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Role of multi zinc finger in gene and creation of its architecture

多锌指在基因中的作用及其结构的创建

基本信息

批准号：
14370755
负责人：
SUGIURA Yukio
金额：
$ 9.47万
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：
2002
资助国家：
日本
起止时间：
2002 至 2004
项目状态：
已结题

项目摘要

In gene regulation, the positional finding of a unique target site in the genome by multi-zinc fingers has been widely studied. The custom sets of 6-zinc finger proteins are very practical in medicinal research for their adequate recognition length of DNA and manageability. To assess the possibility of multi-connection of finger domains for understanding of DNA binding mechanisms and gene regulation, the longest artificial zinc finger protein, Sp1ZF15, has been constructed. This zinc finger consists of 5 units of Sp1 zinc finger peptide connected by canonical short linker sequences (TGEKP). Recognition on the 50 successive base pairs of DNA by Sp1ZF15 was determined by gel mobility shift assays and DNase I footprinting analyses. Furthermore, capable bindings to shorter targets were also examined. Sequence alterations of the GCG triplet to ATA at a target site clearly showed that Sp1ZF15 alters its DNA binding mode depending on the target sequences. Of special interest is the fact that Sp1ZF15 controls the number of the finger domains active in DNA binding corresponding to the length and sequence of the target DNA. These results show the zinc finger protein to be an effective applicant for a binding module to long DNA sequence for the development of gone regulation. In addition, the present evidence suggest that a vast collection of distinct transcription factors by varying the choice, number and order, in multi zinc finger proteins, might be produced in a cell.

在基因调控中，通过多锌指在基因组中定位独特的靶位点已经被广泛研究。定制的6-锌指蛋白具有足够的DNA识别长度和特异性，在医学研究中具有重要的实用价值。为了研究锌指结构域的多重连接对理解DNA结合机制和基因调控的可能性，构建了最长的人工锌指蛋白Sp1 ZF 15。该锌指由5个Sp1锌指肽单元组成，这些单元通过典型短接头序列（TGEKP）连接。通过凝胶迁移率变动分析和DNA酶I足迹分析确定Sp1 ZF 15对DNA的50个连续碱基对的识别。此外，还检查了与较短靶标的能力结合。GCG三联体在靶位点处与ATA的序列改变清楚地表明Sp1 ZF 15根据靶序列改变其DNA结合模式。特别令人感兴趣的是，Sp1 ZF 15控制与靶DNA的长度和序列相对应的DNA结合中有活性的指结构域的数量。这些结果表明，锌指蛋白是一个有效的申请人的结合模块的长DNA序列的发展去调节。此外，目前的证据表明，通过改变多锌指蛋白中的选择、数量和顺序，可以在细胞中产生大量不同的转录因子。