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.     

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.     

A-Tract bending: insights into experimental structures by computational models

While solution structures of adenine tract (A-tract) oligomers have indicated a unique bend direction equivalent to negative global roll (commonly termed "minor-groove bending"), crystallographic data have not unambiguously characterized the bend direction; nevertheless, many features are shared by all A-tract crystal and solution structures (e.g. propeller twisting, narrow minor grooves, and localized water spines). To examine the origin of bending and to relate findings to the crystallographic and solution data, we analyze molecular dynamics trajectories of two solvated A-tract dodecamers: 1D89, d(CGCGA(6)CG), and 1D98, d(CGCA(6)GCG), using a new general global bending framework for analyzing bent DNA and DNA/protein complexes. It is significant that the crystallographically-based initial structures are converted from dissimilar to similar bend directions equivalent to negative global roll, with the average helical-axis bend ranging from 10.5 degrees to 14.1 degrees. The largest bend occurs as positive roll of 12 degrees on the 5' side of the A-tracts (supporting a junction model) and is reinforced by gradual curvature at each A-tract base-pair (bp) step (supporting a wedge model). The precise magnitude of the bend is subtly sequence dependent (consistent with a curved general sequence model). The conversion to negative global roll only requires small local changes at each bp, accumulated over flexible moieties both outside and inside the A-tract. In contrast, the control sequence 1BNA, d(CGCGA(2)TTCGCG), bends marginally (only 6.9 degrees ) with no preferred direction. The molecular features that stabilize the bend direction in the A-tract dodecamers include propeller twisting of AT base-pairs, puckering differences between A and T deoxyriboses, a narrow minor groove, and a stable water spine (that extends slightly beyond the A-tract, with lifetimes approaching 0.2 ns). The sugar conformations, in particular, are proposed as important factors that support bent DNA. It is significant that all these curvature-stabilizing features are also observed in the crystallographic structures, but yield overall different bending paths, largely due to the effects of sequences outside the A-tract. These results merge structural details reported for A-tract structures by experiment and theory and lead to structural and dynamic insights into sequence-dependent DNA flexibility, as highlighted by the effect of an A-tract variant of a TATA-box element on bending and flexibility required for TBP binding.     

Preferential counterion binding to A-tract DNA oligomers

The free solution mobility of four 20 bp DNA oligomers, with and without A-tracts, has been measured by capillary electrophoresis in Tris-acetate buffer, to test the hypothesis that site-specific binding of monovalent counterions can occur in the narrow minor groove of A-tract DNAs. Preferential counterion binding has been proposed to cause A-tract bending because of asymmetric charge neutralization and collapse of the helix backbone toward the minor groove. Preferential counterion binding in A-tract DNAs should be manifested by a decrease in the electrophoretic mobility observed in free solution, compared to that of non-A-tract DNAs of the same size. Of the four sequences studied here, the slowest absolute mobility, indicative of the greatest counterion binding, was observed for a 20 bp oligomer containing two runs of A3T3 in phase with the helix repeat. A 20-mer containing phased CACA sequences migrated with the fastest mobility; 20-mers containing phased A5 tracts or phased runs of T3A3 migrated with intermediate mobilities. Very similar mobility differences were observed when 1-20 mM NaCl was added to the buffer. The results suggest that preferential counterion binding occurs in A-tract DNAs, especially those containing the AnTn sequence motif.     

Locations and contexts of sequences that hybridize to poly(dG-dT).(dC-dA) in mammalian ribosomal DNAs and two X-linked genes.

