Comparison of DNA-binding properties between BTEB and Sp1

We have expressed truncated forms of BTEB and Sp1 in Escherichia coli and investigated the DNA-binding properties of the two proteins. The two proteins as well as their chimeric proteins protected the same DNA region in the BTE sequence (a GC box in the P-4501A1 gene) as examined by ortho-phenanthroline-Cu footprinting. The region overlapped nearly perfectly with the GC box consensus sequence. Methylation interference footprinting revealed that all the guanines within the region and two other guanines in the close vicinity interacted with the proteins. Competitive gel mobility shift assay using various synthetic oligonucleotides of the GC box sequences as the competitors demonstrated that BTEB and Spl have similar sequence specificities for DNA binding. We have purified the bacterially expressed BTEB and measured the dissociation constant of the BTEB-BTE complex using gel mobility shift assay. The dissociation constant was (3.0±1.0)×l0^-10M and was comparable to that of Spl binding to a GC box. Taken together, these findings indicate that the binding modes of BTEB and Sp1 to the GC box are similar to each other.