Architectures of Transcriptional Regulation : Creation and Functional Analysis of Multi-Zinc Fingers

转录调控的架构：多锌指的创建和功能分析

基本信息

批准号：
12470505
负责人：
SUGIURA Yukio
金额：
$ 10.5万
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：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12470505/
关键词：
Transcription factor Zinc finger Architecture DNA binding DNA bending Multifinger Gene regulation Kinetic

项目摘要

Proteins control most biological reactions and the disorder of their expression level causes many diseases. The advent of genomic sequencing and the availability of the complete sequences of several genomes provide new opportunities to study biology and to develop therapeutic strategies through specific modulation of the transcription of target genes. Therefore, regulation of the transcription level by "artificial repressers" is of special importance. Of the DNA-binding motifs that have been manipulated by design or selection, Cys_2-His_2 zinc finger proteins have demonstrated the greatest potential for manipulation into general and specific transcription factors. Many transcription factors are known to induce DNA bending and support the formation of specific DNA architectures. The protein-induced DNA bending is helpful for many combinations of protein-protein and/or - DNA interactions that are necessary for various biological reactions. The kinetic stability of a bent DNA-protein complex has a significant influence on transcriptional efficiency, and hence, such regulation of the kinetic stability is a new con-cept for transcriptional regulation. We created six-zinc finger proteins [SplZF6(Gly) 10,SplZF6(GR)4,and SplZF6(GE)4] by connecting two DNA binding domains of transcription factor Spl with different charged linkers consisting of 10 amino acid residues. The phasing analyses suggested that these three zinc finger proteins induced DNA bending in an analogous manner. On the basis of the surface plasmon resonance experiments, however, specific differences in the kinetic properties of DNA binding among these proteins were demonstrated. Such DNA-bending six-zinc finger proteins with different stabilities for the bent DNA-protein complexes may be useful as new tools for the kinetic regulation of sequence specific transcription.

蛋白质控制大多数生物学反应及其表达水平的障碍会导致许多疾病。基因组测序的出现以及几种基因组的完整序列的可用性为研究生物学和通过特定调节靶基因转录而开发治疗策略提供了新的机会。因此，“人工压抑者”对转录水平的调节非常重要。在设计或选择操纵的DNA结合基序中，CYS_2-HIS_2锌指蛋白表明，将操纵和特定转录因子操纵的最大潜力。已知许多转录因子诱导DNA弯曲并支持特定DNA架构的形成。蛋白质诱导的DNA弯曲对各种生物反应所必需的蛋白质蛋白质和/或DNA相互作用的许多组合有助于。弯曲DNA-蛋白质复合物的动力学稳定性对转录效率具有显着影响，因此，这种调节动力学稳定性是转录调控的新概念。我们通过将两个转录因子SPL的DNA结合结构域与由10个氨基酸残基的不同电荷链接器连接起来，从而创建了六锌指蛋白[SPLZF6（GLY）10，SPLZF6（GR）4和SPLZF6（GE）4]。相位分析表明，这三种锌指蛋白以类似的方式诱导DNA弯曲。然而，根据表面等离子体共振实验，这些蛋白质中DNA结合的动力学特性的具体差异已证明。这种弯曲的DNA六锌指蛋白具有不同稳定性的弯曲DNA-蛋白质复合物的稳定性，可以作为序列特异性转录动力学调节的新工具。