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.

On the basis of the DNA recognition mode and the structural features unique to the Cysie D22-Hisi D22 class of nuclear acid binding, approaches to link Cysie D22-Hisi D22 zinc fingers with other functional modules such as DNA binding domains to generate artificial chimeric peppers with long binding sites, and DNA-cleavage modules to produce novel sequence specific nuclearses, performed.从统计学上讲，假设基本分布，独特的16或18碱基对序列只会在43亿或680亿个核动肽中发生一次，即人类基因组的大小相同或大尺寸。尽管已经从序列中推断出含有锌指的长聚二甲基阵列的天然蛋白质，但尚未证明锌指蛋白可以结合如此长的连续DNA序列。因此，特别感兴趣的是，创建了六个或九个指的肽并证明以序列特定方式结合18或27个连续的DNA基碱对。诱导DNA组成变化的人工蛋白作为特定基因的转录调节剂有趣。我们通过将转录因子SP1的两个DNA结合结构域与柔性多甘氨酸肽接头连接到转录因子SP1的两个DNA结合结构域来创建6-锌指的蛋白Sp1ZF6（Gly）N（n = 4,7,10），并将其对DNA结构的影响与天然3-ZINC SP1的效果进行了比较（530-623）。特别感兴趣的是，新设计的6指Petides SP1ZF（Gly）7和SP1ZF（Gly）10可以在两个不同的结合位点的间隔区域诱导DNA弯曲，并且在两个3- Zinc指的小指图之间具有至关重要的长度在整个DNA弯曲方向上具有至关重要的效应。这种聚二酰基锌指肽应是新型转基因植物和动物的发展。