Creation and Function of Novel Gene Regulation Molecules Based on Zinc Finger Motif

On the basis of the DNA recognition mode and the structural features unique to the Cys I D 22 ii D 2-His I D 22 ii class of nucleic acid binding，approaches to link Cys ii D 22 ii D 2 zinc fingers with other functional modules such as DNA binding domains to generate artificial chimeric peptides with long binding sites，and DNA-cleavage modules to produce novel sequence specificial chimeric peptides with long binding sites，and DNA-cleavage modules to produce novel sequence specificial chimeric peptides with long binding sites，and DNA-cleavage modules to produce novel sequence specificial chimeric peptides with long binding sites.Statistically，assuming random base distribution，a unique 16-or 18-base pair sequence will occur only once in 4.3 billion or 68billion nucleotides，roughly the same or big size of a human genome.Although natural proteins containing long polydactyl arrayl arrays of zinc finger do-mains have been inferred from sequence，no zinc finger proteins have been demonstrated to bind such a long，contiguous DNA sequence。Therefore，it is of special interest that six-or nine-fingered peptide was created and demonstrated to bind 18 or 27 contiguous base pairs of DNA in a sequence specific fashion.An artificial protein that induces a DNA conformational change is interesting as a transcriptional regulator of a specific gene.We created 6-zinc finger proteins，Sp1ZF6(Gly)n(n=4，7，10)，by connecting two DNA binding domains of transcription factor Sp1with flexible polyglycine pide linkers，and their effects on DNA structure were compared with that of native3-zinc finger Sp1(530-623)。Of special interest are the fact that newly designed6-finger peptides Sp1ZF(Gly)7and Sp1ZF(Gly)10can induce DNA bending at the intervening region of the two distal binding sites and that the liker length between two3-zinc finger motifs has a crucial effect on the entire DNA bending direction.Such polydactyl zinc finger peptides should be the development of novel transgenic plants and animals.

On the basis of the DNA recognition mode and the structural features unique to the CysィイD22ィエD2-HisィイD22ィエD2 class of nucleic acid binding, approaches to link CysィイD22ィエD2-HisィイD22ィエD2 zinc fingers with other functional modules such as DNA binding domains to generate artificial chimeric peptides with long binding sites, and DNA-cleavage modules to produce novel sequence specific nucleases, ィイD14-5ィエD1 have been performed. Statistically, assuming random base distribution, a unique 16-or 18-base pair sequence will occur only once in 4.3 billion or 68 billion nucleotides, roughly the same or big size of a human genome. Although natural proteins containing long polydactyl arrayl arrays of zinc finger do-mains have been inferred from sequence, no zinc finger proteins have been demonstrated to bind such a long, contiguous DNA sequence. Therefore, it is of special interest that six-or nine-fingered peptide was created and demonstrated to bind 18 or 27 contiguous base pairs of DNA in a sequence specific fashion. An artificial protein that induces a DNA conformational change is interesting as a transcriptional regulator of a specific gene. We created 6-zinc finger proteins, Sp1ZF6(Gly)n(n=4,7,10), by connecting two DNA binding domains of transcription factor Sp1 with flexible polyglycine peptide linkers, and their effects on DNA structure were compared with that of native 3-zinc finger Sp1(530-623). Of special interest are the fact that newly designed 6-finger peptides Sp1ZF(Gly)7 and Sp1ZF(Gly)10 can induce DNA bending at the intervening region of the two distal binding sites and that the liker length between two 3-zinc finger motifs has a crucial effect on the entire DNA bending direction. Such polydactyl zinc finger peptides should be the development of novel transgenic plants and animals.