Site-specific inhibition of transcription factor binding to DNA by a metallointercalator

The metallointercalator Lambda-1-Rh(MGP)(2)phi(5+) binds tightly and specif ically to the site 5'-CATATG-3' in the major groove of double helical DNA b y a combination of direct readout and shape selection. To examine competiti ve interactions between this small metal complex and a DNA-binding transcri ption factor,the preferred binding site for Lambda-1-Rh(MGP)(2)phi(5+) was engineered irate the AP-1 recognition element (ARE) of the major-groove bin ding bZIP transcription factor yAP-1, the yeast analogue of mammalian AP-1. Binding experiments confirmed that the modified ARE retained normal yAP-1 binding affinity. Photocleavage experiments demonstrated that the modified ARE contained a high-affinity binding site for Lambda-1-Rh(MGP)(2)phi(5+), whereas the native ARE showed no interaction. Competition experiments using gel shift mobility assays demonstrated that Lambda-1-Rh(MGP)(2)phi(5+) at 120 nM competes 50% of yAP-1 binding to the 5'-CATATG-3' containing oligonu cleotide. In contrast, competitive disruption of protein binding to the nat ive ARE requires 3 mu M Lambda-1-Rh(MGP)(2)phi(5+). Metallointercalator der ivatives, including geometric isomers of Lambda-1-Rh(MGP)(2)phi(5+), show I lo specific binding to the target site and show no inhibition of yAP-1/DNA complexes at concentrations as high as 20 mu M. Thus, metallointercalators can be tuned to show selectivity for major groove sites on DNA comparable t o transcription factors and indeed can inhibit transcription factor binding site selectively.