Structural and mechanistic insights into Helicobacter pylori NikR activation

NikR is a transcriptional metalloregulator central in the mandatory response to acidity of Helicobacter pylori that controls the expression of numerous genes by binding to specific promoter regions. NikR/DNA interactions were proposed to rely on protein activation by Ni(II) binding to high-affinity (HA) and possibly secondary external (X) sites. We describe a biochemical characterization of HpNikR mutants that shows that the HA sites are essential but not sufficient for DNA binding, while the secondary external (X) sites and residues from the HpNikR dimer–dimer interface are important for DNA binding. We show that a second metal is necessary for HpNikR/DNA binding, but only to some promoters. Small-angle X-ray scattering shows that HpNikR adopts a defined conformation in solution, resembling the cis-conformation and suggests that nickel does not trigger large conformational changes in HpNikR. The crystal structures of selected mutants identify the effects of each mutation on HpNikR structure. This study unravels key structural features from which we derive a model for HpNikR activation where: (i) HA sites and an hydrogen bond network are required for DNA binding and (ii) metallation of a unique secondary external site (X) modulates HpNikR DNA binding to low-affinity promoters by disruption of a salt bridge

Local Renyi entropic profiles of DNA sequences

<p>Abstract</p> <p>Background</p> <p>In a recent report the authors presented a new measure of continuous entropy for DNA sequences, which allows the estimation of their randomness level. The definition therein explored was based on the Rényi entropy of probability density estimation (pdf) using the Parzen's window method and applied to Chaos Game Representation/Universal Sequence Maps (CGR/USM). Subsequent work proposed a fractal pdf kernel as a more exact solution for the iterated map representation. This report extends the concepts of continuous entropy by defining DNA sequence entropic profiles using the new pdf estimations to refine the density estimation of motifs.</p> <p>Results</p> <p>The new methodology enables two results. On the one hand it shows that the entropic profiles are directly related with the statistical significance of motifs, allowing the study of under and over-representation of segments. On the other hand, by spanning the parameters of the kernel function it is possible to extract important information about the scale of each conserved DNA region. The computational applications, developed in Matlab m-code, the corresponding binary executables and additional material and examples are made publicly available at <url>http://kdbio.inesc-id.pt/~svinga/ep/</url>.</p> <p>Conclusion</p> <p>The ability to detect local conservation from a scale-independent representation of symbolic sequences is particularly relevant for biological applications where conserved motifs occur in multiple, overlapping scales, with significant future applications in the recognition of foreign genomic material and inference of motif structures.</p

SIGffRid: A tool to search for sigma factor binding sites in bacterial genomes using comparative approach and biologically driven statistics

<p>Abstract</p> <p>Background</p> <p>Many programs have been developed to identify transcription factor binding sites. However, most of them are not able to infer two-word motifs with variable spacer lengths. This case is encountered for RNA polymerase Sigma (<it>σ</it>) Factor Binding Sites (SFBSs) usually composed of two boxes, called -35 and -10 in reference to the transcription initiation point. Our goal is to design an algorithm detecting SFBS by using combinational and statistical constraints deduced from biological observations.</p> <p>Results</p> <p>We describe a new approach to identify SFBSs by comparing two related bacterial genomes. The method, named SIGffRid (SIGma Factor binding sites Finder using R'MES to select Input Data), performs a simultaneous analysis of pairs of promoter regions of orthologous genes. SIGffRid uses a prior identification of over-represented patterns in whole genomes as selection criteria for potential -35 and -10 boxes. These patterns are then grouped using pairs of short seeds (of which one is possibly gapped), allowing a variable-length spacer between them. Next, the motifs are extended guided by statistical considerations, a feature that ensures a selection of motifs with statistically relevant properties. We applied our method to the pair of related bacterial genomes of <it>Streptomyces coelicolor </it>and <it>Streptomyces avermitilis</it>. Cross-check with the well-defined SFBSs of the SigR regulon in <it>S. coelicolor </it>is detailed, validating the algorithm. SFBSs for HrdB and BldN were also found; and the results suggested some new targets for these <it>σ </it>factors. In addition, consensus motifs for BldD and new SFBSs binding sites were defined, overlapping previously proposed consensuses. Relevant tests were carried out also on bacteria with moderate GC content (i.e. <it>Escherichia coli</it>/<it>Salmonella typhimurium </it>and <it>Bacillus subtilis</it>/<it>Bacillus licheniformis </it>pairs). Motifs of house-keeping <it>σ </it>factors were found as well as other SFBSs such as that of SigW in <it>Bacillus </it>strains.</p> <p>Conclusion</p> <p>We demonstrate that our approach combining statistical and biological criteria was successful to predict SFBSs. The method versatility autorizes the recognition of other kinds of two-box regulatory sites.</p

The role of FIS in trans activation of stable RNA operons of E. coli.

