MUTATIONS ON THE DNA-BINDING SURFACE OF TATA-BINDING PROTEIN CAN SPECIFICALLY IMPAIR THE RESPONSE TO ACIDIC ACTIVATORS IN-VIVO

The TATA-binding protein (TBP) contains a concave surface that interac ts specifically with TATA promoter elements and a convex surface that mediates protein-protein interactions with general and gene-specific t ranscription factors, Biochemical experiments suggest that interaction s between activator proteins and TBP are important in stimulating tran scription by the RNA polymerase II machinery. To gain insight into the role of TBP in mediating transcriptional activation in vivo, we imple mented a genetic strategy in Saccharomyces cerevisiae that involved th e use of a TBP derivative with altered specificity for TATA elements. By genetically screening a set of TBP mutant libraries that were biase d to the convex surface that mediates protein-protein interactions, we identified TBP derivatives that are impaired in the response to three acidic activators (Gcn4, Ga14, and Ace1) but appear normal for consti tutive polymerase II transcription, A genetic complementation assay in dicates that the activation-defective phenotypes reflect specific func tional properties of the TBP derivatives rather than an indirect effec t on transcription. Surprisingly, three of the four activation-defecti ve mutants affect residues that directly contact DNA. Moreover, all fo ur mutants are defective for TATA element binding, but they interact n ormally with an acidic activation domain and TFIIB. In addition, we sh ow that a subset of TBP derivatives with mutations on the DNA-binding surface of TBP are also compromised in their responses to acidic activ ators in vivo. These observations suggest that interactions at the TBP -TATA element interface can specifically affect the response to acidic activator proteins in vivo.