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

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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.