The thrU(tufB) operon of Escherichia coli is endowed with a cis-acting region upstream of the promoter, designated UAS for Upstream Activator Sequence. A protein fraction has been isolated that binds specifically to DNA fragments of the UAS, thus forming three protein-DNA complexes corresponding to three binding sites on the UAS. It stimulates in vitro transcription of the operon by facilitating the binding of the RNA polymerase to the promoter. All three protein-DNA complexes contain one and the same protein. Dissociation constants for the three complexes have been determined, the lowest being in the sub-nanomolar range. The protein also binds to the UAS of the tyrT operon and to the UAS upstream of the P1 promoter of the rrnB operon, suggesting that transcription of the three operons, if not of more stable RNA operons, is activated by a common trans activator. We demonstrate that the E.coli protein FIS (Factor for Inversion Stimulation) also binds to the UAS of the thrU(tufB) operon forming three protein-DNA complexes. A burst of UAS- and FIS-dependent promoter activity is observed after reinitiation of growth of stationary cultures in fresh medium

Synthetic lethals in HIV: ways to avoid drug resistance: Running title: Preventing HIV resistance

International audienceBackground : RNA viruses rapidly accumulate genetic variation, which can give rise to synthetic lethal (SL) and deleterious (SD) mutations. Synthetic lethal mutations (non-lethal when alone but lethal when combined in one genome) have been studied to develop cancer therapies. This principle can also be used against fast-evolving RNA-viruses. Indeed, targeting protein sites involved in SD + SL interactions with a drug would render any mutation of such sites, lethal. Results : Here, we set up a strategy to detect intragenic pairs of SL and SD at the surface of the protein to predict less escapable drug target sites. For this, we detected SD + SL, studying HIV protease (PR) and reverse transcriptase (RT) sequence alignments from two groups of VIH+ individuals: treated with drugs (T) or not (NT). Using a series of statistical approaches, we were able to propose bona fide SD + SL couples. When focusing on spatially close co-variant SD + SL couples at the surface of the protein, we found 5 SD + SL groups (2 in the protease and 3 in the reverse transcriptase), which could be good candidates to form pockets to accommodate potential drugs. Conclusions : Thus, designing drugs targeting these specific SD + SL groups would not allow the virus to mutate any residue involved in such groups without losing an essential function. Moreover, we also show that the selection pressure induced by the treatment leads to the appearance of new mutations, which change the mutational landscape of the protein. This drives the existence of differential SD + SL couples between the drug-treated and non-treated groups. Thus, new anti-viral drugs should be designed differently to target such groups

FIS-dependent trans activation of stable RNA operons of Escherichia coli under various growth conditions.

In Escherichia coli transcription of the tRNA operon thrU (tufB) and the rRNA operon rrnB is trans-activated by the protein FIS. This protein, which stimulates the inversion of various viral DNA segments, binds specifically to a cis-acting sequence (designated UAS) upstream of the promoter of thrU (tufB) and the P1 promoter of the rrnB operon. There are indications that this type of regulation is representative for the regulation of more stable RNA operons. In the present investigation we have studied UAS-dependent transcription activation of the thrU (tufB) operon in the presence and absence of FIS during a normal bacterial growth cycle and after a nutritional shift-up. In early log phase the expression of the operon rises steeply in wild-type cells, whereafter it declines. Concomitantly, a peak of the cellular FIS concentration is observed. Cells in the stationary phase are depleted of FIS. The rather abrupt increase of transcription activation depends on the nutritional quality of the medium. It is not seen in minimal medium. After a shift from minimal to rich medium, a peak of transcription activation and of FIS concentration is measured. This peak gets higher as the medium gets more strongly enriched. We conclude that a correlation between changes of the UAS-dependent activation of the thrU (tufB) operon and changes of the cellular FIS concentration under a variety of experimental conditions exists. This correlation strongly suggests that the production of FIS responds to environmental signals, thereby trans-activating the operon. Cells unable to produce FIS (fis cells) also show an increase of operon transcription in the early log phase and after a nutritional shift-up, albeit less pronounced than that wild-type cells. Presumably it is controlled by the ribosome feedback regulatory system. cis activation of the operon by the upstream activator sequence is apparent in the absence of FIS. This activation is constant throughout the entire growth cycle and is independent of nutritional factors. The well-known growth rate-dependent control, displayed by exponentially growing cells studied under various nutritional conditions, is governed by two regulatory mechanisms: repression, presumably by ribosome feedback inhibition, and stimulation by trans activation. FIS allows very fast bacterial growth