The yeast FACT complex has a role in transcriptional initiation.
A crucial step in eukaryotic transcriptional initiation is recognition of the promoter TATA by the TATA-binding protein (TBP), which then allows TFIIA and TFIIB to be recruited. However, nucleosomes block the interaction between TBP and DNA. We show that the yeast FACT complex (yFACT) promotes TBP binding to a TATA box in chromatin both in vivo and in vitro. The SPT16 gene encodes a subunit of yFACT, and we show that certain spt16 mutations are synthetically lethal with TBP mutants. Some of these genetic defects can be suppressed by TFIIA overexpression, strongly suggesting a role for yFACT in TBP-TFIIA complex formation in vivo. Mutations in the TOA2 subunit of TFIIA that disrupt TBP-TFIIA complex formation in vitro are also synthetically lethal with spt16. In some cases this spt16 toa2 lethality is suppressed by overexpression of TBP or the Nhp6 architectural transcription factor that is also a component of yFACT. The Spt3 protein in the SAGA complex has been shown to regulate TBP binding at certain promoters, and we show that some spt16 phenotypes can be suppressed by spt3 mutations. Chromatin immunoprecipitations show TBP binding to promoters is reduced in single spt16 and spt3 mutants but increases in the spt16 spt3 double mutant, reflecting the mutual suppression seen in the genetic assays. Finally, in vitro studies show that yFACT promotes TBP binding to a TATA sequence within a reconstituted nucleosome in a TFIIA-dependent manner. Thus, yFACT functions in establishing transcription initiation complexes in addition to the previously described role in elongation.
Biswas
Debabrata
D
Department of Pathology, University of Utah Health Sciences Center, 30 North 1900 East, Salt Lake City, Utah 84132-2501, USA.
Yu
Yaxin
Y
Prall
Matthew
M
Formosa
Tim
T
Stillman
David J
DJ
D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D047369:Chromatin Immunoprecipitation; D017353:Gene Deletion; D005804:Genes, Lethal; D009154:Mutation; D009707:Nucleosomes / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*; D013489:Suppression, Genetic; D016385:TATA Box / Q000235:genetics; D035181:TATA-Box Binding Protein / Q000378:metabolism*; D035561:Transcription Factor TFIIA / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D014158:Transcription, Genetic / Q000235:genetics / Q000502:physiology*; D035602:Transcriptional Elongation Factors
D018797:Cell Cycle Proteins; D009707:Nucleosomes; C071238:SPT16 protein, S cerevisiae; C075582:SPT3 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D035181:TATA-Box Binding Protein; C441520:TOA2 protein, S cerevisiae; D035561:Transcription Factor TFIIA; D014157:Transcription Factors; D035602:Transcriptional Elongation Factors