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寻找独特靶位点的位置已被广泛研究。定制的 6-锌指蛋白组因其足够的 DNA 识别长度和可管理性而在医学研究中非常实用。为了评估指结构域多重连接以了解 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 Sp1ZF15。该锌指由 5 个 Sp1 锌指肽单元组成，通过规范短接头序列 (TGEKP) 连接。 Sp1ZF15 对 DNA 50 个连续碱基对的识别通过凝胶迁移率变化测定和 DNase I 足迹分析来确定。此外，还检查了与较短靶标的有效结合。 GCG 三联体在靶位点对 ATA 的序列改变清楚地表明，Sp1ZF15 根据靶序列改变其 DNA 结合模式。特别令人感兴趣的是，Sp1ZF15 控制着与目标 DNA 的长度和序列相对应的 DNA 结合中活跃的指状结构域的数量。这些结果表明，锌指蛋白是长DNA序列结合模块的有效申请人，以促进失控调控的发展。此外，目前的证据表明，通过改变多锌指蛋白的选择、数量和顺序，可以在细胞中产生大量不同的转录因子。