Artificial DNA-bending six-zinc finger peptides with different charged linkers: distinct kinetic properties of DNA bindings.
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 concept for transcriptional regulation. We created six-zinc finger proteins [Sp1ZF6(Gly)10, Sp1ZF6(GR)4, and Sp1ZF6(GE)4] by connecting two DNA binding domains of transcription factor Sp1 with different charged linkers consisting of 10 amino acid residues. Gel mobility shift assays and methylation interference assays revealed that these artificial proteins recognized the expected DNA sequence and that the DNA binding specificities of these three proteins were similar regardless of the difference in the charges or flexibility of the linkers. 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. Of special interest is the fact that the dissociation rate of Sp1ZF6(Gly)10 was faster than that of Sp1ZF6(GR)4 despite the similarities in the DNA binding mode, the induced DNA structural change, and the association rate. 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.
Imanishi
Miki
M
Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan.
Sugiura
Yukio
Y
D001483:Base Sequence; D004247:DNA / Q000737:chemistry*; D007700:Kinetics; D009690:Nucleic Acid Conformation; D016329:Sp1 Transcription Factor / Q000737:chemistry; D020349:Surface Plasmon Resonance; D016335:Zinc Fingers
D016329:Sp1 Transcription Factor; D004247:DNA