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.

蛋白质控制着大部分的生物反应，其表达水平的紊乱会导致许多疾病。基因组测序的出现和多个基因组完整序列的可用性为研究生物学和通过靶基因转录的特异性调节来制定治疗策略提供了新的机会。因此，“人工阻遏物”对转录水平的调控尤为重要。在通过设计或选择操纵的 DNA 结合基序中，Cys_2-His_2 锌指蛋白已证明具有操纵一般和特定转录因子的最大潜力。已知许多转录因子可诱导 DNA 弯曲并支持特定 DNA 结构的形成。蛋白质诱导的 DNA 弯曲有助于蛋白质-蛋白质和/或-DNA 相互作用的许多组合，这些相互作用是各种生物反应所必需的。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，这种动力学稳定性的调节是转录调节的新概念。我们通过将转录因子 Spl 的两个 DNA 结合域与由 10 个氨基酸残基组成的不同带电接头连接起来，创建了六锌指蛋白 [SplZF6(Gly) 10、SplZF6(GR)4 和 SplZF6(GE)4]。定相分析表明这三种锌指蛋白以类似的方式诱导 DNA 弯曲。然而，基于表面等离振子共振实验，这些蛋白质之间 DNA 结合的动力学特性的特定差异被证明。这种对弯曲DNA-蛋白质复合物具有不同稳定性的DNA弯曲六锌指蛋白可能可用作序列特异性转录的动力学调节的新工具。