Promoter-specific activation defects by a novel yeast TBP mutant compromised for TFIIB interaction

TFIIB is an RNA polymerase II general transcription factor (GTF) that has a lso been implicated in the mechanism of action of certain promoter-specific activators (see, for examples, [1-11]). TFIIB enters the preinitiation com plex (PIC) primarily through contact with the TATA box binding protein (TBP ), an interaction mediated by three TBP residues [12-14]. To study the role of TFIIB in transcription activation in vivo, we randomly mutagenized thes e three residues in yeast TBP and screened for promoter-specific activation mutants. One mutant bearing a single conservative substitution, TBP-E186D, is the focus of this study. As expected, TBP-E186D binds normally to the T ATA box but fails to support the entry of TFIIB into the PIC. Cells express ing TBP-E186D are viable but have a severe slow-growth phenotype. Whole-gen ome expression analysis indicates that transcription of 17% of yeast genes are compromised by this mutation. Chimeric promoter analysis indicates that the region of the gene that confers sensitivity to the TBP-E186D mutation is the UAS (upstream activating sequence), which contains the activator bin ding sites. Most interestingly, other TBP mutants that interfere with diffe rent interactions (TFIIB, TFIIA, or the TATA box) and a TFIIB mutant defect ive for interaction with TBP all manifest distinct and selective promoter-s pecific activation defects. Our results implicate the entry of TFIIB into t he PIC as a critical step in the activation of certain promoters and reveal diverse mechanisms of transcription activation.