ENERGY-TRANSFER ANALYSIS OF FOS-JUN DIMERIZATION AND DNA-BINDING

The protooncogenes fos and jun encode proteins that bind to DNA as dim eric complexes and regulate gene expression. Protein dimerization is m ediated by a leucine zipper and results in juxtaposition of regions of each protein rich in basic amino acids that comprise a bimolecular DN A binding domain. We have developed an approach based on resonance ene rgy transfer for the quantitative analysis of dimerization and DNA bin ding by Fos and Jun in solution. Fos-(118-211) and Jun-(225-334) polyp eptides were labeled with either 5-iodoacetamidofluorescein or rhodami ne X iodoacetamide on unique cysteine residues located in their DNA bi nding domains. Formation of heterodimeric complexes between the labele d proteins allowed resonance energy transfer between the donor fluores cein and the acceptor rhodamine fluorophores. DNA binding induced a co nformational transition that increased the efficiency of resonance ene rgy transfer. This increase was consistent with a 3-Angstrom reduction in the distance between the fluorophores. Using this assay, we determ ined the affinity of the Fos-Jun interaction and examined the kinetics of dimerization and DNA binding as well as the rate of subunit exchan ge. Dimerization and DNA binding by Fos and Jun were rapid, with half- times of <10 s. In the absence of DNA, Fos and Jun subunits exchanged rapidly, with a half-time of <10 s. In contrast, in the presence of DN A, the complex was extremely stable. Thus, leucine zipper-containing t ranscription factors may exchange submits readily when free in solutio n, but not when bound to DNA.