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.     

Nearest-neighbor non-additivity versus long-range non-additivity in TATA-box structure and its implications for TBP-binding mechanism

TBP recognizes its target sites, TATA boxes, by recognizing their sequence-dependent structure and flexibility. Studying this mode of TATA-box recognition, termed ‘indirect readout’, is important for elucidating the binding mechanism in this system, as well as for developing methods to locate new binding sites in genomic DNA. We determined the binding stability and TBP-induced TATA-box bending for consensus-like TATA boxes. In addition, we calculated the individual information score of all studied sequences. We show that various non-additive effects exist in TATA boxes, dependent on their structural properties. By several criterions, we divide TATA boxes to two main groups. The first group contains sequences with 3–4 consecutive adenines. Sequences in this group have a rigid context-independent cooperative structure, best described by a nearest-neighbor non-additive model. Sequences in the second group have a flexible, context-dependent conformation, which cannot be described by an additive model or by a nearest-neighbor non-additive model. Classifying TATA boxes by these and other structural rules clarifies the different recognition pathways and binding mechanisms used by TBP upon binding to different TATA boxes. We discuss the structural and evolutionary sources of the difficulties in predicting new binding sites by probabilistic weight-matrix methods for proteins in which indirect readout is dominant.     

On the Information Content of Protein Sequences

Abstract

TATA-box binding protein (TBP) in a monomelic form and the complexes it forms with DNA have been elucidated with molecular dynamics simulations. Large TBP domain motions (bend and twist) are detected in the monomer as well as in the DNA complexes; these motions can be important for TBP binding of DNA. TBP interacts with guanine bases flanking the TATA element in the simulations of the complex; these interactions may explain the preference for guanine observed at these DNA positions. Side chains of some TBP residues at the binding interface display significant dynamic flexibility that results in ‘flipflop’ contacts involving multiple base pairs of the DNA. We discuss the possible functional significance of these observations.     

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.