Selected base sequence outside the target binding site of zinc finger protein Sp1

Nucleic Acids Res. 2001 Dec 15;29(24):4920-9. doi: 10.1093/nar/29.24.4920.


Human transcription factor Sp1 contains three contiguous repeats of the C2H2-type zinc finger motif and binds to the decanucleotide sequence 5'-(G/T)GGGCGG(G/A)(G/A)(C/T)-3' (GC box). In order to determine whether the three-zinc finger peptide Sp1(530-623) has selectivity for sequence outside the GC box, we used a selection and amplification of binding experiment. The high affinity sequence generated from this selection is 5'-GGGTGGGCGTGGC-3' (s-GC box), which is flanked by a novel conserved guanine triplet on the 5'-side of the core decanucleotide. Gel mobility shift assays reveal that Sp1(530-623) binds to the s-GC box with 2.3-fold higher affinity than to the wild-type GC box, 5'-GGGGCGGGGC-3' (c-GC box). DNase I and hydroxyl radical footprinting analyses show that the area of the s-GC box protected by binding of Sp1(530-623) is wider by 1 nt than that of the c-GC box. On the other hand, alkylation interference analyses demonstrate that Sp1(530-623) forms only one special base contact at the guanine triplet. With respect to cleavage of the c-GC and s-GC boxes by the 1,10-phenanthroline-copper complex (OP-Cu), binding of Sp1(530-623) has no effect on the cleavage pattern of the s-GC box, whereas OP-Cu actually enhances cleavage of the c-GC box. Additionally, the extent of cleavage of the s-GC box by DNase I and OP-Cu is clearly different from that of the c-GC box under peptide-free conditions. The results strongly indicate that: (i) the conformation of the s-GC box is evidently distinct from that of the c-GC box; (ii) Sp1(530-623) binds to the s-GC box without induction of a conformational change in DNA detectable by cleavage with OP-Cu. The present study provides useful information for the design of multi-zinc finger proteins with various sequence specificities.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • Binding Sites / genetics
  • Binding, Competitive
  • DNA / genetics
  • DNA / metabolism*
  • DNA Footprinting / methods
  • Electrophoretic Mobility Shift Assay
  • Humans
  • Molecular Sequence Data
  • Protein Binding
  • Sequence Homology, Amino Acid
  • Sp1 Transcription Factor / genetics
  • Sp1 Transcription Factor / metabolism*
  • Zinc Fingers / genetics*


  • Sp1 Transcription Factor
  • DNA