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

通过结构研究，为理解RNA聚合酶调控启动子依赖性转录水平的分子机制奠定必要的结构基础。这一过程在生理学上是重要的，但由于缺乏结构信息而鲜为人知。首先，研究了蓝藻RNA聚合酶β′亚基中特异性存在的影响转录起始复合物构象变化的插入结构域CBID结构域。CBID晶胞参数为a=185.9, b=185.9, c=276.5（埃），a=β=90,γ=120（度），属于P3_1（或P3_2）空间群。晶体的衍射很差，完全是孪生的。该晶体含有10多个CBID分子（分子分子量为70 kDa），利用该晶体进行结构测定不成功。尽管我们努力改进晶体，但所有得到的晶体都表现出相同的晶体学特征，我们无法确定三级结构。其次，为了确定转录起始复合物（由RNA聚合酶和启动子组成的开放复合物和成功合成长转录物的延伸复合物之间的中间物）的结构，我们从大肠杆菌中制备了RNA聚合酶并进行了结晶试验。虽然无法获得目标晶体，但两个研究组分别报道了酵母RNA聚合酶起始复合物的晶体结构。这些相关结构表明，RNA链的伸长稳定了DNA-RNA杂合体，并影响了杂合体在酶活性中心的空间排列。我们假设DNA-RNA杂化在活性中心的紊乱会使起始复合物变成活性复合物或非活性复合物，这部分可以用这些晶体结构来解释。

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.