The TATA-Binding Protein Core Domain in Solution Variably Bends TATA Sequences via a Three-Step Binding Mechanism

• Published in: Biochemistry (Easton) 2009-03, Vol.48 (8), p.1801-1809
• Main Authors: Delgadillo, Roberto F, Whittington, JoDell E, Parkhurst, Laura K, Parkhurst, Lawrence J
• Format: Article
• Language: English
• Subjects: Base Sequence; Entropy; Fluorescence Resonance Energy Transfer; Humans; Kinetics; Nucleic Acid Conformation; Protein Binding; Protein Structure, Tertiary; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins - chemistry; Saccharomyces cerevisiae Proteins - metabolism; Solutions; TATA Box - genetics; TATA-Box Binding Protein - chemistry; TATA-Box Binding Protein - metabolism; Time Factors
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi8018724

Studies of the binding and bending of the AdMLP TATA sequence (TATAAAAG) by the core domain of yeast TBP allow quantitation of the roles of the N-terminal domains of yeast and human TBP. All three proteins bind DNA via a three-step mechanism with no evidence for an initially bound but unbent DNA. The large enthalpy and entropy of activation for the first step in y TBP binding can now be assigned to movement of the NTD from the DNA binding pocket and not to energetics of DNA bending. The energetic patterns for h TBP and c TBP suggest that the 158-amino acid NTD in h TBP does not initially occupy the DNA binding pocket. Despite the appearance of similar energetics for h TBP and c TBP, order of magnitude differences in rate constants lead to differing populations of intermediates during DNA binding. We find that the NTDs destabilize the three bound forms of DNA for both y TBP and h TBP. For all three proteins, the DNA bend angle (θ) depends on the TATA sequence, with θ for c TBP and h TBP being greater than that for y TBP. For all three proteins, θ for the G6 variant (TATAAGAG) varies with temperature and increases in the presence of osmolyte to be similar to that of AdMLP. Crystallographic studies of c TBP binding to a number of variants had shown no dependence of DNA bending on sequence. The results reported here reveal a clear structural difference for the bound DNA in solution versus the crystal; we attribute the difference to the presence of osmolytes in the crystals.