Molecular Anatomy of Transcription Apparatus

大肠杆菌RNA聚合酶启动子的选择性是通过与转录因子分子相互作用的两个步骤来决定的：(1)核心酶与各种sigma亚基中的一个结合，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种辅助转录因子中的一种结合。采用定量免疫印迹法测定细胞内各西格玛亚基浓度。结果表明，sigma亚基的浓度及其对核心酶的亲和力受细胞生长条件的控制。为了揭示RNA聚合酶特异性的第二步调控，我们确定了20多个转录因子在RNA聚合酶上的接触位点。结果表明，每个因子与RNA聚合酶在一个狭窄的由大约10个氨基酸残基组成的区域相互作用，并且接触位点聚集在每个亚基的调控区域内。用i类因子对α亚基上的接触点进行了精细映射。此外，在α亚基上发现i类因子的接触面参与结合DNA增强子元件。

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.