Regulation of cellular genetic function by new DNA bending fingers

DNA弯曲在各种生物反应中都是非常重要的。特别是，已知许多转录因子可诱导DNA弯曲并支持转录起始复合物的形成。我们创造了新的人工6-锌指肽，可以有效地诱导DNA弯曲。转录因子Sp1的两个DNA结合域通过聚甘氨酸连接体连接。获得的6-锌指蛋白Sp1ZF6(Gly)n （n= 4,7,10）与天然3-锌指肽Sp1在DNA弯曲能力方面进行了比较。凝胶电泳结果显示，Sp1ZF6(Gly)_7和Sp1ZF6(Gly)_<10>与两个远端GC盒结合，导致DNA弯曲。相位分析强烈表明，诱导的DNA弯曲是指向主槽的，Sp1ZF6(Gly)_7引起的DNA弯曲方向变化最剧烈。特别有趣的是，两个3-锌指基序之间的连接体长度对整个DNA弯曲方向有至关重要的影响。此外，Sp1ZF6(Gly)_<10>的柔性中性连接体也转化为含精氨酸和谷氨酸残基的带电连接体。为了阐明DNA弯曲能力与转录活性之间的关系，我们还报道了基因检测。实验结果表明，人造锌指肽通过DNA弯曲激活基因表达。这种DNA弯曲指可能在不久的将来作为一种基于DNA结构变化的基因表达调节器是可行的。

DNA bending is very important for various biological reactions. In particular, many transcription factors have been known to induce DNA bending and to support formation of the transcriptional initiation complex. We crated new artificial 6-zinc finger peptides that effectively induced DNA bending. The two DNA binding domains of transcription factor Sp1 were connected through polyglycine linker. The obtained 6-zinc finger proteins Sp1ZF6(Gly)n (n=4, 7, 10) were compared with native 3-zinc finger peptide Sp1 in the respect of DNA bending ability. Gel electrophoretic methods revealed that Sp1ZF6(Gly)_7 and Sp1ZF6(Gly)_<10> bound to two distal GC boxes and resulted in DNA bending. The phasing assays strongly suggested that the induced DNA bending was directed toward the major groove and that Sp1ZF6(Gly)_7 caused the most drastic directional change in DNA bending. Of special interest is the fact that the linker length between two 3-zinc finger motifs has a crucial effect on the entire DNA bending direction. In addition, the flexible and neutral linker of Sp1ZF6(Gly)_<10> was also converted to charged linkers including arginine and glutamate residues. In order to clarify the relation between the DNA bending ability and the transcriptional activity, we also reporter gene assays. The experimental results led to the activation of gene expression by the DNA bending due to artificial zinc finger peptide. Such DNA bending fingers may be feasible for use as a gene expression regulator based on the structural change in DNA in the near future.