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

我们想弄清楚产气荚膜梭菌中编码磷酸酶C的plc gane启动子上游的弯曲DNA（定相A区）如何通过其C末端结构域（α CTD，79 aa）与RNA聚合酶（RNAP）α亚基（315 aa）结合。将编码RNAP α亚基的rpaA基因和编码α CTD的grace基因分别克隆到pET 16 b中，构建了重组质粒pET 16 b。将每个基因在大肠杆菌BL 21（DE 3）pLysS中过表达。然后，将两种His标记的蛋白质纯化至可用于结晶的程度。但纯化蛋白的量只有几毫克。在50 mg时读数不足以进行结晶。约书亚萨孔博士（美国阿肯色州大学）指出相控A束与α亚基或α CTD的复合物在25℃（一定的低温）下的稳定性是非常重要的，应加以考察。因为我们还没有考试 关于我们 由于目前还没有建立起稳定性和大规模纯化过量蛋白质的方法，因此，相控A束DNA与RNAP α亚基或α CTD的共结晶还没有建立起来，同时，我们尝试建立RNAP的重组体系（α2，β，α '，σ），以鉴定参与定相A-束和α CTD之间相互作用的氨基酸残基。β、β '和σ亚基在适当的大肠杆菌菌株中过量产生，并成为包涵体。将它们溶解在6 M尿素或盐酸胍中。这些溶解的蛋白质和His-标记的α亚基或其N-末端结构域（α NTD，228 aa）在RNAP的重构中混合。重组的RNAP（含His-α亚基或His-α NTD）具有相同水平的轻微活性。这表明，我们在α CTD中引入突变后，对α CTD的活性没有任何影响，而RNAP的重组将为研究α CTD对转录的调控做出重要贡献。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