Transcriptional activation by TFIIB mutants that are severely impaired in interaction with promoter DNA and acidic activation domains.
Biochemical experiments indicate that the general transcription factor IIB (TFIIB) can interact directly with acidic activation domains and that activators can stimulate transcription by increasing recruitment of TFIIB to promoters. For promoters at which recruitment of TFIIB to promoters is limiting in vivo, one would predict that transcriptional activity should be particularly sensitive to TFIIB mutations that decrease the association of TFIIB with promoter DNA and/or with activation domains; i.e., such TFIIB mutations should exacerbate a limiting step that occurs in wild-type cells. Here, we describe mutations on the DNA-binding surface of TFIIB that severely affect both TATA-binding protein (TBP)-TFIIB-TATA complex formation and interaction with the VP16 activation domain in vitro. These TFIIB mutations affect the stability of the TBP-TFIIB-TATA complex in vivo because they are synthetically lethal in combination with TBP mutants impaired for TFIIB binding. Interestingly, these TFIIB derivatives support viability, and they efficiently respond to Gal4-VP16 and natural acidic activators in different promoter contexts. These results suggest that in vivo, recruitment of TFIIB is not generally a limiting step for acidic activators. However, one TFIIB derivative shows reduced transcription of GAL4, suggesting that TFIIB may be limiting at a subset of promoters in vivo.
Chou
S
S
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Struhl
K
K
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics; D005800:Genes, Fungal; D017405:Herpes Simplex Virus Protein Vmw65 / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D015345:Oligonucleotide Probes / Q000235:genetics; D011401:Promoter Regions, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D035181:TATA-Box Binding Protein; D035581:Transcription Factor TFIIB; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015533:Transcriptional Activation
D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C052130:GAL4 protein, S cerevisiae; D017405:Herpes Simplex Virus Protein Vmw65; D015345:Oligonucleotide Probes; D029701:Saccharomyces cerevisiae Proteins; D035181:TATA-Box Binding Protein; D035581:Transcription Factor TFIIB; D014157:Transcription Factors