Topological effects of the TATA box binding protein on minicircle DNA and a possible thermodynamic linkage to chromatin remodeling

DNA ring closure experiments on short restriction fragments ( approximately 160 bp) bound by the TATA box binding protein (TBP) have demonstrated the formation of negative topoisomers, consistent with crystallographically observed TBP-induced DNA untwisting but in contrast to most previous results on topological effects in plasmid DNA. The difference may be due to the high free energy cost of substantial writhe in minicircles. A speculative mechanism for the loss of TBP-induced writhe suggests that TBP is capable of inducing DeltaTw between 0 and -0.3 in minicircles, via loss of out-of-plane bending upon retraction of intercalating Phe stirrups, and that TBP can thus act as a "supercoil shock absorber". The proposed biological relevance of these observations is that they may model the behavior of DNA in constrained chromatin environments. Irrespective of the detailed mechanism of TBP-induced supercoiling, its existence suggests that chromatin remodeling and enhanced TBP binding are thermodynamically linked. Remodeling ATPases or histone acetylases release some of the negative supercoiling previously restrained by the nucleosome. When TBP takes up the supercoiling, its binding should be enhanced transiently until the unrestrained supercoiling is removed by diffusion or topoisomerases. The effect is predicted to be independent of local remodeling-induced changes in TATA box accessibility.     

Signals for TBP/TATA box recognition

The TATA box-binding protein (TBP) recognizes its target sites (TATA boxes) by indirectly reading the DNA sequence through its conformation effects (indirect readout). Here, we explore the molecular mechanisms underlying indirect readout of TATA boxes by TBP by studying the binding of TBP to adenovirus major late promoter (AdMLP) sequence variants, including alterations inside as well as in the sequences flanking the TATA box. We measure here the dissociation kinetics of complexes of TBP with AdMLP targets and, by phase-sensitive assay, the intrinsic bending in the TATA box sequences as well as the bending of the same sequence induced by TBP binding. In these experiments we observe a correlation of the kinetic stability to sequence changes within the TATA recognition elements. Comparison of the kinetic data with structural properties of TATA boxes in known crystalline TBP/TATA box complexes reveals several "signals" for TATA box recognition, which are both on the single base-pair level, as well as larger DNA tracts within the TATA recognition element. The DNA bending induced by TBP on its binding sites is not correlated to the stability of TBP/TATA box complexes. Moreover, we observe a significant influence on the kinetic stability of alteration in the region flanking the TATA box. This effect is limited however to target sites with alternating TA sequences, whereas the AdMLP target, containing an A tract, is not influenced by these changes.     

How Proteins Recognize the TATA Box

The crystal structure of a complex of human TATA-binding protein with TATA-sequence DNA has been solved, complementing earlier TBP/DNA analyses fromSaccharomyces cerevisiaeandArabidopsis thaliana. Special insight into TATA box specificity is provided by considering the TBP/DNA complex, not as a protein molecule with bound DNA, but as a DNA duplex with a particularly large minor groove ligand. This point of view provides explanations for: (1) why T·A base-pairs are required rather than C·G; (2) why an alternation of T and A bases is needed; (3) how TBP recognizes the upstream and downstream ends of the TATA box in order to bind properly; and (4) why the second half of the TATA box can be more variable than the first.     

DNA and protein footprinting analysis of the modulation of DNA binding by the N-terminal domain of the Saccharomyces cerevisiae TATA binding protein

Recombinant full-length Saccharomyces cerevisiae TATA binding protein (TBP) and its isolated C-terminal conserved core domain (TBPc) were prepared with measured high specific DNA-binding activities. Direct, quantitative comparison of TATA box binding by TBP and TBPc reveals greater affinity by TBPc for either of two high-affinity sequences at several different experimental conditions. TBPc associates more rapidly than TBP to TATA box bearing DNA and dissociates more slowly. The structural origins of the thermodynamic and kinetic effects of the N-terminal domain on DNA binding by TBP were explored in comparative studies of TBPc and TBP by "protein footprinting" with hydroxyl radical (*OH) side chain oxidation. Some residues within TBPc and the C-terminal domain of TBP are comparably protected by DNA, consistent with solvent accessibility changes calculated from core domain crystal structures. In contrast, the reactivity of some residues located on the top surface and the DNA-binding saddle of the C-terminal domain differs between TBP and TBPc in both the presence and absence of bound DNA; these results are not predicted from the crystal structures. A strikingly different pattern of side chain oxidation is observed for TBP when a nonionic detergent is present. Taken together, these results are consistent with the N-terminal domain actively modulating TATA box binding by TBP and nonionic detergent modulating the interdomain interaction.     

Dimer dissociation and thermosensitivity kinetics of the Saccharomyces cerevisiae and human TATA binding proteins.

A kinetic analysis of dimer dissociation, TATA DNA binding, and thermal inactivation of the yeast Saccharomyces cerevisiae and human TATA binding proteins (TBP) was conducted. We find that yeast TBP dimers, like human TBP dimers, are slow to dissociate in vitro (t(1/2) approximately 20 min). Mild mutations in the crystallographic dimer interface accelerate the rate of dimer dissociation, whereas severe mutations prevent dimerization. In the presence of excess TATA DNA, which measures the entire active TBP population, dimer dissociation represents the rate-limiting step in DNA binding. These findings provide a biochemical extension to genetic studies demonstrating that TBP dimerization prevents unregulated gene expression in yeast [Jackson-Fisher, A. J., Chitikila, C., Mitra, M., and Pugh, B. F. (1999) Mol. Cell 3, 717-727]. In the presence of vast excesses of TBP over TATA DNA, which measures only a very small fraction of the total TBP, the monomer population in a monomer/dimer equilibrium binds DNA rapidly, which is consistent with a simultaneous binding and bending of the DNA. Under conditions where other studies failed to detect dimers, yeast TBP's DNA binding activity was extremely labile in the absence of TATA DNA, even at temperatures as low as 0 degrees C. Kinetic analyses of TBP instability in the absence of DNA at 30 degrees C revealed that even under fairly stabilizing solution conditions, TBP's DNA binding activity rapidly dissipated with t(1/2) values ranging from 6 to 26 min. TBP's stability appeared to vary with the square root of the TBP concentration, suggesting that TBP dimerization helps prevent TBP inactivation.