Enhancement of TBP binding by activators and general transcription factors

Eukaryotic transcriptional activators function, at least in part, by promot ing assembly of the preinitiation complex(1-3), which comprises RNA polymer ase II and its general transcription factors (GTFs). Activator-mediated sti mulation of the assembly of the preinitiation complex has been studied in v itro but has been relatively refractory to in vivo analysis, Here we use a DNA-crosslinking/immunoprecipitation assay to study in living cells the fir st step in the assembly of the preinitiation complex, the interaction betwe en the TATA-box-binding protein (TBP) and its binding site, the TATA box. A nalysis of a variety of endogenous yeast genes, and of a series of activato rs of differing strength, reveals a general correlation between TBP binding and transcriptional activity. Using mutant yeast strains, we show that Mot 1 prevents the binding of TBP to inactive promoters and that activator-medi ated stimulation of TBP binding requires additional GTFs, including TFIIB a nd Srb4. Taken together, our results indicate that TBP binding in vivo is s tringently controlled, and that the ability of activators to stimulate this step in the assembly of the preinitiation complex is a highly cooperative process involving multiple transcription factors.