Artificial nine zinc-finger peptide with 30 base pair binding sites.
It is well-known that DNA binding of native nine zinc-finger protein TEIIIA is dominated by interaction of select few fingers. Newly designed zinc-finger peptide Sp1ZF9 containing nine Cys2-His2 type motifs has been manipulated. The DNA-binding property of Sp1ZF9 was compared with those of native three zinc-finger Sp1(530-623) and artificial six zinc-finger Sp1ZF6 peptides. Although the equilibrium time was less than 0.5 h for Sp1(530-623)-DNA complex, Sp1ZF6 and Sp1ZF9 required approximately 48 and 72 h, respectively, for full complex formation. Evidently, the footprinting analysis demonstrated that Sp1ZF9 and Sp1ZF6 bind at least 27 and 18 contiguous base pairs of DNA sequence, respectively. Sp1ZF9 showed two step bindings to DNA, namely first the recognition of GC (5'-GGG-GCG-GGGCC-3') sequence by the N-terminal Sp1 domain and next the recognition of the corresponding target sequences by the middle and C-terminal Sp1 domains. In contrast with unimolecular binding of Sp1ZF9 and Sp1ZF6, two Sp1(530-623) molecules bind to one GCIII (5'-GGG-GCG-GGG-GGG-GCG-GGG-GGG++ +-GCG-GGGCC-3') site region. Semispecific complex formed at the beginning of Sp1ZF9-DNA interaction has also been characterized by kinetic analysis using surface plasmon resonance. Interestingly, the association rate constants for GC and GCIII complexes of Sp1ZF9 are smaller than those of the corresponding Sp1(530-623) complexes. Of special interest is the fact that new nine zinc-finger peptide Sp1ZF9 can bind to DNA sequence of approximately 30 base pairs. Such multi zinc-finger peptides may be useful as genome-specific transcriptional switches in future.
Kamiuchi
T
T
Institute for Chemical Research, Kyoto University, Japan.
Abe
E
E
Imanishi
M
M
Kaji
T
T
Nagaoka
M
M
Sugiura
Y
Y
D020029:Base Pairing; D001665:Binding Sites / Q000235:genetics; D004268:DNA-Binding Proteins / Q000138:chemical synthesis / Q000235:genetics* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005813:Genes, Synthetic; D006801:Humans; D017874:Immediate-Early Proteins / Q000378:metabolism; D008562:Membrane Glycoproteins; D010446:Peptide Fragments / Q000737:chemistry; D010455:Peptides / Q000138:chemical synthesis / Q000235:genetics* / Q000378:metabolism; D016329:Sp1 Transcription Factor / Q000138:chemical synthesis / Q000737:chemistry; D015534:Trans-Activators; D014759:Viral Envelope Proteins; D014764:Viral Proteins; D016335:Zinc Fingers / Q000235:genetics*
D004268:DNA-Binding Proteins; D017874:Immediate-Early Proteins; D008562:Membrane Glycoproteins; D010446:Peptide Fragments; D010455:Peptides; D016329:Sp1 Transcription Factor; D015534:Trans-Activators; C077406:UL115 protein, Human herpesvirus 5; D014759:Viral Envelope Proteins; D014764:Viral Proteins; C071440:glycoprotein H, Cytomegalovirus; C499358:glycoprotein H, Human cytomegalovirus; C439068:glycoprotein O, cytomegalovirus; C499353:glycoprotein gCIII, Cytomegalovirus