| Project/Area Number |
13480222
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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 | Yokohama City University |
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Principal Investigator |
NISHIMURA Yoshifumi Yokohama City University, Graduate school of integrated Science, Professor, 総合理学研究科, 教授 (70107390)
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| Project Period (FY) |
2001 – 2003
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| Project Status |
Completed (Fiscal Year 2003)
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| Budget Amount *help |
¥11,200,000 (Direct Cost: ¥11,200,000)
Fiscal Year 2003: ¥3,200,000 (Direct Cost: ¥3,200,000)
Fiscal Year 2002: ¥3,200,000 (Direct Cost: ¥3,200,000)
Fiscal Year 2001: ¥4,800,000 (Direct Cost: ¥4,800,000)
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| Keywords | transcription factor / telomeres / DNA-binding protein / TRF1 / TRF2 / NMR / TFIIE / Zn binding domain / 転写因子 / Rap1 / Myb / DNA認識 / テロメア結合タンパク質 / DNA結合ドメイン / 表面構造 / hTRF1 / hRap1 |
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| Research Abstract |
The zinc finger domain in the large subunit of TFIIE is phylogenetically conserved and is essential for transcription. We have determined its NMR structure. It consists of one alpha helix and six beta strands showing novel features distinct from previously determined zinc-binding structures. We created point mutants in this domain and examined their binding abilities to other general transcription factors as well as their transcription activities. Interesting functional asymmetry of Zn^<2+>-ligand mutants was observed: the N-terminal two mutants remained approximately 20% activity on a supercoiled template. CD and NMR studies showed that those two equilibrate mainly with the random coil structure, although all four mutants possessed partially folded characteristic structures coordinating Zn^<2+>-. And also, highly conserved D 164 mutants (D 164A and D 164K) were found to increase transcription.
In addition, we have determined the solution structure of the DNA binding domain of hTRF2 bound to a telomeric double-stranded DNA with the sequence of GTTAGGGTTAGGG and compared it with the corresponding DNA complex structure of hTRF1. Telomeres are the ends of eukaryotic linear chromosomes consisting of repetitive G-rich sequence and telomeric repeat binding factors. In mammalian telomeres, TRF1 and TRF2 bind to double-stranded telomeric DNA. Both contain a central TRF-homology (TRFH) domain and a C-terminal DNA binding domain. Both DNA-bound structures are very close to each other, however, small but significant structural differences are observed. Based on the present structure of hTRF2 we could make several mutants of hTRF2, which have a stronger binding ability to telomeric DNA rather than the wild type.
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