The Molecular Activation Mechanism of RNA Polymerase

本研究旨在从结构的角度阐明亚基与转录因子的相互作用模式和原核RNA聚合酶的激活机制。首先利用核磁共振技术确定了大肠杆菌RNA聚合酶α亚基n端结构域（α ctd）的二级结构。它含有3个α螺旋和2个β片，与x射线测定的晶体结构相当一致。然后通过突变工作，我们确定了α-β和α-β'相互作用所必需的α亚基氨基酸残基。它们都落在αNTD上。此外，我们还确定了β-β′相互作用的重要残基。我们克隆了嗜热热菌HB8的α亚基基因，表达了该蛋白，并测定了其c端结构域（αCTD）的溶液结构。它的三级结构与大肠杆菌接近，热稳定性更强，但比大肠杆菌短14个残基。在一些特定的基因如rrnBP1中，αCTD与UP元件DNA相互作用并激活转录。我们制备了几种含有丰富A区的UP元件部分序列的DNA低聚物。这种相互作用的特异性不是50，但αCTD倾向于内在弯曲的DNA，并从小凹槽侧与UP元件相互作用。αCTD也可能是CRP等转录因子蛋白的接触区。然而，我们不能删除任何证据直接相互作用的两个组成系统。我们仅在CRP结合位点DNA存在的情况下观察到两者之间的相互作用，并将声波延伸到启动子的下端，从而为αCTD提供空间。启动子DNA的弯曲可以很好地解释这种激活，这可能有助于RNA聚合酶的闭合复合体。α - ctd可以将弯曲的DNA保存在复合物中。

The aim of this research is to clarify the mode of the subunit interaction with transcription factors from the structural view points and the activation mechanism of prokaryotic RNA polymerase. First of all we have determined the secondary structures of the N-terminal domain (αCTD) of E.coli RNA polymerase α subunit by using NMR. It contains 3 α helices and 2 βsheets and quite consistent with that determined by X-ray crystal structure. Then by mutational works we identified the amino acid residues of the α subunit which are essential for α-β and α-β' interaction. They all fall on αNTD. Moreover we also determined the residues which are Important for β-β' interaction. We cloned the αsubunit gene of Thermus thermophilus HB8, expressed the protein and determined the solution structure of the C-terminal domain (αCTD). Its tertiary structure is close to that of E.coli and is more heat stable, although it is shorter by 14 residues than that of E.coli. In some specific gene like rrnBP1 αCTD interacts with the UP element DNA and activates transcription. We prepared several DNA oligomers containing the partial sequence of the UP elements which is rich of the A tract. The interaction is not 50 specific, but αCTD prefers the intrinsically bent DNA and interacts with the UP element from the minor groove side. αCTD is also raid to be contact region for transcription factor protein like CRP. However we could not deled any evidence for direct interaction for the two component system. We only observed the interaction between then in the presence of the CRP binding site DNA with sonic extension to down siream of the promoter which provides the space for αCTD. The activation is well explained by the bending of promoter DNA which may facilities the closed complex of RNA polymerase. αCTD may haul the bent DNA and keep it in the complex.