Structural study of promoter-dependent transcription level control by the transcription initiator complex

进行结构研究以建立理解RNA聚合酶依赖于启动子的转录水平控制的分子机制所必需的结构基础。这一过程在生理学上很重要，但由于缺乏结构信息而了解较少。首先，对蓝藻RNA聚合酶β'亚基上的一个插入结构域CBID结构域进行了结晶，推测其可能影响转录起始复合物的构象变化。CBID晶体的晶胞参数为a=185.9，B=185.9，c=276.5（埃），a=β=90，γ=120（度），属于空间群P3_1（或P3_2）。这些晶体衍射很差，而且是完美的孪生。晶体含有超过10个CBID分子（分子量为70 kDa），使用晶体的结构测定是不成功的。尽管我们努力改进晶体，但所有获得的晶体都表现出相同的晶体学特征，并且我们不能确定三级结构。第二，为了确定转录起始复合物的结构，我们从大肠杆菌中制备了RNA聚合酶，它是由RNA聚合酶和启动子组成的开放复合物与延伸复合物之间的中间产物，成功地合成了长转录物。大肠杆菌中进行了结晶试验。虽然未能获得目标晶体，但两个研究小组分别报道了酵母RNA聚合酶起始复合物的晶体结构。这些相关结构表明，RNA链的延长稳定了DNA-RNA杂合体，并影响了杂合体在酶活性中心的空间排列。这些晶体结构部分地解释了我们的假设，即DNA-RNA杂化物在活性中心的无序将起始复合物转换为活性或非活性复合物。

Structural study was conducted to establish structural basis necessary to understand molecular mechanism of promoter-dependent transcription level control by RNA polymerase. This process is physiologically important, but less understood due to lack of structural information. Firstly, CBID domain, an insertion domain specifically found in theβ' subunit of the RNA polymerase of cyanobacteria and supposed to affect conformation changes of transcription initiating complex, was crystallized. The CBID crystal had cell parameters of a=185.9, b=185.9, c=276.5(angstroms), a=β=90,γ=120(degrees), and belonging to space group P3_1(or P3_2). The crystals diffracted poorly and were perfectly twinning. The crystals contain more than 10 CBID molecules(molecular weight of the molecule is 70 kDa), and structure determination using the crystals was unsuccessful. Despite of our efforts for improving the crystals, all the obtained crystals exhibited the same crystallographic characters, and we could not determine the tertiary structure. Secondly, to determine the structure of transcription initiation complex, an intermediate between an open complex composed of RNA polymerase and promoter and an elongation complex successfully synthesizes long transcript, we prepared RNA polymerase from E. coli and tried crystallization. The target crystal could not be obtained, but the crystal structures of the initiation complex of yeast RNA polymerase were reported from two research groups respectively. These related structures showed that elongation of RNA chain stabilizes DNA-RNA hybrid and this affects spatial arrangement of the hybrid in active center of the enzyme. Our hypothesis supposing disorder of DNA-RNA hybrid in active center switches the initiating complex to active or inactive complex, is partly explained by these crystal structures.