Sequences located several kilobases both 5' and 3' of the stably transcribed portion of several genes hybridize to radio-labeled pure fragments of the alternating sequence poly (dG-dT) (dC-dA) ["poly(GT)"]. The genes include the ribosomal DNA of mouse, rat, and human, and also human glucose-6-phosphate dehydrogenase (G6PD) and mouse hypoxanthine-guanine phosphoribosyl transferase (HPRT). HPRT has additional hybridizing sequences in introns. Fragments that include the hybridizing sequences and up to 300 bp of adjoining DNA show perfect runs of poly(GT) (greater than 30bp) in all but the human 5' region of rDNA, which shows a somewhat different alternating purine:pyrimidine sequence, poly(GTAT) (36bp). Within 150 bp of these sequences in various instances are found a number of other sequences reported to affect DNA conformation in model systems. Most marked is an enhancement of sequences matching at least 67% to the consensus binding sequence for topoisomerase II. Two to ten-fold less of such sequences were found in other sequenced portions of the nontranscribed spacer or in the transcribed portion of rDNA. The conservation of the locations of tracts of alternating purine:pyrimidine between evolutionarily diverse species is consistent with a possible functional role for these sequences.     

Flanking A·T Basepairs Destabilize the B? Conformation of DNA A-Tracts

Capillary electrophoresis has been used to characterize the interaction of monovalent cations with 26-basepair DNA oligomers containing A-tracts embedded in flanking sequences with different basepair compositions. A 26-basepair random-sequence oligomer was used as the reference; lithium and tetrabutylammonium (TBA⁺) ions were used as the probe ions. The free solution mobilities of the A-tract and random-sequence oligomers were identical in solutions containing <∼100 mM cation. At higher cation concentrations, the A-tract oligomers migrated faster than the reference oligomer in TBA⁺ and slower than the reference in Li⁺. Hence, cations of different sizes can interact very differently with DNA A-tracts. The increased mobilities observed in TBA⁺ suggest that the large hydrophobic TBA⁺ ions are preferentially excluded from the vicinity of the A-tract minor groove, increasing the effective net charge of the A-tract oligomers and increasing the mobility. By contrast, Li⁺ ions decrease the mobility of A-tract oligomers because of the preferential localization of Li⁺ ions in the narrow A-tract minor groove. Embedding the A-tracts in AT-rich flanking sequences markedly alters preferential interactions of monovalent cations with the B^∗ conformation. Hence, A-tracts embedded in genomic DNA may or may not interact preferentially with monovalent cations, depending on the relative number of A·T basepairs in the flanking sequences.     

Sequence analysis of a PM2-DNA anti-Z-IgG-binding region

An anti-Z-antibody-binding region between PM2-DNA map units 0.05 and 0.18, containing approx. 25% of the bound PM2 antibody molecules (1,2) has been sequenced. Analysis of this PM2 DNA sequence from map units 0.00 to 0.175 demonstrates that alternating purine/pyrimidine tracts capable of adopting the left-handed conformation are present within this antibody-binding region. Longer (GC)n-rich tracts are clustered together and comprise seven alternating purine/pyrimidine-rich areas (48%–84%) ranging from 19 to 142 nucleotides in length. The DNA located between these alternating purine/pyrimidine-rich areas exhibit a low level (0%–19%) of this sequence arrangement. There is a very strong correlation between the alternating purine/pyrimidine-rich areas and the anti-Z-DNA-IgG-binding sites. Nucleotides 1461–1583 of the PM2-DNA genome encode the bacteriophage capsid protein IV. One of the PM2 left-handed sites is located within this protein-coding sequence; a B-to-Z transition within this site may be involved in protein-IV gene regulation in vivo.     

An element with palindromic structure is required for the expression of TBP (TATA box-binding protein) gene in Drosophila melanogaster

Previously we showed that the 5'-flanking regions between -261 and -207 of the Drosophila melanogaster TBP (TATA box binding protein) gene is important for its expression. We further made serial deletion mutants in this region and analyzed their promoter activities using the transient transfection assay. We found that the 16 bp deletion from -261 to -245 greatly reduces the promoter activity of the Drosophila TBP gene. The 16 bp DNA element contains half of a 11 bp long palindromic sequence, CTTTT-GAAAAG. Disruption of the palindromic sequence by site-directed mutagenesis severely affected promoter activity. In addition, the electrophoretic mobility shift assay showed that the oligonucleotide containing the palindromic sequence can make specific DNA/protein complexes when it was mixed with the Drosophila nuclear extract, suggesting that it interacts with nuclear protein(s). Our data suggest that the palindromic sequence has a critical role in the expression of the Drosophila TBP gene.     

