Binding of transcription factor Sp1 to GC box DNA revealed by footprinting analysis: different contact of three zinc fingers and sequence recognition mode.

Transcription factor Sp1 has three tandem repeats of a Cys2His2-type zinc finger motif and specifically binds to GC box DNA. We investigated the interaction of Sp1 with GC box DNA by several footprinting techniques. Methylation of four guanine bases in the sequence 5'-GGGCG-3' is strongly protected by Sp1 binding, whereas a guanine base flanked at the 5' end by the above sequence is extremely hypermethylated. Methylation interference experiments explicitly show that four guanine bases from the guanine-rich strand, and one from the cytosine-rich strand, in the sequence 5'-GGGCG-3' are crucially required for GC box recognition by Sp1. In footprinting using the 1,10-phenanthroline-copper complex, binding of Sp1 clearly alters the cleavage patterns by the metal complex. Footprints of the protein did not cover the full length of each GC box sequence, and the protein strongly masked scission in the sequence 5'-GCGG(A/G)(G/A)-3'. In cleavage of GC box DNA by the bleomycin-iron complex, Sp1 binding induces new cutting at a 5'-GA-3' site within the box. The results indicate that (i) the three zinc fingers do not contribute equivalently to the binding of Sp1 to the GC box, namely, important base contacts arise from the second and third fingers, and (ii) the protein binding induces local but significant structural distortion of the 3' region of the guanine-rich strand in the GC box. These features are clearly distinct from those of Zif268 and Krox20, which are three-zinc-finger proteins closely related to Sp1.