3D-DART: a DNA structure modelling server

There is a growing interest in structural studies of DNA by both experimental and computational approaches. Often, 3D-structural models of DNA are required, for instance, to serve as templates for homology modeling, as starting structures for macro-molecular docking or as scaffold for NMR structure calculations. The conformational adaptability of DNA when binding to a protein is often an important factor and at the same time a limitation in such studies. As a response to the demand for 3D-structural models reflecting the intrinsic plasticity of DNA we present the 3D-DART server (3DNA-Driven DNA Analysis and Rebuilding Tool). The server provides an easy interface to a powerful collection of tools for the generation of DNA-structural models in custom conformations. The computational engine beyond the server makes use of the 3DNA software suite together with a collection of home-written python scripts. The server is freely available at http://haddock.chem.uu.nl/dna without any login requirement

ProBiS algorithm for detection of structurally similar protein binding sites by local structural alignment

Motivation: Exploitation of locally similar 3D patterns of physicochemical properties on the surface of a protein for detection of binding sites that may lack sequence and global structural conservation

Different sequence signatures in the upstream regions of plant and animal tRNA genes shape distinct modes of regulation

In eukaryotes, the transcription of tRNA genes is initiated by the concerted action of transcription factors IIIC (TFIIIC) and IIIB (TFIIIB) which direct the recruitment of polymerase III. While TFIIIC recognizes highly conserved, intragenic promoter elements, TFIIIB binds to the non-coding 5′-upstream regions of the tRNA genes. Using a systematic bioinformatic analysis of 11 multicellular eukaryotic genomes we identified a highly conserved TATA motif followed by a CAA-motif in the tRNA upstream regions of all plant genomes. Strikingly, the 5′-flanking tRNA regions of the animal genomes are highly heterogeneous and lack a common conserved sequence signature. Interestingly, in the animal genomes the tRNA species that read the same codon share conserved motifs in their upstream regions. Deep-sequencing analysis of 16 human tissues revealed multiple splicing variants of two of the TFIIIB subunits, Bdp1 and Brf1, with tissue-specific expression patterns. These multiple forms most likely modulate the TFIIIB–DNA interactions and explain the lack of a uniform signature motif in the tRNA upstream regions of animal genomes. The anticodon-dependent 5′-flanking motifs provide a possible mechanism for independent regulation of the tRNA transcription in various human tissues

The Saccharomyces cerevisiae RNA polymerase III recruitment factor subunits Brf1 and Bdp1 impose a strict sequence preference for the downstream half of the TATA box

Association of the TATA-binding protein (TBP) with its cognate site within eukaryotic promoters is key to accurate and efficient transcriptional initiation. To achieve recruitment of Saccharomyces cerevisiae RNA polymerase III, TBP is associated with two additional factors, Brf1 and Bdp1, to form the initiation factor TFIIIB. Previous data have suggested that the structure or dynamics of the TBP–DNA complex may be altered upon entry of Brf1 and Bdp1 into the complex. We show here, using the altered specificity TBP mutant TBPm3 and an iterative in vitro selection assay, that entry of Brf1 and Bdp1 into the complex imposes a strict sequence preference for the downstream half of the TATA box. Notably, the selected sequence (TGTAAATA) is a perfect match to the TATA box of the RNA polymerase III-transcribed U6 small nuclear RNA (SNR6) gene. We suggest that the selected T•A base pair step at the downstream end of the 8 bp TBP site may provide a DNA flexure that promotes TFIIIB-DNA complex formation

Two distinct DNA sequences recognized by transcription factors represent enthalpy and entropy optima

Most transcription factors (TFs) can bind to a population of sequences closely related to a single optimal site. However, some TFs can bind to two distinct sequences that represent two local optima in the Gibbs free energy of binding (Delta G). To determine the molecular mechanism behind this effect, we solved the structures of human HOXB13 and CDX2 bound to their two optimal DNA sequences, CAATAAA and TCGTAAA. Thermodynamic analyses by isothermal titration calorimetry revealed that both sites were bound with similar Delta G. However, the interaction with the CAA sequence was driven by change in enthalpy (Delta H), whereas the TCG site was bound with similar affinity due to smaller loss of entropy (Delta S). This thermodynamic mechanism that leads to at least two local optima likely affects many macromolecular interactions, as Delta G depends on two partially independent variables Delta H and Delta S according to the central equation of thermodynamics, Delta G = Delta H - T Delta S.Peer reviewe

Cross-validated methods for promoter/transcription start site mapping in SL trans-spliced genes, established using the Ciona intestinalis troponin I gene

