Influence of the N-Terminal Domain and Divalent Cations on Self-Association and DNA Binding by the Saccharomyces cerevisiae TATA Binding Protein

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 fulllength protein.The initiation of gene transcription in eukaryotes requires assembly of a multiprotein preinitiation complex (PIC)1 that recruits RNA polymerase to a promoter. The binding of the TATA binding protein (TBP) to a TATA box sequence is a key step in PIC assembly at many promoters (1). Crystallographic and solution studies have established the formation of a 1:1 complex of TBP and TATA box (e.g., refs 2-5). TBP consists of a 180-residue C-terminal domain (TBPc) that is ~80% identical in sequence among all eukaryotes and a N-terminal domain that varies in both length and sequence (2). The shortest N-terminal domain consists of four residues in Pyrococcus woesei TBP (6) with the 158 residues of human TBP being the longest (7). The crystal structures of yeast, human, Arabidopsis, and archael TBP molecules that have been determined either lack or have rudimentary N-terminal domains (6,(8)(9)(10).

DNA and Protein Footprinting Analysis of the Modulation of DNA Binding by the N-Terminal Domain of the Saccharomyces cerevisiae TATA Binding Protein

“…The binding affinity difference between TBP and TBPc for the TATA box is as large as an order of magnitude in direct comparisons under identical experimental conditions (Figure 4). A smaller differential is observed in studies utilizing a 14 bp oligonucleotide, suggesting a contribution by the DNA flanking the TATA box to the difference observed by footprinting (51).…”

“…That this is not the case suggests that modulation of TATA box binding by TBP involves processes in addition to competition between DNA and the N-terminal domain for the surface of the saddle. The possibility that the N-terminal domain directly contacts the DNA is opposed by other structural studies (51). Changes in the structure of the C-terminal domain upon deletion of the N-terminal polypeptide is considered at the end of the discussion (51).…”

“…The possibility that the N-terminal domain directly contacts the DNA is opposed by other structural studies (51). Changes in the structure of the C-terminal domain upon deletion of the N-terminal polypeptide is considered at the end of the discussion (51).…”

“…The protein footprinting studies described in this paper report on the interaction of the C-and N-terminal domains. An indication of differences in the internal dynamics of the C-terminal domain with and without the N-terminal polypeptide comes from a TBP vs TBPc comparative analysis of the quenching of the intrinsic fluorescence of the tyrosines embedded within the C-terminal core domain together with FRET analysis of DNA bending (15,51,71). These studies have shown that the core domain is more compact in TBPc compared to the full-length protein both free in solution and complexed with DNA.…”

Salt-Mediated Electrostatics in the Association of TATA Binding Proteins to DNA: A Combined Molecular Mechanics/Poisson-Boltzmann Study

“…By measuring the binding affinity, K obs , of TBP to DNA at different salt concentrations, and estimating the coefficient, SK obs , from a linear fit of log K obs versus the logarithm of the instantaneous salt concentration of the solution, SK obs ¼ dlogK obs /dlog [salt], two independent groups reported differences in DNA-binding behavior for the mesophilic and halophilic TBPs (12)(13)(14).…”