Energy transfer analysis of Fos-Jun dimerization and DNA binding.
The protooncogenes fos and jun encode proteins that bind to DNA as dimeric complexes and regulate gene expression. Protein dimerization is mediated by a leucine zipper and results in juxtaposition of regions of each protein rich in basic amino acids that comprise a bimolecular DNA binding domain. We have developed an approach based on resonance energy transfer for the quantitative analysis of dimerization and DNA binding by Fos and Jun in solution. Fos-(118-211) and Jun-(225-334) polypeptides were labeled with either 5-iodoacetamidofluorescein or rhodamine X iodoacetamide on unique cysteine residues located in their DNA binding domains. Formation of heterodimeric complexes between the labeled proteins allowed resonance energy transfer between the donor fluorescein and the acceptor rhodamine fluorophores. DNA binding induced a conformational transition that increased the efficiency of resonance energy transfer. This increase was consistent with a 3-A reduction in the distance between the fluorophores. Using this assay, we determined the affinity of the Fos-Jun interaction and examined the kinetics of dimerization and DNA binding as well as the rate of subunit exchange. 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 DNA, the complex was extremely stable. Thus, leucine zipper-containing transcription factors may exchange subunits readily when free in solution, but not when bound to DNA.
Patel
L R
LR
Roche Institute of Molecular Biology, Nutley, NJ 07110.
Curran
T
T
Kerppola
T K
TK
D001704:Biopolymers; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism*; D004735:Energy Transfer; D004926:Escherichia coli; D016760:Proto-Oncogene Proteins c-fos / Q000378:metabolism*; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism*
D001704:Biopolymers; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D004247:DNA