Evidence that a minor groove-binding peptide and a major groove-binding protein can simultaneously occupy a common site on DNA.

Affinity cleaving proteins have been synthesized based on the DNA-binding domain of the yeast transcriptional activator GCN4 with the DNA cleaving moiety Fe.EDTA attached at the NH2 terminus [Oakley, M. G., & Dervan, P. B. (1990) Science 248, 847]. Cleavage patterns generated by Fe-EDTA-GCN4(226-281) bound to the DNA sites 5'-CTGACTAAT-3' and 5'-ATGACTCTT-3' reveal that the NH2 termini of the GCN4 DNA-binding domain are located in the major groove of DNA, 9-10 base pairs apart, consistent with a Y-shaped dimeric structure. 1-Methylimidazole-2-carboxamide netropsin (2-ImN) is a designed synthetic peptide which binds in the minor groove of DNA at 5'-TGACT-3' sites as an antiparallel, side-by-side dimer [Mrksich, M., Wade, W. S., Dwyer, T. J., Geierstanger, B. H., Wemmer, D.E., & Dervan, P. B. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 7586]. Through the use of Fe.EDTA-GCN4(226-281) as a sequence-specific footprinting agent, it is shown that the dimeric protein GCN4-(226-281) and the dimeric peptide 2-ImN can simultaneously occupy their common binding site in the major and minor grooves of DNA, respectively. The association constants for 2-ImN in the presence and in the absence of Fe.EDTA-GCN4(226-281) are found to be similar, suggesting that the binding of the two dimers is not cooperative.