1998 Fiscal Year Final Research Report Summary
The Molecular Activation Mechanism of RNA Polymerase
| Project/Area Number |
09480176
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Biophysics
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| Research Institution | Osaka University |
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Principal Investigator |
KYOGOKU Yoshimasa Institute for Protein Research, Osaka University, Professor, 蛋白質研究所, 教授 (90012632)
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| Co-Investigator(Kenkyū-buntansha) |
FUJITA Nobuyuki National Institute of Genetics, Instructor, 助手 (90173434)
YAMAZAKI Toshio Institute for Protein Research, Osaka University, Associate Professor, 蛋白質研究所, 助教授 (60273710)
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| Project Period (FY) |
1997 – 1998
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| Keywords | RNA polymerase / E.coli / Thermus thermophilus HB8 / αCTD / αNTD / NMR / solution structure / activation mechanism |
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| Research Abstract |
The aim of this research is to clarify the mode of the subunit interaction with transcription factors from the structural view points and the activation mechanism of prokaryotic RNA polymerase. First of all we have determined the secondary structures of the N-terminal domain (αCTD) of E.coli RNA polymerase α subunit by using NMR. It contains 3 α helices and 2 βsheets and quite consistent with that determined by X-ray crystal structure. Then by mutational works we identified the amino acid residues of the α subunit which are essential for α-β and α-β' interaction. They all fall on αNTD. Moreover we also determined the residues which are Important for β-β' interaction. We cloned the αsubunit gene of Thermus thermophilus HB8, expressed the protein and determined the solution structure of the C-terminal domain (αCTD). Its tertiary structure is close to that of E.coli and is more heat stable, although it is shorter by 14 residues than that of E.coli. In some specific gene like rrnBP1 αCTD interacts with the UP element DNA and activates transcription. We prepared several DNA oligomers containing the partial sequence of the UP elements which is rich of the A tract. The interaction is not 50 specific, but αCTD prefers the intrinsically bent DNA and interacts with the UP element from the minor groove side. αCTD is also raid to be contact region for transcription factor protein like CRP. However we could not deled any evidence for direct interaction for the two component system. We only observed the interaction between then in the presence of the CRP binding site DNA with sonic extension to down siream of the promoter which provides the space for αCTD. The activation is well explained by the bending of promoter DNA which may facilities the closed complex of RNA polymerase. αCTD may haul the bent DNA and keep it in the complex.
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