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-DNA AdMLP reaction is characterized by high affinity (K d ‫؍‬ 5 nM), simultaneous binding and DNA bending, and rapid formation of a stable human TBP-DNA complex having DNA bent ϳ100°. 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 hTBP wt -induced bend is more severe. This directly measured hTBP wt -DNA AdMLP interaction differs fundamentally from a recently published hTBP wt -DNA AdMLP model characterized by low affinity ( M) 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.The recognition and binding of promoter TATA sequences (DNA TATA ) 1 by the TATA-binding protein (TBP) is central to the initiation and regulation of gene transcription in eukaryotes. The importance of the TBP-DNA TATA interaction has prompted extensive biochemical and biophysical investigations, which have revealed complex binding mechanisms and dramatic TBP-induced helical distortion, including DNA TATA bending and unwinding (see Ref. 1 and references therein and Refs. 2-11). The extent of the DNA TATA bending is TATA sequence-dependent in solution (8,9,12,13). Because these collective results have been obtained using different TBPs, full-length and truncated proteins, and diverse experimental conditions, key issues relating to the behavior and structure of TBP-promoter complexes remain unresolved.The 180-residue C-terminal DNA binding domains (CTD) of the TBPs from yeast (yTBP), human (hTBP), and Arabidopsis thaliana (aTBP) are highly conserved, with greater than 80% homology and with generally conservative substitutions in the remainder. The pseudo-symmetric TBP CTD contacts the duplex along the distorted minor groove via interactions that are largely non-polar and hydrophobic. The amino acid residues involved in these DNA contacts are nearly 100% conserved, with only a single variation in which Arg-204 in hTBP changes to Lys-110 in yTBP and Lys-68 in aTBP (2). The N-terminal domain is, in contrast, heterogenous between the yeast (60 amino acids (14)) and human (159 amino acids (15)) proteins and severely truncated in the A. thaliana protein (18 amino acids (16)).How these similarities and di...

TFIIA Changes the Conformation of the DNA in TBP/TATA Complexes and Increases their Kinetic Stability

“…This value is in agreement with the bend angle of 102°that Parkhurst and colleagues determined for human TBP bound to a TATA box of the same sequence. 18 Human TFIIA changes the conformation of the DNA in the TBP/TATA complex We next asked whether the association of the general transcription factor TFIIA with the TBP/TATA-14 complex alters the conformation of the TATA DNA. Solution-based FRET assays were performed on the TATA-14 construct with saturating concentrations of human TBP (25 nM) and human TFIIA (50 nM).…”

“…FRET has been used to study the bending of DNA by yeast and human TBP. 17,18 To begin to assess the effect of other transcription factors on the TBPinduced DNA bend, we established FRET assays. We initially probed TBP binding to the TATA-14 construct shown in Figure 1(a).…”

“…Using this approach, we confirmed earlier results, which showed that human TBP bends the TATA DNA by 102°when evaluated with the twokink bend model. 18 It is interesting to note that yeast TBP bends TATA DNA by 80°, 41 which is significantly less than that for human TBP, indicating that human TBP and yeast TBP have different properties in binding and bending TATA DNA.…”

“…Fluorescence resonance energy transfer (FRET) studies by Parkhurst and colleagues have shown that yeast TBP bends DNA 80°whereas human TBP bends the DNA 102°in solution, as determined using a two-kink model in which the net bend angle from linear is the sum of the two equal bend angles at each kink. [17][18][19] The bend angles calculated from these studies are thought to be the average between the bent and unbent populations, rather than one static bend angle. Therefore, at saturating TBP concentrations, the average calculated bend angle is dictated by the equilibrium constant for bending/unbending.…”

Basic statistical recipes for the emergence of biochemical discernment

“…The situation is more controversial for the eukaryotic DNAbending protein TATA-binding protein (TBP). The bending of its target DNA motif, the TATA-box, could follow an initial association of TBP with linear DNA (Zhao and Herr, 2002), but alternatively DNA bending and binding have also been proposed to occur simultaneously (Masters et al, 2003). The kinetic rational of simultaneous binding and folding has however been questioned (Hammes et al, 2009).…”