Wigneshweraraj Siva R, Kuznedelov Konstantin, Severinov Konstantin, Buck Martin
Department of Biological Sciences, Imperial College of Science, Technology and Medicine, Sir Alexander Fleming Building, Imperial College Road, London SW7 2AZ, United Kingdom.
J Biol Chem. 2003 Jan 31;278(5):3455-65. doi: 10.1074/jbc.M209442200. Epub 2002 Nov 6.
Recent determinations of the structures of the bacterial RNA polymerase (RNAP) and promoter complex thereof establish that RNAP functions as a complex molecular machine that contains distinct structural modules that undergo major conformational changes during transcription. However, the contribution of the RNAP structural modules to transcription remains poorly understood. The bacterial core RNAP (alpha(2)beta beta'omega; E) associates with a sigma (sigma) subunit to form the holoenzyme (E sigma). A mutation removing the beta subunit flap domain renders the Escherichia coli sigma(70) RNAP holoenzyme unable to recognize promoters. sigma(54) is the major variant sigma subunit that utilizes enhancer-dependent promoters. Here, we determined the effects of beta flap removal on sigma(54)-dependent transcription. Our analysis shows that the role of the beta flap in sigma(54)-dependent and sigma(70)-dependent transcription is different. Removal of the beta flap does not prevent the recognition of sigma(54)-dependent promoters, but causes multiple defects in sigma(54)-dependent transcription. Most importantly, the beta flap appears to orchestrate the proper formation of the E sigma(54) regulatory center at the start site proximal promoter element where activator binds and DNA melting originates.
近期对细菌RNA聚合酶（RNAP）及其启动子复合物结构的测定表明，RNAP作为一种复杂的分子机器发挥作用，它包含不同的结构模块，这些模块在转录过程中会发生重大的构象变化。然而，RNAP结构模块对转录的贡献仍知之甚少。细菌核心RNAP（α₂ββ′ω；E）与一个σ（sigma）亚基结合形成全酶（Eσ）。去除β亚基的侧翼结构域的突变会使大肠杆菌σ⁷⁰ RNAP全酶无法识别启动子。σ⁵⁴是利用增强子依赖性启动子的主要变体σ亚基。在此，我们确定了去除β侧翼结构域对σ⁵⁴依赖性转录的影响。我们的分析表明，β侧翼结构域在σ⁵⁴依赖性转录和σ⁷⁰依赖性转录中的作用不同。去除β侧翼结构域并不妨碍对σ⁵⁴依赖性启动子的识别，但会在σ⁵⁴依赖性转录中导致多种缺陷。最重要的是，β侧翼结构域似乎在起始位点近端启动子元件处协调Eσ⁵⁴调控中心的正确形成，激活剂在此结合且DNA解链起始。
