TATA-binding protein mutants that are lethal in the absence of the Nhp6 high-mobility-group protein.
The Saccharomyces cerevisiae Nhp6 protein is related to the high-mobility-group B family of architectural DNA-binding proteins that bind DNA nonspecifically but bend DNA sharply. Nhp6 is involved in transcriptional activation by both RNA polymerase II (Pol II) and Pol III. Our previous genetic studies have implicated Nhp6 in facilitating TATA-binding protein (TBP) binding to some Pol II promoters in vivo, and we have used a novel genetic screen to isolate 32 new mutations in TBP that are viable in wild-type cells but lethal in the absence of Nhp6. The TBP mutations that are lethal in the absence of Nhp6 cluster in three regions: on the upper surface of TBP that may have a regulatory role, near residues that contact Spt3, or near residues known to contact either TFIIA or Brf1 (in TFIIIB). The latter set of mutations suggests that Nhp6 becomes essential when a TBP mutant compromises its ability to interact with either TFIIA or Brf1. Importantly, the synthetic lethality for some of the TBP mutations is suppressed by a multicopy plasmid with SNR6 or by an spt3 mutation. It has been previously shown that nhp6ab mutants are defective in expressing SNR6, a Pol III-transcribed gene encoding the U6 splicing RNA. Chromatin immunoprecipitation experiments show that TBP binding to SNR6 is reduced in an nhp6ab mutant. Nhp6 interacts with Spt16/Pob3, the yeast equivalent of the FACT elongation complex, consistent with nhp6ab cells being extremely sensitive to 6-azauracil (6-AU). However, this 6-AU sensitivity can be suppressed by multicopy SNR6 or BRF1. Additionally, strains with SNR6 promoter mutations are sensitive to 6-AU, suggesting that decreased SNR6 RNA levels contribute to 6-AU sensitivity. These results challenge the widely held belief that 6-AU sensitivity results from a defect in transcriptional elongation.
Eriksson
Peter
P
Department of Pathology, University of Utah Health Sciences Center, Salt Lake City, UT 84132, USA.
Biswas
Debabrata
D
Yu
Yaxin
Y
Stewart
James M
JM
Stillman
David J
DJ
D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D024001:HMGN Proteins; D006609:High Mobility Group Proteins / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D020836:Protein Structure, Quaternary; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000502:physiology; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D035181:TATA-Box Binding Protein / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015533:Transcriptional Activation
D004268:DNA-Binding Proteins; D024001:HMGN Proteins; D006609:High Mobility Group Proteins; C086471:NHP6A protein, S cerevisiae; C086472:NHP6B protein, S cerevisiae; D009687:Nuclear Proteins; D029701:Saccharomyces cerevisiae Proteins; D035181:TATA-Box Binding Protein