title: Functional silencing of TATA-binding protein (TBP) by a covalent linkage of the N-terminal domain of TBP-associated factor 1.
abstract: General transcription factor TFIID is comprised of TATA-binding protein (TBP) and TBP-associated factors (TAFs), together playing critical roles in regulation of transcription initiation. The TAF N-terminal domain (TAND) of yeast TAF1 containing two subdomains, TAND1 (residues 10-37) and TAND2 (residues 46-71), is sufficient to interact with TBP and suppress the TATA binding activity of TBP. However, the detailed structural analysis of the complex between yeast TBP and TAND12 (residues 6-71) was hindered by its poor solubility and stability in solution. Here we report a molecular engineering approach where the N terminus of TBP is fused to the C terminus of TAND12 via linkers of various lengths containing (GGGS)(n) sequence, (n = 1, 2, 3). The length of the linker within the TAND12-TBP fusion has a significant effect on solubility and stability (SAS). The construct with (GGGS)(3) linker produces the best quality single-quantum-coherence (HSQC) NMR spectrum with markedly improved SAS. In parallel to these observations, the TAND12-TBP fusion exhibits marked reduction of TBP function in binding to TAF1 as well as temperature sensitivity in in vivo yeast cell growth. Remarkably, the temperature sensitivity was proportional to the length of the linker in the fusions: the construct with (GGGS)(3) linker did not grow at 20 degrees C, while those with (GGGS)(1) and (GGGS)(2) linkers did. These results together indicate that the native interaction between TBP and TAND12 is well maintained in the TAND12-(GGGS)(3)-TBP fusion and that this fusion approach provides an excellent model system to investigate the structural detail of the TBP-TAF1 interaction.
authors.affiliation: Division of Signaling Biology, Ontario Cancer Institute, Department of Medical Biophysics, University of Toronto, Toronto Medical Discovery Towers, Toronto, Ontario, Canada.
authors.forename: Tapas K
authors.initials: TK
authors.lastname: Mal
authors.forename: Shinya
authors.initials: S
authors.lastname: Takahata
authors.forename: Sewon
authors.initials: S
authors.lastname: Ki
authors.forename: Le
authors.initials: L
authors.lastname: Zheng
authors.forename: Tetsuro
authors.initials: T
authors.lastname: Kokubo
authors.forename: Mitsuhiko
authors.initials: M
authors.lastname: Ikura
mesh_terms: D031541:Fluorescence Resonance Energy Transfer; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D035381:TATA-Binding Protein Associated Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D035181:TATA-Box Binding Protein / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013816:Thermodynamics; D035362:Transcription Factor TFIID / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D035381:TATA-Binding Protein Associated Factors; D035181:TATA-Box Binding Protein; D035362:Transcription Factor TFIID