Architecture of Protein and DNA Contacts within the TFIIIB-DNA Complex

The RNA polymerase III factor TFIIIB forms a stable complex with DNA and can promote multiple rounds of initiation by polymerase. TFIIIB is composed of three subunits, the TATA binding protein (TBP), TFIIB-related factor (BRF), and B". Chemical footprinting, as well as mutagenesis of TBP, BRF, and promoter DNA, was used to probe the architecture of TFIIIB subunits bound to DNA. BRF bound to TBP-DNA through the nonconserved C-terminal region and required 15 bp downstream of the TATA box and as little as 1 bp upstream of the TATA box for stable complex formation. In contrast, formation of complete TFIIIB complexes required 15 bp both upstream and downstream of the TATA box. Hydroxyl radical footprinting of TFIIIB complexes and modeling the results to the TBP-DNA structure suggest that BRF and B" surround TBP on both faces of the TBP-DNA complex and provide an explanation for the exceptional stability of this complex. Competition for binding to TBP by BRF and either TFIIB or TFIIA suggests that BRF binds on the opposite face of the TBP-DNA complex from TFIIB and that the binding sites for TFIIA and BRF overlap. The positions of TBP mutations which are defective in binding BRF suggest that BRF binds to the top and N-terminal leg of TBP. One mutation on the N-terminal leg of TBP specifically affects the binding of the B" subunit.     

A real-time study of the interaction of TBP with a TATA box-containing duplex identical to an ancestral or minor allele of human gene LEP or TPI

It is known that only a single-nucleotide substitution (SNP: a single nucleotide polymorphism) in the sequence of a TATA box can influence the affinity of the interaction of TBP with the TATA box and contribute to the pathogenesis of complex hereditary human diseases and sometimes may be a cause of monogenic diseases (for instance, β-thalassemia). In the present work, we studied the interaction of human TBP with a double-stranded oligodeoxyribonucleotide (ODN) 15 or 26 bp long identical to a TATA box of promoters of a real-life human gene, TPI or LEP, and labeled with fluorophores TAMRA and FAM. To analyze the interaction of TBP with a TATA box of an ancestral or minor allele (SNP in the TATA box) in real time, we used the stopped-flow method with detection of a Förster resonance energy transfer (FRET) signal. The nature of the resulting kinetic curves reflecting changes in the FRET signal (and therefore of DNA conformation during the interaction with TBP) pointed to a multistage mechanism of the formation of the TBP complex with the TATA-containing ODN. The results showed that with the increasing concentration and length of the ODN, heterogeneity of conformational changes (taking place during the first second of the interaction with TBP) in DNA also increases. In contrast to the initial nonspecific interaction, the subsequent phases strictly depend on TBP concentration: at the TBP:ODN ratio of 10:1, the velocity of change of the FRET signal increases approximately 100-fold.     

Analysis of co-crystal structures to identify the stereochemical determinants of the orientation of TBP on the TATA box.

Possible stereochemical determinants of the orientation of TBP on the TATA box are discussed using the crystal coordinates of TBP-TATA complexes, which have been determined by other groups. The C-terminal half of the TBP beta-sheet interacts with the TATA site of the DNA, and the N-terminal half with the A-rich site, so that the two sites with distinct curvatures produce a unique fit. Although chemical contacts take place between one side of the beta-sheet and the DNA minor groove, the interaction seems to be facilitated indirectly by the characteristics of the other side of the beta-sheet and the DNA major groove. Thus, Ala71, Leu162 and Pro190 differentiate the curvature of the beta-sheet in the N- and C-halves. The methyl positions in the DNA major groove modulate the bendability of the two DNA sites by using differences in the rolling capacity of TA and AT compared with PyT, and in the shifting capacity of AT compared with TT. The deformations of the first steps (TA and PyT) in the two sites are the largest and thus are important for the overall bending of the DNA. The differences between the two DNA sites are greatest at the second steps (AT and TT) and so these are important for determining the orientation of TBP.