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

蛋白质控制着大多数的生物反应，其表达水平的紊乱导致了许多疾病。基因组测序的出现和几个基因组完整序列的出现为研究生物学和通过对靶基因转录的特定调控来开发治疗策略提供了新的机会。因此，“人工抑制物”对转录水平的调控具有特别重要的意义。在通过设计或选择而操纵的DNA结合基序中，Cys_2-His_2锌指蛋白被证明是最有潜力操纵成通用和特定转录因子的。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，这是各种生物反应所必需的。弯曲的DNA-蛋白质复合体的动力学稳定性对转录效率有重要影响，因此，这种动力学稳定性的调节是转录调控的一个新概念。我们将转录因子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，构建了六锌指蛋白[SplZF6(Gly)10、SplZF6(GR)4和SplZF6(Ge)4]。时相分析表明，这三种锌指蛋白以相似的方式诱导DNA弯曲。然而，在表面等离子体共振实验的基础上，这些蛋白质之间的DNA结合动力学性质的特定差异被证明。这种对DNA-蛋白质复合体具有不同稳定性的DNA弯曲六锌指蛋白可能成为序列特异性转录动力学调控的新工具。

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.