| Activation of transcription of a protein coding gene requires the assembly of a large preinitiation complex (PIC) of general transcription factors (GTFs) and RNA polymerase II at the promoter. How specific activators can positively influence this process and thus "activate" gene transcription is a central question in the study of eukaryotic gene regulation. The multi-subunit factor TFIID, which contains the TATA-binding protein (TBP) and several TBP-associated factors (TAFs), must bind to the promoter DNA before a functional PIC can assemble. A large number of activator proteins have been shown to interact with the GTFs and many, including CREB, have been shown to bind to specific TAFs within the TFIID complex. These facts lead to our hypothesis that the mechanism of gene activation by the CREB CAD involves binding to one of the TAFs and recruiting TFIID to the promoter, thus, facilitating the formation of the remainder of the PIC and increasing the overall rate of initiation of transcription.; The cAMP response element binding protein (CREB) contains two separate and independent activation domains, a kinase inducible domain (KID) and a constitutive activation domain (CAD). The identification of an interaction between the CREB CAD and a subunit of the TFIID complex, TAF110, prompted us to investigate whether interaction with this TAF, and recruitment of TFIID to the promoter, is the mechanism of transcriptional activation by the CREB CAD. Using both two-hybrid and reverse two-hybrid systems in yeast, together with a PCR-based random mutagenesis technique, we mapped the amino acid residues in the CAD involved in direct interaction with TAF110 to specific hydrophobic residues in a central hydrophobic cluster (HC). With a co-immunoprecipitation assay, we showed that CAD mutants, that are defective for binding to TAF110 in yeast, are also impaired for binding to TAF110 in vitro compared to wild type. An in vitro recruitment assay, which demonstrated that the CREB CAD is sufficient to recruit an RNA polymerase II complex to the promoter, showed that CAD mutants that are compromised for TAF110 binding are also defective for recruitment of the complex. Using transient transfection experiments in mammalian cells in culture, we showed that the CAD mutants are also completely impaired for activation of transcription in vivo. Using a similar system, we have also shown that TAF110 mutant proteins, that cannot interact with the CAD, or that cannot integrate into the TFIID complex, cannot serve as a coactivator for transcription by the CAD in vivo.; The results presented here support a mechanism for gene activation by the CREB CAD in which the CAD interacts with the TAF110 subunit of TFIID, recruiting TFIID to the promoter, thereby facilitating the formation of the remainder of the RNA polymerase II preinitiation complex, thus, increasing the rate of initiation of transcription of the gene. |
