Sequence-specific recognition of DNA by zinc-finger peptides derived from the transcription factor Sp1.
We have overexpressed and purified two peptide fragments of Sp1 that contain the three "zinc-finger" domains necessary for specific Sp1 DNA binding. These peptides assume a stable, folded conformation in solution in the presence of Zn2+ as shown by DNA binding assays and NMR spectroscopy. Mobility-shift assays demonstrate that the Sp1 peptides recognize a number of different Sp1 DNA binding sites (GC boxes, with the core sequence GGGCGG). The dissociation constant for a 92-amino acid peptide binding to the GGGGCGGGGC sequence (Kd approximately 10 nM) and the relative affinities for several other DNA sequences definitively demonstrate Sp1-like binding properties. The thermodynamic binding site for Sp1-Zn92 has been mapped using the primer-extension/mobility-shift assay revealing that the 5' portion of the GC box DNA sequence (GGG GCG) contributes more strongly to the total binding energy than the 3' portion (GGGC). These findings are interpreted in the context of the Sp1 amino acid sequence in comparison with the structurally characterized Zif-268/DNA complex. A model is proposed that offers a structural explanation for the ability of Sp1 to recognize a diverse array of DNA sequences in terms of the individual (and different) DNA binding properties of each of the three zinc-finger domains.
Kriwacki
R W
RW
Department of Chemistry, Yale University, New Haven, CT 06511.
Schultz
S C
SC
Steitz
T A
TA
Caradonna
J P
JP
D000595:Amino Acid Sequence; D001483:Base Sequence; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D016325:HIV Long Terminal Repeat / Q000235:genetics; D066298:In Vitro Techniques; D008668:Metallothionein / Q000235:genetics; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D011401:Promoter Regions, Genetic; D013539:Simian virus 40 / Q000235:genetics; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics; D014157:Transcription Factors / Q000378:metabolism; D016335:Zinc Fingers
D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D010455:Peptides; D016329:Sp1 Transcription Factor; D014157:Transcription Factors; D004247:DNA; D008668:Metallothionein; D013762:Tetrahydrofolate Dehydrogenase