Dynamics of Fos-Jun-NFAT1 complexes

Transcription initiation in eukaryotes is controlled by nucleoprotein compl exes formed through cooperative interactions among multiple transcription r egulatory proteins. These complexes may be assembled via stochastic collisi ons or defined pathways, We investigated the dynamics of Fos-Jun-NFAT1 comp lexes by using a multicolor fluorescence resonance energy transfer assay. F os-Jun heterodimers can bind to AP-I sites in two opposite orientations, on ly one of which is populated in mature Fos-Jun-NFAT1 complexes. We studied the reversal of Fos-Jun binding orientation in response to NFAT1 by measuri ng the efficiencies of energy transfer from donor fluorophores linked to op posite ends of an oligonucleotide to an acceptor fluorophore linked to one subunit of the heterodimer. The reorientation of Fos-Jun by NFAT1 was not i nhibited by competitor oligonucleotides or heterodimers. The rate of Fos-Ju n reorientation was faster than the rate of heterodimer dissociation at som e binding sites. The facilitated reorientation of Fos-Jun heterodimers ther efore can enhance the efficiency of Fos-Jun-NFAT1 complex formation. We als o examined the influence of the preferred orientation of Fos-Jun binding on the stability and transcriptional activity of Fos-Jun-NFAT1 complexes, Com plexes formed at sites where Fos-Jun favored the same binding orientation i n the presence and absence of NFAT1 exhibited an B-fold slower dissociation rate than complexes formed at sites where Fos-Jun favored the opposite bin ding orientation. Fos-Jun-NFAT1 complexes also exhibited greater transcript ion activation at promoter elements that favored the same orientation of Fo s-Jun binding in the presence and absence of NFAT1, Thus, the orientation o f heterodimer binding can influence both the dynamics and promoter selectiv ity of multiprotein transcription regulatory complexes.