In conventionally-expressed eukaryotic genes, transcription start sites (TSSs) can be identified by mapping the mature mRNA 5′-terminal sequence onto the genome. However, this approach is not applicable to genes that undergo pre-mRNA 5′-leader trans-splicing (SL trans-splicing) because the original 5′-segment of the primary transcript is replaced by the spliced leader sequence during the trans-splicing reaction and is discarded. Thus TSS mapping for trans-spliced genes requires different approaches. We describe two such approaches and show that they generate precisely agreeing results for an SL trans-spliced gene encoding the muscle protein troponin I in the ascidian tunicate chordate Ciona intestinalis. One method is based on experimental deletion of trans-splice acceptor sites and the other is based on high-throughput mRNA 5′-RACE sequence analysis of natural RNA populations in order to detect minor transcripts containing the pre-mRNA’s original 5′-end. Both methods identified a single major troponin I TSS located ∼460 nt upstream of the trans-splice acceptor site. Further experimental analysis identified a functionally important TATA element 31 nt upstream of the start site. The two methods employed have complementary strengths and are broadly applicable to mapping promoters/TSSs for trans-spliced genes in tunicates and in trans-splicing organisms from other phyla

Minimal components of the RNA polymerase II transcription apparatus determine the consensus TATA box

In Saccharomyces cerevisiae, multiple approaches have arrived at a consensus TATA box sequence of TATA(T/A)A(A/T)(A/G). TATA-binding protein (TBP) affinity alone does not determine TATA box function. To discover how a minimal set of factors required for basal and activated transcription contributed to the sequence requirements for a functional TATA box, we performed transcription reactions using highly purified proteins and CYC1 promoter TATA box mutants. The TATA box consensus sequence is a good predictor of promoter activity. However, several nonconsensus sequences are almost fully functional, indicating that mechanistic requirements are not the only selective pressure on the TATA box. We also found that the effect of a mutation at a certain position is often dependent on other bases within a particular TATA box. Although activators and coactivators strongly influence TBP recruitment and stability at promoters, neither Mediator, the activator Gal4-V16, nor TFIID specifically compensate for the low transcription levels of the weak TATA boxes. The addition of Mediator to purified transcription reactions did, however, increase the functional selectivity for certain consensus TATA sequences. Transcription in whole-cell extracts or in vivo with these TATA box mutants indicated that factors, other than those in our purified system, may help initiate transcription from weak TATA boxes

Comprehensive Study in the Inhibitory Effect of Berberine on Gene Transcription, Including TATA Box

Berberine (BBR) is an established natural DNA intercalator with numerous pharmacological functions. However, currently there are neither detailed reports concerning the distribution of this alkaloid in living cells nor reports concerning the relationship between BBR's association with DNA and the function of DNA. Here we report that the distribution of BBR within the nucleus can be observed 30 minutes after drug administration, and that the content of berberine in the nucleus peaks at around 4 µmol, which is twelve hours after drug administration. The spatial conformation of DNA and chromatin was altered immediately after their association with BBR. Moreover, this association can effectively suppress the transcription of DNA in living cell systems and cell-free systems. Electrophoretic mobility shift assays (EMSA) demonstrated further that BBR can inhibit the association between the TATA binding protein (TBP) and the TATA box in the promoter, and this finding was also attained in living cells by chromatin immunoprecipitation (ChIP). Based on results from this study, we hypothesize that berberine can suppress the transcription of DNA in living cell systems, especially suppressing the association between TBP and the TATA box by binding with DNA and, thus, inhibiting TATA box-dependent gene expression in a non-specific way. This novel study has significantly expanded the sphere of knowledge concerning berberine's pharmacological effects, beginning at its paramount initial interaction with the TATA box

Electrostatic hot spot on DNA-binding domains mediates phosphate desolvation and the pre-organization of specificity determinant side chains

A major obstacle towards elucidating the molecular basis of transcriptional regulation is the lack of a detailed understanding of the interplay between non-specific and specific protein–DNA interactions. Based on molecular dynamics simulations of C2H2 zinc fingers (ZFs) and engrailed homeodomain transcription factors (TFs), we show that each of the studied DNA-binding domains has a set of highly constrained side chains in preset configurations ready to form hydrogen bonds with the DNA backbone. Interestingly, those domains that bury their recognition helix into the major groove are found to have an electrostatic hot spot for Cl− ions located on the same binding cavity as the most buried DNA phosphate. The spot is characterized by three protein hydrogen bond donors, often including two basic side chains. If bound, Cl− ions, likely mimicking phosphates, steer side chains that end up forming specific contacts with bases into bound-like conformations. These findings are consistent with a multi-step DNA-binding mechanism in which a pre-organized set of TF side chains assist in the desolvation of phosphates into well defined sites, prompting the re-organization of specificity determining side chains into conformations suitable for the recognition of their cognate sequence