The transcription factor CREB binds to a DNA element known as the cAMP-regulated enhancer (CRE). CREB is activated through phosphorylation by protein kinase A (PKA), but precisely how phosphorylation stimulates CREB function is unknown. One model is that phosphorylation may allow the recruitment of coactivators which then interact with basal transcription factors. We have previously identified a nuclear protein of M(r)265K, CBP, that binds specifically to the PKA-phosphorylated form of CREB. We have used fluorescence anisotropy measurements to define the equilibrium binding parameters of the phosphoCREB:CBP interaction and report here that CBP can activate transcription through a region in its carboxy terminus. The activation domain of CBP interacts with the basal transcription factor TFIIB through a domain that is conserved in the yeast coactivator ADA-1 (ref. 8). Consistent with its role as a coactivator, CBP augments the activity of phosphorylated CREB to activate transcription of cAMP-responsive genes.

How eukaryotic promoter-specific activator proteins (activators) stimulate transcription is a central question. We have previously shown that an acidic activator can directly interact with the general transcription factor TFIIB and increase its stable assembly into a preinitiation complex. We have proposed that this increase in TFIIB assembly is at least part of the mechanism by which an acidic activator functions. A prediction of this hypothesis is that a TFIIB mutant unable to interact with an acidic activator could not support activated transcription, and here we present experiments that verify this prediction. In conjunction with previous studies, our results argue that interaction between an acidic activator and TFIIB is necessary for transcriptional activation.

Transcription factor IIB (TFIIB) plays a pivotal role in the formation of transcription-competent initiation complexes. TFIIB was found to interact with the TATA-binding protein, the small subunit of TFIIF, and RNA polymerase II. These interactions require distinct domains in THIB. Using the gel mobility-shiit assay, it was found that the amino terminus of TFIIB was necessary for the formation of complexes containing RNA polymerase II and TFIIF, whereas the carboxy-terminal domain, which is composed of two imperfect direct repeats and includes a putative amphipathic a-helix, was sufficient for the formation of complexes containing the TATA-binding protein and TFIIB (DB complex). Protein-protein interaction analyses demonstrate that the amphipathic a-helix in TFIIB is important for the interaction with the TATA-binding protein. Specific residues mapping to the carboxyl terminus of the second direct repeat were found to be crucial for the interaction of TFIIB and RNA polymerase II. The interaction with the small subunit of TFIIF was mapped to the amino terminus of TFIIB, which includes a zinc finger. Identification of the protein factors that govern transcription initiation by RNA polymerase II has led to the delineation of two functional groups of transcription factors. One group, the general transcription factors (GTFs), catalyze basal transcription through the TATA and initiator DNA elements in a multiprotein complex with RNA polymerase II {RNAPII} (for review, see Weis and Reinberg 1992~ Zawel and Reinberg 1992}. The second group, the specific factors, is composed of DNA-binding proteins that bind independently of RNAPII to specific DNA sequences upstream of the TATA and initiator DNA elements (for review, see Johnson and McKnight 1989~ Mitchell andTjian 1989}. The specific transcription factors are thought to participate directly in regulatory networks that mediate either stimulation or repression of basal transcription of specific genes. It is likely that the specific transcription factors exert their modulatory actions on basal transcription by interacting directly with the GTFs and RNAPII {for review, see aCorresponding author.

Transcriptional activator proteins (activators) function, at least in part, by increasing preinitiation complex assembly (for reviews see refs 1-4). Previous studies have shown that an acidic activator forms a contact with the general transcription factor TFIIB (refs 5-7) and recruits it into the preinitiation complex. Mutational studies indicate that this interaction between the acidic activator and TFIIB is required for transcriptional stimulation. We show here that the acidic activator-TFIIB interaction has an additional function in preinitiation complex assembly. We provide evidence that in native TFIIB the amino- and carboxy-terminal domains are engaged in an intramolecular interaction. The acidic activator disrupts this intramolecular interaction to expose binding sites for general transcription factors that enter the preinitiation complex through association with TFIIB. Thus, the acidic activator induces a conformational change in TFIIB that drives preinitiation complex assembly forward.