Studies on the law temperature-dependent enhancement of transcription by a bent DNA

我们希望弄清楚产气荚膜梭菌编码磷脂酶C的plc基因启动子上游的弯曲DNA（阶段性a -束）是如何通过其C端结构域（α CTD, 79 aa）与RNA聚合酶(RNAP)α亚基（315 aa）结合的。为了确定其结构，我们将编码RNAP α亚基的rpaA基因和编码α CTD的grace基因克隆到pET16b中，确定了33 bp的阶段性a束DNA与RNAP α亚基或α CTD的共结晶。每个基因在大肠杆菌BL21(DE3)pLysS中均过表达。然后，这两种His标记的蛋白质都被尽可能多地纯化用于结晶。但纯化后的蛋白质含量只有几毫克，在50毫克的读数不足以进行结晶。美国阿肯色大学joshua Sakon博士指出，相a束与α亚基或α CTD配合物在25℃（一定低温）下的稳定性是非常重要的，应该进行检测。由于我们还没有对其稳定性和大规模提取过剩蛋白的方法进行研究，因此还没有确定相a束DNA与RNAP α亚基或α CTD的共结晶。同时，我们试图建立RNAP（α2, β, β′，σ）的重构体系，以鉴定与α CTD相互作用相关的氨基酸残基。β、β′和σ亚基在适当的大肠杆菌菌株中过量产生，形成包涵体。将它们溶解于6 M尿素或盐酸胍中。这些溶解蛋白与His标记的α亚基或其n端结构域（α NTD, 228 aa）混合在RNAP的重构中。含有His-α亚基或His-α NTD的两种重组rnap在相同水平上具有轻微的活性。这表明我们可以在α CTD中引入突变而不影响其活性，并且RNAP的重构将对α CTD调控转录的研究有很大的帮助。少

We would like to make clear the structure how the bent DNA (phased A-tracts), upstream the promoter of plc gane encoding the phospholpase C in Clostridium perfringens, binds to the RNA polymerase (RNAP)α subunit (315 aa) through its C-terminai domain (α CTD, 79 aa). To determine the structrue, the co-crystallization of the phased A-tracts DNA (33 bp) and RNAP α subunit or α CTD is necessary rpaA gene encoding the RNAP α subunit and the grace encoding the α CTD were cloned into pET16b. Each gene was overexpreessed in E.coli BL21(DE3)pLysS. Then both His, tagged proteins were purified as much as available for crystallization. But the amounts of the purified proteins were only a few mg. They did not reads at 50 mg enough for crystallization. Dr.Joshua Sakon (University of Arkansas, U.S.A.) pointed out that the stability of the complex of the phased A-tracts and the α subunit or the α CTD at 25℃ (a certain low temperature) was very important, and should be examined. Since we have not exami … More ned the stability and the method by which the overproduced proteins were purled in a large scale have been not established yet, the co-crystallization of the phased A-tracts DNA and the RNAP α subunit or the α CTD has not been tied.In parallel, we tried to establish the system of the reconstitution of the RNAP(α2, β, β', σ) to identify the residues of amino-acids involved in the interaction between the phased A-tracts and the α CTD. The β, β', and σ subunits were overproduced in appropriate E.coli strains, and became inclusion bodies. They were solubilized in 6 M urea or guanidine hydrochloride. These solubilized proteins and the His- tagged α subunit or its N-terminal domain (α NTD, 228 aa) were mixed in the reconstitution of the RNAP. The both reconstituted RNAPs, containing the His-α subunit or the His-α NTD, have slight activities at the same level. This suggested that we night introduce the mutations in the α CTD without any influence on the activity, and that the reconstitution of the RNAP would contribute greatly to the studies on the regulation of transcription through the α CTD. Less