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Creation and Function of Novel Gene Regulation Molecules Based on Zinc Finger Motif

基于锌指基序的新型基因调控分子的创建和功能

基本信息

批准号：
10470493
负责人：
SUGIURA Yukio
金额：
$ 7.81万
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：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10470493/
关键词：
Zinc finger Transcription factor Gene target DNA recognition Binding motif Gene regulation Metal peptide Molecular disign 転写調節モチーフ

项目摘要

On the basis of the DNA recognition mode and the structural features unique to the CysィイD22ィエD2-HisィイD22ィエD2 class of nucleic acid binding, approaches to link CysィイD22ィエD2-HisィイD22ィエD2 zinc fingers with other functional modules such as DNA binding domains to generate artificial chimeric peptides with long binding sites, and DNA-cleavage modules to produce novel sequence specific nucleases, ィイD14-5ィエD1 have been performed. Statistically, assuming random base distribution, a unique 16-or 18-base pair sequence will occur only once in 4.3 billion or 68 billion nucleotides, roughly the same or big size of a human genome. Although natural proteins containing long polydactyl arrayl arrays of zinc finger do-mains have been inferred from sequence, no zinc finger proteins have been demonstrated to bind such a long, contiguous DNA sequence. Therefore, it is of special interest that six-or nine-fingered peptide was created and demonstrated to bind 18 or 27 contiguous base pairs of DNA in a sequence specific fashion. An artificial protein that induces a DNA conformational change is interesting as a transcriptional regulator of a specific gene. We created 6-zinc finger proteins, Sp1ZF6(Gly)n(n=4,7,10), by connecting two DNA binding domains of transcription factor Sp1 with flexible polyglycine peptide linkers, and their effects on DNA structure were compared with that of native 3-zinc finger Sp1(530-623). Of special interest are the fact that newly designed 6-finger peptides Sp1ZF(Gly)7 and Sp1ZF(Gly)10 can induce DNA bending at the intervening region of the two distal binding sites and that the liker length between two 3-zinc finger motifs has a crucial effect on the entire DNA bending direction. Such polydactyl zinc finger peptides should be the development of novel transgenic plants and animals.

基于DNA识别模式和Cys寡核苷酸D22寡核苷酸D2-His寡核苷酸D22寡核苷酸D2类核酸结合所特有的结构特征，将Cys寡核苷酸D22寡核苷酸D2-His寡核苷酸D22寡核苷酸D2锌指与其他功能模块如DNA结合结构域连接以产生具有长结合位点的人工嵌合肽，以及将DNA切割模块连接以产生新的序列特异性核酸酶的方法，已进行了第14 -5天第1天的试验。从统计学上讲，假设随机碱基分布，一个独特的16或18个碱基对序列在43亿或680亿个核苷酸中只会出现一次，这与人类基因组的大小大致相同或更大。虽然已经从序列中推断出含有长的多指阵列锌指结构域的天然蛋白质，但是没有锌指蛋白质被证明结合如此长的连续DNA序列。因此，特别令人感兴趣的是，六指或九指肽被产生并被证明以序列特异性方式结合DNA的18或27个连续碱基对。诱导DNA构象变化的人工蛋白作为特定基因的转录调节因子是令人感兴趣的。我们将转录因子Sp1的两个DNA结合结构域用柔性多聚甘氨酸肽接头连接起来，构建了6-锌指蛋白Sp1 ZF 6（Gly）n（n= 4，7，10），并将其与天然3-锌指蛋白Sp1（530-623）对DNA结构的影响进行了比较。特别有趣的是，新设计的6-指肽Sp1 ZF（Gly）7和Sp1 ZF（Gly）10可以在两个远端结合位点的中间区域诱导DNA弯曲，并且两个3-锌指基序之间的连接体长度对整个DNA弯曲方向具有至关重要的影响。这类多指锌指肽应该是开发新型转基因植物和动物的基础。