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

Research Project

Project/Area Number	21770125
Research Category	Grant-in-Aid for Young Scientists (B)
Allocation Type	Single-year Grants
Research Field	Structural biochemistry
Research Institution	National Institute of Genetics
Principal Investigator	ITOU Hiroshi 国立遺伝学研究所, 構造遺伝学研究センター, 助教 (10390626)
Project Period (FY)	2009 – 2011
Project Status	Completed (Fiscal Year 2011)
Budget Amount *help	¥4,420,000 (Direct Cost: ¥3,400,000、Indirect Cost: ¥1,020,000) Fiscal Year 2011: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000) Fiscal Year 2010: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000) Fiscal Year 2009: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000)
Keywords	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
Research Abstract	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.