2003 Fiscal Year Final Research Report Summary
Regulation of cellular genetic function by new DNA bending fingers
| Project/Area Number |
13557210
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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 | 展開研究 |
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| Research Field |
医薬分子機能学
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| Research Institution | KYOTO UNIVERSITY |
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Principal Investigator |
SUGIURA Yukio Kyoto Univ., Inst.Chem.Res., Prof., 化学研究所, 教授 (40025698)
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| Co-Investigator(Kenkyū-buntansha) |
NAGAOKA Makoto Kyoto Univ., Inst.Chem.Res., Assistant Prof., 化学研究所, 助手 (60314275)
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| Project Period (FY) |
2001 – 2003
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| Keywords | Zinc finger / Artificial protein / DNA recognition / DNA motif / DNA bending / Gene target / Artificial restriction enzyme / Artificial repressor |
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| Research Abstract |
DNA bending is very important for various biological reactions. In particular, many transcription factors have been known to induce DNA bending and to support formation of the transcriptional initiation complex. We crated new artificial 6-zinc finger peptides that effectively induced DNA bending. The two DNA binding domains of transcription factor Sp1 were connected through polyglycine linker. The obtained 6-zinc finger proteins Sp1ZF6(Gly)n (n=4, 7, 10) were compared with native 3-zinc finger peptide Sp1 in the respect of DNA bending ability. Gel electrophoretic methods revealed that Sp1ZF6(Gly)_7 and Sp1ZF6(Gly)_<10> bound to two distal GC boxes and resulted in DNA bending. The phasing assays strongly suggested that the induced DNA bending was directed toward the major groove and that Sp1ZF6(Gly)_7 caused the most drastic directional change in DNA bending. Of special interest is the fact that the linker length between two 3-zinc finger motifs has a crucial effect on the entire DNA bending direction. In addition, the flexible and neutral linker of Sp1ZF6(Gly)_<10> was also converted to charged linkers including arginine and glutamate residues. In order to clarify the relation between the DNA bending ability and the transcriptional activity, we also reporter gene assays. The experimental results led to the activation of gene expression by the DNA bending due to artificial zinc finger peptide. Such DNA bending fingers may be feasible for use as a gene expression regulator based on the structural change in DNA in the near future.
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