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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，利用多个锌指定位基因组中唯一的靶点已经得到了广泛的研究。定制的6-锌指蛋白组合在医学研究中非常实用，因为它们具有足够的DNA识别长度和可管理性。为了评估多个指状域连接的可能性，以了解DNA结合机制和基因调控，构建了最长的人工锌指蛋白Sp1ZF15。该锌指由5个单位的Sp1锌指多肽组成，这些多肽通过规范的短连接序列(TGEKP)相连。通过凝胶迁移率改变分析和DNase I足迹分析，确定Sp1ZF15对DNA的50个连续碱基对的识别能力。此外，还研究了与较短靶标的有效结合。从GCG三联体到ATA在靶部位的序列改变清楚地表明，Sp1ZF15的DNA结合模式取决于靶序列。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结合中活跃的指状域的数量。这些结果表明，锌指蛋白是一种有效的与长DNA序列结合的模块，用于开发GONE调控。此外，目前的证据表明，在多锌指蛋白中，通过改变选择、数量和顺序，可能会在一个细胞中产生大量不同的转录因子。