Unique DNA binding mode of the N-terminal zinc finger of transcription factor Sp1.
Transcription factor Sp1 has three tandem repeats of a Cys2His2-type zinc finger motif and specifically binds to GC box DNA. Although the DNA binding mode of the three zinc fingers of Sp1 is predicted to be similar to that of Zif268, this model does not explain the DNA binding property of the N-terminal zinc finger (finger 1). To understand the DNA recognition mode of Sp1, we have performed detailed analyses for the contribution of finger 1 to the high-affinity binding to the GC box DNA and for the interaction mechanism between finger 1 and DNA. Results of electrophoretic analyses using finger-deleted mutants of Sp1 and GC box mutants in the finger-contacting subsite demonstrate that the contribution of finger 1 to the total DNA binding affinity is lower than that of the C-terminal finger 3 but is dispensable for the high-affinity binding. The DNA sequence selectivity of finger 1 at the 3'-portion of the GC box is lower than that of fingers 2 and 3 at the 5'-portion. Alanine scanning mutagenesis in the alpha-helix of finger 1 reveals that Lys-1 immediately preceding the helix is important for the recognition of the two guanine bases, but other putative key amino acids do not affect the DNA binding. These results demonstrate that (1) the contribution of finger 1 to the DNA binding affinity and the sequence selectivity of Sp1 is smaller than that of fingers 2 and 3 and (2) the interaction mechanism between finger 1 and DNA is different from the Zif268 model. DNA interaction of Sp1 finger 1 has also been discussed in connection with that of TFIIIA or WT1.
Yokono
M
M
Institute for Chemical Research, Kyoto University, Japan.
Saegusa
N
N
Matsushita
K
K
Sugiura
Y
Y
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000596:Amino Acids / Q000235:genetics / Q000378:metabolism; D016384:Consensus Sequence / Q000235:genetics; D004247:DNA / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding / Q000235:genetics; D017384:Sequence Deletion; D016329:Sp1 Transcription Factor / Q000235:genetics / Q000378:metabolism*; D016335:Zinc Fingers / Q000235:genetics*
D000596:Amino Acids; D016329:Sp1 Transcription Factor; D004247:DNA