Binding of TFIID to the CYC1 TATA boxes in yeast occurs independently of upstream activating sequences.
Functional transcription initiation complexes can be assembled in vitro without the aid of regulatory factors that bind to upstream activating sequences. However, promoters that lack upstream activating sequences are transcribed poorly if at all in vivo, suggesting that regulatory factors are necessary for the assembly of transcription initiation complexes in cells. To test this possibility, we asked whether the general transcription factor TFIID can bind to a promoter in yeast that lacks upstream activating sequences and is transcriptionally inactive. Analysis of an inactive CYC1 core promoter by high-resolution genomic footprinting revealed efficient binding of TFIID to either of two TATA box elements. Addition of a heat shock element rendered this promoter highly responsive to induction of transcription by heat shock but did not alter the TATA box footprints in the core promoter. Inactivation of all but one TATA box by site-directed mutagenesis did not prevent TFIID from binding to the remaining wild-type TATA box independently of regulatory sequences. These results indicate that upstream regulatory factors are not required for the in vivo binding of TFIID to the CYC1 promoter and that binding of TFIID to DNA is not necessarily a rate-limiting step in the activation of transcription in cells. Differences in chromatin structure may account for why regulatory transcription factors are required for the binding of TFIID to some promoters but not to others.
Chen
J
J
Department of Microbiology and Molecular Genetics, University of Vermont, Burlington 05405.
Ding
M
M
Pederson
D S
DS
D001483:Base Sequence; D001665:Binding Sites; D003574:Cytochrome c Group / Q000235:genetics*; D045304:Cytochromes c; D004271:DNA, Fungal / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000096:biosynthesis; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D016385:TATA Box; D035362:Transcription Factor TFIID; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D001616:beta-Galactosidase / Q000096:biosynthesis
C014059:CYC1 protein, S cerevisiae; D003574:Cytochrome c Group; D004271:DNA, Fungal; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D035362:Transcription Factor TFIID; D014157:Transcription Factors; D045304:Cytochromes c; D001616:beta-Galactosidase