The transcriptional activity of an in vitro assembled human interferon-beta gene 
enhanceosome is highly synergistic. This synergy requires five distinct 
transcriptional activator proteins (ATF2/c-JUN, interferon regulatory factor 1, 
and p50/p65 of NF-kappaB), the high mobility group protein HMG I(Y), and the 
correct alignment of protein-binding sites on the face of the DNA double helix. 
Here, we investigate the mechanisms of enhanceosome-dependent transcriptional 
synergy during preinitiation complex assembly in vitro. We show that the 
stereospecific assembly of the enhanceosome is critical for the efficient 
recruitment of TFIIB into a template-committed TFIID-TFIIA-USA (upstream 
stimulatory activity complex) and for the subsequent recruitment of the RNA 
polymerase II holoenzyme complex. In addition, we provide evidence that 
recruitment of the holoenzyme by the enhanceosome is due, at least in part, to 
interactions between the enhanceosome and the transcriptional coactivator CREB, 
cAMP responsive element binding protein (CBP). These studies reveal a unique 
role of enhanceosomes in the cooperative assembly of the transcription machinery 
on the human interferon-beta promoter.