Determination of binding constant of transcription factor AP‐1 and DNA

Abstract

The equilibrium binding and association kinetics of the fos-jun dimer (basic and leucine zipper domain) to the AP-1 DNA were studied using a quantitative assay. The basic-region and leucine zipper (bZip) domain of c-fos was expressed as a fusion protein with glutathione S-transferase, and it was bound to glutathione-agarose. The GST-fused fos bZip region was allowed to form a heterodimer with the bZip domain of c-jun, to which radiolabeled AP-1 nucleotides were added. After thorough washing, the gel-bound radioactivity was counted. The binding and dissociation rate constants (k(1) and k-(1)) of the fos-jun dimer and DNA could be obtained from a time-course experiment. The association binding constant (K(1)) was determined using both a thermodynamic equation and kinetic parameters. Nordihydroguaiaretic acid (NDGA), momordin I, natural product inhibitors of the fos-jun/DNA complex formation, was applied to this jun-GST-fused fos system and it was found to decrease the apparent equilibrium binding of dimer and DNA. The thermodynamic constant of dimer and inhibitor binding was also determined.