title: Reevaluation of transcriptional regulation by TATA-binding protein oligomerization: predominance of monomers.
abstract: The TATA-binding protein (TBP) plays an important role in transcriptional initiation by all three nuclear RNA polymerases. TBP contains a conserved C-terminal domain (cTBP) that binds DNA. Crystallographic studies of cTBP (i.e., TBP without the N-terminal domain) from various species and molecular biology studies of cTBP and mixed cTBP/TBP species have led to the view that DNA binding by TBP is regulated by TBP dimerization. Using sedimentation equilibrium, we show that yeast cTBP forms dimers in solution at 5 degrees C with a dissociation constant of 7 +/- 1 microM. This observation of cTBP dimers in solution is in accord with the dimeric state observed in crystal structures of cTBP. In contrast, physiologically relevant, full-length yeast TBP is monomeric at 5 degrees C and forms dimers at 30 degrees C with a dissociation constant of 51 +/- 16 microM. This dissociation constant precludes formation of stable full-length TBP dimers at physiological concentrations. In addition, we tested for yeast TBP oligomerization in the presence of TBP-associated factors in the context of TFIID. No evidence for TBP oligomers was found using immunoprecipitation techniques from yeast whole-cell extracts. We conclude that yeast TBP is predominantly monomeric under physiological conditions, arguing against a role for TBP dimerization in the regulation of transcriptional initiation.
authors.affiliation: Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado 80523-1870, USA.
authors.forename: K M
authors.initials: KM
authors.lastname: Campbell
authors.forename: R T
authors.initials: RT
authors.lastname: Ranallo
authors.forename: L A
authors.initials: LA
authors.lastname: Stargell
authors.forename: K J
authors.initials: KJ
authors.lastname: Lumb
mesh_terms: D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D008970:Molecular Weight; D009994:Osmolar Concentration; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011108:Polymers / Q000737:chemistry* / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics; D016385:TATA Box / Q000235:genetics; D035181:TATA-Box Binding Protein; D014157:Transcription Factors / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D014461:Ultracentrifugation
chemical_list: D004268:DNA-Binding Proteins; D010446:Peptide Fragments; D011108:Polymers; D035181:TATA-Box Binding Protein; D014157:Transcription Factors