Selected base sequence outside the target binding site of zinc finger protein Sp1
AUTOR(ES)
Nagaoka, Makoto
FONTE
Oxford University Press
RESUMO
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.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=97581Documentos Relacionados
- Sp1 and the subfamily of zinc finger proteins with guanine-rich binding sites.
- Importance of a flanking AT-rich region in target site recognition by the GC box-binding zinc finger protein MIG1.
- Targeting of Sp1 to a non-Sp1 site in the human neurofilament (H) promoter via an intermediary DNA-binding protein.
- Toward rules relating zinc finger protein sequences and DNA binding site preferences.
- Evi-1, a murine zinc finger proto-oncogene, encodes a sequence-specific DNA-binding protein.