title: Native human TATA-binding protein simultaneously binds and bends promoter DNA without a slow isomerization step or TFIIB requirement.
abstract: The association of TATA-binding protein (TBP) with promoter DNA is central to the initiation and regulation of eukaryotic protein synthesis. Our laboratory has previously conducted detailed investigations of this interaction using yeast TBP and seven consensus and variant TATA sequences. We have now investigated this key interaction using human TBP and the TATA sequence from the adenovirus major late promoter (AdMLP). Recombinant native human protein was used together with fluorescently labeled DNA, allowing real time data acquisition in solution. We find that the wild-type hTBP-DNAAdMLP reaction is characterized by high affinity (Kd < or = 5 nm), simultaneous binding and DNA bending, and rapid formation of a stable human TBP-DNA complex having DNA bent approximately 100 degrees. These data allow, for the first time, a direct comparison of the reactions of the full-length, native human and yeast TBPs with a consensus promoter, studied under identical conditions. The general reaction characteristics are similar for the human and yeast proteins, although the details differ and the hTBPwt-induced bend is more severe. This directly measured hTBPwt-DNAAdMLP interaction differs fundamentally from a recently published hTBPwt-DNAAdMLP model characterized by low affinity (microM) binding and an unstable complex requiring either a 30-min isomerization or TFIIB to achieve DNA bending. Possible sources of these significant differences are discussed.
authors.affiliation: Department of Chemistry, University of Nebraska, Lincoln, Nebraska 68588-0304, USA.
authors.forename: Kristina M
authors.initials: KM
authors.lastname: Masters
authors.forename: Kay M
authors.initials: KM
authors.lastname: Parkhurst
authors.forename: Margaret A
authors.initials: MA
authors.lastname: Daugherty
authors.forename: Lawrence J
authors.initials: LJ
authors.lastname: Parkhurst
mesh_terms: D004268:DNA-Binding Proteins / Q000378:metabolism*; D006801:Humans; D007536:Isomerism; D009690:Nucleic Acid Conformation; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D035181:TATA-Box Binding Protein / Q000378:metabolism*; D035581:Transcription Factor TFIIB / Q000378:metabolism*
chemical_list: D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D035181:TATA-Box Binding Protein; D035581:Transcription Factor TFIIB