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 also been implicated in the mechanism of action of certain promoter-specific activators (see, for examples, [1-11]). TFIIB enters the preinitiation complex (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 these 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 TATA box but fails to support the entry of TFIIB into the PIC. Cells expressing TBP-E186D are viable but have a severe slow-growth phenotype. Whole-genome 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 binding sites. Most interestingly, other TBP mutants that interfere with different interactions (TFIIB, TFIIA, or the TATA box) and a TFIIB mutant defective for interaction with TBP all manifest distinct and selective promoter-specific activation defects. Our results implicate the entry of TFIIB into the PIC as a critical step in the activation of certain promoters and reveal diverse mechanisms of transcription activation.
Virbasius
C M
CM
Howard Hughes Medical Institute, Programs in Gene Function and Expression, University of Massachusetts Medical School, 373 Plantation Street, Worcester, MA 01605, USA.
Holstege
F C
FC
Young
R A
RA
Green
M R
MR
D002352:Carrier Proteins; D016213:Cyclins / Q000235:genetics; D003574:Cytochrome c Group / Q000235:genetics; D045304:Cytochromes c; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D008668:Metallothionein / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics; D016385:TATA Box; D035181:TATA-Box Binding Protein; D035581:Transcription Factor TFIIB; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation
C082721:CLN2 protein, S cerevisiae; C500769:CUP1-1 protein, S cerevisiae; C014059:CYC1 protein, S cerevisiae; D002352:Carrier Proteins; D016213:Cyclins; D003574:Cytochrome c Group; D004268:DNA-Binding Proteins; D029701:Saccharomyces cerevisiae Proteins; D035181:TATA-Box Binding Protein; D035581:Transcription Factor TFIIB; D014157:Transcription Factors; C007121:copper thionein; D045304:Cytochromes c; D008668:Metallothionein