TATA-binding protein and the Gal4 transactivator do not bind to promoters cooperatively

The yeast Ga14 protein, like many activators, binds TATA-binding protein (T BP) directly in vitro. It has been speculated that this protein-protein int eraction is important for Ga14p-mediated activation of transcription, but l ittle work has been done to test specific models involving this interaction , In this study, the effect of Ga14p on TBP-TATA binding is addressed. Spec ifically, it is asked if the Ga14p-TBP interaction can support cooperative binding of the two factors to promoters. It is easy to see how such an even t could stimulate transcription, particularly horn promoters with a non-con sensus TATA box. In vitro, however, a derivative of GaMp (Ga14(1-93+768-881 )) containing the DNA-binding, dimerization, and activation domains does no t bind to promoter DNA cooperatively with either recombinant, purified TBP, or with protein from a yeast crude extract. In vivo, reporter gene experim ents using promoters with differing TBP affinities reveal no major GaMp-med iated stimulation of TBP function from weak TATA boxes, as would be predict ed if the proteins bind cooperatively. Furthermore, native Gal4p and a pote nt Ga14p-based artificial activator lacking a TBP-binding activation domain support similar ratios of transcription from a series of promoters identic al except for mutations in the TATA box. It is concluded that Ga14p and TBP do not bind cooperatively to promoters and that this mechanism does not co ntribute substantially to Ga14p-mediated transcriptional activation.