of core enzyme with one of various sigma subunits, each recognizing a group of unique promoters, to constitute different forms of holoenzyme ; and (2) association of holoenzyme with one of about 100 species of accessory transcription factors.The intracellular concentration of each sigma subunit was determined using a quantitative immunoblot method.Results indicate that the concentration of sigma subunits and their affinity to core enzyme are subject to control depending on the cell growth conditions.In order to reveal the second step modulation of RNA polymerase specificity, the contact site on RNA polymerase was determined for more than 20 transcription factors.Results indicate that each factor interacts with RNA polymerase at a narrow region consisting of about 10 amino acid residues and that the contact sites are clustered within a regulatory domain of each subunit.Fine mapping has been done for the contact sites on alpha subunit with class-I factors.Furthermore, the contact surface of the class-I factor on the alpha subunit was found to participate in binding DNA enhancer element.