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CysD22D2-HisD22类核酸结合特有的结构特征，将CysD22D2-His D22锌指与DNA结合结构域等其他功能模块连接起来，生成具有长结合的人工嵌合肽位点和 DNA 切割模块以产生新的序列特异性核酸酶，从统计上看，假设随机碱基分布，独特的 16 或 18 碱基对序列只会在 43 亿或 680 亿个核苷酸中出现一次，这与人类基因组的大小大致相同或很大。已经从序列中推断出含有长的锌指结构域的多指阵列，但尚未证明锌指蛋白能够结合如此长的、连续的DNA序列。特别令人感兴趣的是，六指或九指肽被创建并被证明能够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质作为特定的转录调节剂很有趣。我们通过使用灵活的聚甘氨酸肽接头连接转录因子 Sp1 的两个 DNA 结合域，创建了 6-锌指蛋白 Sp1ZF6(Gly)n(n=4,7,10)，以及将它们对 DNA 结构的影响与天然 3 锌指 Sp1(530-623) 进行了比较，特别令人感兴趣的是新设计的 6 指肽 Sp1ZF(Gly)7 和 Sp1ZF(Gly)10 可以诱导 DNA 弯曲。在两个远端结合位点的中间区域，两个 3-锌指基序之间的相似长度对整个 DNA 弯曲方向具有至关重要的影响。手指肽应开发新型转基因植物和动物。