Influence of the N-terminal domain and divalent cations on self-association and DNA binding by the Saccharomyces cerevisiae TATA binding protein

Biochemistry. 2007 Apr 24;46(16):4876-87. doi: 10.1021/bi061651w. Epub 2007 Mar 23.

Abstract

The localization of a single tryptophan to the N-terminal domain and six tyrosines to the C-terminal domain of TBP allows intrinsic fluorescence to separately report on the structures and dynamics of the full-length TATA binding protein (TBP) of Saccharomyces cerevisiae and its C-terminal DNA binding domain (TBPc) as a function of self-association and DNA binding. TBPc is more compact than the C-terminal domain within the full-length protein. Quenching of the intrinsic fluorescence by DNA and external dynamic quenchers shows that the observed tyrosine fluorescence is due to the four residues surrounding the "DNA binding saddle" of the C-terminal domain. TBP's N-terminal domain unfolds and changes its position relative to the C-terminal domain upon DNA binding. It partially shields the DNA binding saddle in octameric TBP, shifting upon dissociation to monomers to expose the saddle to DNA. Structure-energetic correlations were obtained by comparing the contribution that electrostatic interactions make to DNA binding by TBP and TBPc; DNA binding by TBPc is more hydrophobic than that by TBP, suggesting that the N-terminal domain either interacts with bound DNA directly or screens a part of the C-terminal domain, diminishing its electronegativity. The competition between divalent cations, K+, and DNA is not straightforward. Divalent cations strengthen binding of TBP to DNA and do so more strongly for TBPc. We suggest that divalent cations affect the structure of the bound DNA perhaps by stabilizing its distorted conformation in complexes with TBPc and TBP and that the N-terminal domain mimics the effects of divalent cations. These data support an autoinhibitory mechanism in which competition between the N-terminal domain and DNA for the saddle diminishes the DNA binding affinity of the full-length protein.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Acrylamide / chemistry
  • Calcium / pharmacology
  • DNA / chemistry*
  • Fluorescence Polarization
  • Fluorescence Resonance Energy Transfer
  • Magnesium / pharmacology
  • Potassium Iodide / chemistry
  • Protein Structure, Tertiary
  • Saccharomyces cerevisiae / chemistry
  • Saccharomyces cerevisiae Proteins / chemistry*
  • Spectrometry, Fluorescence
  • Static Electricity
  • TATA-Box Binding Protein / chemistry*
  • Tyrosine / chemistry

Substances

  • Saccharomyces cerevisiae Proteins
  • TATA-Box Binding Protein
  • Potassium Iodide
  • Acrylamide
  • Tyrosine
  • DNA
  • Magnesium
  • Calcium