THE MECHANISM OF TRANSCRIPTIONAL SYNERGY OF AN IN-VITRO ASSEMBLED INTERFERON-BETA ENHANCEOSOME

A functional interferon-beta gene enhanceosome was assembled in vitro using the purified recombinant transcriptional activator proteins ATF2 /c-JUN, IRF1, and p50/p65 of NF-kappa B. Maximal levels of transcripti onal synergy between these activators required the specific interactio ns with the architectural protein HMG IM and the correct helical phasi ng of the binding sites of these proteins on the DNA helix. Analyses o f the in vitro assembled enhanceosome revealed that the transcriptiona l synergy is due, at least in part, to the cooperative assembly and st ability of the complex. Reconstitution experiments showed that the for mation of a stable enhanceosome-dependent preinitiation complex requir es cooperative interactions between the enhanceosome; the general tran scription factors TFIID, TFIIA, and TFIIB; and the cofactor USA. These studies provide a direct biochemical demonstration of the importance of the structure and function of natural multicomponent transcriptiona l enhancer complexes in gene regulation.