Role of sequence encoded κB DNA geometry in gene regulation by Dorsal

Many proteins of the Rel family can act as both transcriptional activators and repressors. However, mechanism that discerns the ‘activator/repressor’ functions of Rel-proteins such as Dorsal (Drosophila homologue of mammalian NFκB) is not understood. Using genomic, biophysical and biochemical approaches, we demonstrate that the underlying principle of this functional specificity lies in the ‘sequence-encoded structure’ of the κB-DNA. We show that Dorsal-binding motifs exist in distinct activator and repressor conformations. Molecular dynamics of DNA-Dorsal complexes revealed that repressor κB-motifs typically have A-tract and flexible conformation that facilitates interaction with co-repressors. Deformable structure of repressor motifs, is due to changes in the hydrogen bonding in A:T pair in the ‘A-tract’ core. The sixth nucleotide in the nonameric κB-motif, ‘A’ (A6) in the repressor motifs and ‘T’ (T6) in the activator motifs, is critical to confer this functional specificity as A6 → T6 mutation transformed flexible repressor conformation into a rigid activator conformation. These results highlight that ‘sequence encoded κB DNA-geometry’ regulates gene expression by exerting allosteric effect on binding of Rel proteins which in turn regulates interaction with co-regulators. Further, we identified and characterized putative repressor motifs in Dl-target genes, which can potentially aid in functional annotation of Dorsal gene regulatory network

Anomalous DNA binding by E2 regulatory protein driven by spacer sequence TATA

We have investigated the anomalously weak binding of human papillomavirus (HPV) regulatory protein E2 to a DNA target containing the spacer sequence TATA. Experiments in magnesium (Mg2+) and calcium (Ca2+) ion buffers revealed a marked reduction in cutting by DNase I at the CpG sequence in the protein-binding site 3′ to the TATA spacer sequence, Studies of the cation dependence of DNA-E2 affinities showed that upon E2 binding the TATA sequence releases approximately twice as many Mg2+ ions as the average of the other spacer sequences. Binding experiments for TATA spacer relative to ATAT showed that in potassium ion (K+) the E2 affinity of the two sequences is nearly equal, but the relative dissociation constant (Kd) for TATA increases in the order K+ < Na+ < Ca2+ < Mg2+. Except for Mg2+, Kd for TATA relative to ATAT is independent of ion concentration, whereas for Mg2+ the affinity for TATA drops sharply as ion concentration increases. Thus, ions of increasing positive charge density increasingly distort the E2 binding site, weakening the affinity for protein. In the case of Mg2+, additional ions are bound to TATA that require displacement for protein binding. We suggest that the TATA sequence may bias the DNA structure towards a conformation that binds the protein relatively weakly

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

Crystal structure of the DNA-bound VapBC2 antitoxin/toxin pair from Rickettsia felis

Besides their commonly attributed role in the maintenance of low-copy number plasmids, toxin/antitoxin (TA) loci, also called ‘addiction modules’, have been found in chromosomes and associated to a number of biological functions such as: reduction of protein synthesis, gene regulation and retardation of cell growth under nutritional stress. The recent discovery of TA loci in obligatory intracellular species of the Rickettsia genus has prompted new research to establish whether they work as stress response elements or as addiction systems that might be toxic for the host cell. VapBC2 is a TA locus from R. felis, a pathogen responsible for flea-borne spotted fever in humans. The VapC2 toxin is a PIN-domain protein, whereas the antitoxin, VapB2, belongs to the family of swapped-hairpin β-barrel DNA-binding proteins. We have used a combination of biophysical and structural methods to characterize this new toxin/antitoxin pair. Our results show how VapB2 can block the VapC2 toxin. They provide a first structural description of the interaction between a swapped-hairpin β-barrel protein and DNA. Finally, these results suggest how the VapC2/VapB2 molar ratio can control the self-regulation of the TA locus transcription

Novel organization of mitochondrial minicircles and guide RNAs in the zoonotic pathogen Trypanosoma lewisi

Abstract Kinetoplastid flagellates are known for several unusual features, one of which is their complex mitochondrial genome, known as kinetoplast (k) DNA, composed of mutually catenated maxi- and minicircles. Trypanosoma lewisi is a member of the Stercorarian group of trypanosomes which is, based on human infections and experimental data, now considered a zoonotic pathogen. By assembling a total of 58 minicircle classes, which fall into two distinct categories, we describe a novel type of kDNA organization in T. lewisi. RNA-seq approaches allowed us to map the details of uridine insertion and deletion editing events upon the kDNA transcriptome. Moreover, sequencing of small RNA molecules enabled the identification of 169 unique guide (g) RNA genes, with two differently organized minicircle categories both encoding essential gRNAs. The unprecedented organization of minicircles and gRNAs in T. lewisi broadens our knowledge of the structure and expression of the mitochondrial genomes of these human and animal pathogens. Finally, a scenario describing the evolution of minicircles is presented

Comprehensive comparison of the interaction of the E2 master regulator with its cognate target DNA sites in 73 human papillomavirus types by sequence statistics

Mucosal human papillomaviruses (HPVs) are etiological agents of oral, anal and genital cancer. Properties of high- and low-risk HPV types cannot be reduced to discrete molecular traits. The E2 protein regulates viral replication and transcription through a finely tuned interaction with four sites at the upstream regulatory region of the genome. A computational study of the E2–DNA interaction in all 73 types within the alpha papillomavirus genus, including all known mucosal types, indicates that E2 proteins have similar DNA discrimination properties. Differences in E2–DNA interaction among HPV types lie mostly in the target DNA sequence, as opposed to the amino acid sequence of the conserved DNA-binding alpha helix of E2. Sequence logos of natural and in vitro selected sites show an asymmetric pattern of conservation arising from indirect readout, and reveal evolutionary pressure for a putative methylation site. Based on DNA sequences only, we could predict differences in binding energies with a standard deviation of 0.64 kcal/mol. These energies cluster into six discrete affinity hierarchies and uncovered a fifth E2-binding site in the genome of six HPV types. Finally, certain distances between sites, affinity hierarchies and their eventual changes upon methylation, are statistically associated with high-risk types

Structure-Function Studies of DNA Binding Domain of Response Regulator KdpE Reveals Equal Affinity Interactions at DNA Half-Sites

Expression of KdpFABC, a K+ pump that restores osmotic balance, is controlled by binding of the response regulator KdpE to a specific DNA sequence (kdpFABCBS) via the winged helix-turn-helix type DNA binding domain (KdpEDBD). Exploration of E. coli KdpEDBD and kdpFABCBS interaction resulted in the identification of two conserved, AT-rich 6 bp direct repeats that form half-sites. Despite binding to these half-sites, KdpEDBD was incapable of promoting gene expression in vivo. Structure-function studies guided by our 2.5 Å X-ray structure of KdpEDBD revealed the importance of residues R193 and R200 in the α-8 DNA recognition helix and T215 in the wing region for DNA binding. Mutation of these residues renders KdpE incapable of inducing expression of the kdpFABC operon. Detailed biophysical analysis of interactions using analytical ultracentrifugation revealed a 2∶1 stoichiometry of protein to DNA with dissociation constants of 200±100 and 350±100 nM at half-sites. Inactivation of one half-site does not influence binding at the other, indicating that KdpEDBD binds independently to the half-sites with approximately equal affinity and no discernable cooperativity. To our knowledge, these data are the first to describe in quantitative terms the binding at half-sites under equilibrium conditions for a member of the ubiquitous OmpR/PhoB family of proteins