DISPLAR: an accurate method for predicting DNA-binding sites on protein surfaces

Structural and physical properties of DNA provide important constraints on the binding sites formed on surfaces of DNA-targeting proteins. Characteristics of such binding sites may form the basis for predicting DNA-binding sites from the structures of proteins alone. Such an approach has been successfully developed for predicting protein–protein interface. Here this approach is adapted for predicting DNA-binding sites. We used a representative set of 264 protein–DNA complexes from the Protein Data Bank to analyze characteristics and to train and test a neural network predictor of DNA-binding sites. The input to the predictor consisted of PSI-blast sequence profiles and solvent accessibilities of each surface residue and 14 of its closest neighboring residues. Predicted DNA-contacting residues cover 60% of actual DNA-contacting residues and have an accuracy of 76%. This method significantly outperforms previous attempts of DNA-binding site predictions. Its application to the prion protein yielded a DNA-binding site that is consistent with recent NMR chemical shift perturbation data, suggesting that it can complement experimental techniques in characterizing protein–DNA interfaces.     

DNA duplex stability as discriminative characteristic for Escherichia coli σ54- and σ28- dependent promoter sequences

The advent of modern high-throughput sequencing has made it possible to generate vast quantities of genomic sequence data. However, the processing of this volume of information, including prediction of gene-coding and regulatory sequences remains an important bottleneck in bioinformatics research. In this work, we integrated DNA duplex stability into the repertoire of a Neural Network (NN) capable of predicting promoter regions with augmented accuracy, specificity and sensitivity. We took our method beyond a simplistic analysis based on a single sigma subunit of RNA polymerase, incorporating the six main sigma-subunits of Escherichia coli. This methodology employed successfully re-discovered known promoter sequences recognized by E. coli RNA polymerase subunits σ²⁴, σ²⁸, σ³², σ³⁸, σ⁵⁴ and σ⁷⁰, with highlighted accuracies for σ²⁸- and σ⁵⁴- dependent promoter sequences (values obtained were 80% and 78.8%, respectively). Furthermore, the discrimination of promoters according to the σ factor made it possible to extract functional commonalities for the genes expressed by each type of promoter. The DNA duplex stability rises as a distinctive feature which improves the recognition and classification of σ²⁸- and σ⁵⁴- dependent promoter sequences. The findings presented in this report underscore the usefulness of including DNA biophysical parameters into NN learning algorithms to increase accuracy, specificity and sensitivity in promoter beyond what is accomplished based on sequence alone.     

Differential affinity and cooperativity functions of the amino-terminal 70 residues of lambda integrase

The site-specific recombinase (Int) of bacteriophage lambda is a heterobivalent DNA-binding protein that binds two different classes of DNA-binding sites within its recombination target sites. The several functions of Int are apportioned between a large carboxy-terminal domain that cleaves and ligates DNA at each of its four "core-type" DNA-binding sites and a small amino-terminal domain, whose primary function is binding to each of its five "arm-type" DNA sites, which are distant from the core region. Int bridges between the two classes of binding sites are facilitated by accessory DNA-bending proteins that along with Int comprise higher-order recombinogenic complexes. We show here that although the 64 amino-terminal residues of Int bind efficiently to a single arm site, this protein cannot form doubly bound complexes on adjacent arm sites. However, 1-70 Int does show the same cooperative binding to adjacent arm sites as the full length protein. We also found that 1-70 Int specifies cooperative interactions with the accessory protein Xis when the two are bound to their adjacent cognate sites P2 and X1, respectively. To complement the finding that these two amino-terminal domain functions (along with arm DNA binding) are all specified by residues 1-70, we determined that Thr75 is the first residue of the minimal carboxy-terminal domain, thereby identifying a specific interdomain linker region. We have measured the affinity constants for Int binding to each of the five arm sites and the cooperativity factors for Int binding to the two pairs of adjacent arm sites, and we have identified several DNA structural features that contribute to the observed patterns of Int binding to arm sites. Taken together, the results highlight several interesting features of arm DNA binding that invite speculation about additional levels of complexity in the regulation of lambda site-specific recombination.     

Coupling σ Factor Conformation to RNA Polymerase Reorganisation for DNA Melting

ATP-driven remodelling of initial RNA polymerase (RNAP) promoter complexes occurs as a major post recruitment strategy used to control gene expression. Using a model-enhancer-dependent bacterial system (σ⁵⁴-RNAP, Eσ⁵⁴) and a slowly hydrolysed ATP analogue (ATPγS), we provide evidence for a nucleotide-dependent temporal pathway leading to DNA melting involving a small set of σ⁵⁴-DNA conformational states. We demonstrate that the ATP hydrolysis-dependent remodelling of Eσ⁵⁴ occurs in at least two distinct temporal steps. The first detected remodelling phase results in changes in the interactions between the promoter specificity σ⁵⁴ factor and the promoter DNA. The second detected remodelling phase causes changes in the relationship between the promoter DNA and the core RNAP catalytic β/β′ subunits, correlating with the loading of template DNA into the catalytic cleft of RNAP. It would appear that, for Eσ⁵⁴ promoters, loading of template DNA within the catalytic cleft of RNAP is dependent on fast ATP hydrolysis steps that trigger changes in the β′ jaw domain, thereby allowing acquisition of the open complex status.