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Regulation of cellular genetic function by new DNA bending fingers

新的DNA弯曲手指调节细胞遗传功能

基本信息

批准号：
13557210
负责人：
SUGIURA Yukio
金额：
$ 8.96万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2003
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13557210/
关键词：
Zinc finger Artificial protein DNA recognition DNA motif DNA bending Gene target Artificial restriction enzyme Artificial repressor マルチフィンガー遺伝子制御転写調節アーキテクチャーリンカー転写因子 DNA結合

项目摘要

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.

DNA弯曲在各种生物反应中起着至关重要的作用。特别是，已知许多转录因子可以诱导DNA弯曲并支持转录起始复合体的形成。我们合成了能有效诱导DNA弯曲的新型人工6-锌指状肽。转录因子Sp1的两个DNA结合域通过聚甘氨酸连接子连接。将得到的6-锌指蛋白Sp1ZF6(Gly)n(n=4，7，10)与天然3-锌指蛋白Sp1的DNA弯曲能力进行比较。凝胶电泳法显示，Sp1ZF6(Gly)_7和Sp1ZF6(Gly)_(10&gt；)与两个远端GC盒结合，导致DNA弯曲。位相分析强烈地表明，诱导的DNA弯曲指向主槽，而Sp1ZF6(Gly)7引起的DNA弯曲的方向性变化最大。特别有趣的是，两个3-锌指基序之间的连接子长度对整个DNA弯曲方向有至关重要的影响。此外，Sp1ZF6(Gly)_&lt；10&gt；的柔性和中性接头也被转化为包括精氨酸和谷氨酸残基在内的带电接头。为了阐明DNA弯曲能力与转录活性之间的关系，我们还进行了报告基因分析。实验结果表明，人工锌指肽引起的DNA弯曲激活了基因的表达。基于DNA结构的变化，这种DNA弯曲手指可能在不久的将来用作基因表达调控因子。