TIT for TAT: the properties of inosine and adenosine in TATA box DNA

The sequence dependent conformation, flexibility and hydration properties of DNA molecules constitute selectivity determinants in the formation of protein-DNA complexes. TATA boxes in which AT basepairs (bp) have been substituted by IC bp (TITI box) allow for probing these selectivity determinants for the complexation with the TATA box-binding protein (TBP) with different sequences but identical chemical surfaces. The reference promoter Adenovirus 2 Major Late Promoter (mlp) is formed by the apposition of two sequences with very different dynamic properties: an alternating TATA sequence and an A-tract. For a comparative study, we carried out molecular dynamics simulations of two DNA oligomers, one containing the mlp sequence (2 ns), and the other an analog where AT basepairs were substituted by IC basepairs (1 ns). The simulations, carried out with explicit solvent and counterinons, yield straight purine tracts, the A-tract being stiffer than the I-tract, an alternating structure for the YRYR tracts, and hydration patterns that differ between the purine tracts and the alternating sequence tracts. A detailed analysis of the proposed interactions responsible for the stiffness of the purine tracts indicates that the stacking between the bases bears the strongest correlation to stiffness. The hydration properties of the minor groove in the two oligomers are distinctly different. Such differences are likely to be responsible for the stronger binding of TBP to mlp over the inosine-substituted variant. The calculations were made possible by the development, described here, of a new set of forcefield parameters for inosine that complement the published CHARMM all-hydrogen nucleic acid parametrization.     

TIT for TAT: The Properties of Inosine and Adenosine in TATA Box DNA

Abstract

The sequence dependent conformation, flexibility and hydration properties of DNA molecules constitute selectivity determinants in the formation of protein-DNA complexes. TATA boxes in which AT basepairs (bp) have been substituted by IC bp (TITI box) allow for probing these selectivity determinants for the complexation with the TATA box-binding protein (TBP) with different sequences but identical chemical surfaces. The reference promoter Adenovirus 2 Major Late Promoter (mlp) is formed by the apposition of two sequences with very different dynamic properties: an alternating TATA sequence and an A-tract. For a comparative study, we carried out molecular dynamics simulations of two DNA oligomers, one containing the mlp sequence (2 ns), and the other an analog where AT basepairs were substituted by IC basepairs (1 ns). The simulations, carried out with explicit solvent and counteri-ons, yield straight purine tracts, the A-tract being stiffer than the I-tract, an alternating structure for the YRYR tracts, and hydration patterns that differ between the purine tracts and the alternating sequence tracts. A detailed analysis of the proposed interactions responsible for the stiffness of the purine tracts indicates that the stacking between the bases bears the strongest correlation to stiffness. The hydration properties of the minor groove in the two oligomers are distinctly different. Such differences are likely to be responsible for the stronger binding of TBP to mlp over the inosine-substituted variant. The calculations were made possible by the development, described here, of a new set of forcefield parameters for inosine that complement the published CHARMM all-hydrogen nucleic acid parametrization.     

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.     

A detailed interpretation of OH radical footprints in a TBP-DNA complex reveals the role of dynamics in the mechanism of sequence-specific binding

The hydroxyl radical footprint of the TATA-binding protein (TBP) bound to the high-affinity sequence TATAAAAG of the adenovirus 2 major late promoter has been quantitatively compared to a 2 ns molecular dynamics simulation of the complex in aqueous solution at room temperature using the CHARMM23 potential. The nucleotide-by-nucleotide analysis of the TBP-TATA hydroxyl radical footprint correlates with the solvent-accessible surface calculated from the dynamics simulation. The results suggest that local reactivity towards OH radicals results from the interplay between the local DNA geometry imposed by TBP binding, and the dynamics of the side-chains contacting the sugar hydrogen atoms. Analysis of the dynamics suggests that, over time, TBP forms stable interactions with the sugar-phosphate backbone through multiple contacts to different partners. This mechanism results in an enthalpic advantage to complex formation at a low entropic cost.     

Interaction of TFIID in the minor groove of the TATA element.

TFIID binding in the minor groove of DNA at the TATA element was demonstrated by methylation interference and hydroxyl radical footprinting assays, and by binding studies with thymine analog substituted oligonucleotides. These results provide an explanation for TFIID-dependent DNA bending at the TATA element. TFIID binding shows phosphate contacts with the same residues that were found to be essential for TFIID interactions by methylation and thymine-specific modification interference assays. Based on previous studies implicating residues conserved between the direct repeats in DNA binding, as well as models of prokaryotic DNA binding proteins, these results also suggest a model in which the direct repeats of TFIID form two basic antiparallel beta ribbon arms that could contact DNA through the minor groove.