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

转录起始子复合物对启动子依赖性转录水平控制的结构研究

• 批准号: 21770125
• 负责人: ITOU Hiroshi
• 金额: $ 2.83万
• 依托单位: National Institute of Genetics
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Young Scientists (B)
• 财政年份: 2009
• 资助国家: 日本
• 起止时间: 2009 至 2011
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-21770125/
• 关键词: X線結晶解析; 蛋白質X線結晶構造解析法; 構造生物学; 転写開始複合体; タンパク質X線結晶構造解析; 結晶中での分子間化学的架橋; RNAポリメラーゼ; 双晶

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.

结构研究为理解RNA聚合酶调控启动子依赖转录水平的分子机制奠定了必要的结构基础。这一过程在生理上很重要，但由于缺乏结构信息，人们对此知之甚少。首先，将蓝藻核糖核酸聚合酶β‘亚基中特异发现的影响转录起始复合体构象变化的插入结构域CBID结构域结晶。晶体晶胞参数为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。晶体的衍射性很差，完全是孪生的。晶体中含有10个以上的CBID分子(分子的相对分子质量为70 kDa)，使用晶体进行结构测定失败。尽管我们对晶体进行了改进，但所有得到的晶体都表现出相同的结晶学特征，我们无法确定其三级结构。其次，为了确定转录起始复合体的结构，它是由RNA聚合酶和启动子组成的开放复合体和成功合成长转录物的延伸复合体之间的中间产物，我们从大肠杆菌中制备了RNA聚合酶并进行了结晶。未能获得目标晶体，但两个研究组分别报道了酵母RNA聚合酶起始复合体的晶体结构。这些相关结构表明，RNA链的延长稳定了DNA-RNA杂交体，从而影响了杂交体在酶活性中心的空间排列。这些晶体结构部分解释了我们的假设，即活性中心的DNA-RNA杂化无序将起始络合物转换为活性或非活性络合物。