Arrangement Of Binding Sites Research Articles

Interactions between permeant and blocking anions inside the CFTR chloride channel pore

Binding of cytoplasmic anionic open channel blockers within the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel is antagonized by extracellular Cl−. In the present work, patch clamp recording was used to investigate the interaction between extracellular Cl− (and other anions) and cytoplasmic Pt(NO2)42− ions inside the CFTR channel pore. In constitutively open (E1371Q-CFTR) channels, these different anions bind to two separate sites, located in the outer and inner vestibules of the pore respectively, in a mutually antagonistic fashion. A mutation in the inner vestibule (I344K) that greatly increased Pt(NO2)42− binding affinity also greatly strengthened antagonistic Cl−:blocker interactions as well as the voltage-dependence of block. Quantitative analysis of ion binding affinity suggested that the I344K mutation strengthened interactions not only with intracellular Pt(NO2)42− ions but also with extracellular Cl−, and that altered blocker Cl−- and voltage-dependence were due to the introduction of a novel type of antagonistic ion:ion interaction inside the pore that was independent of Cl− binding in the outer vestibule. It is proposed that this mutation alters the arrangement of anion binding sites inside the pore, allowing both Cl− and Pt(NO2)42− to bind concurrently within the inner vestibule in a strongly mutually antagonistic fashion. However, the I344K mutation does not increase single channel conductance following disruption of Cl− binding in the outer vestibule in R334Q channels. Implications for the arrangement of ion binding sites in the pore, and their functional consequences for blocker binding and for rapid Cl− permeation, are discussed.

Upstream Binding of Idling RNA Polymerase Modulates Transcription Initiation from a Nearby Promoter

The bacterial gene regulatory regions often demonstrate distinctly organized arrays of RNA polymerase binding sites of ill-defined function. Previously we observed a module of closely spaced polymerase binding sites upstream of the canonical promoter of the Escherichia coli fis operon. FIS is an abundant nucleoid-associated protein involved in adjusting the chromosomal DNA topology to changing cellular physiology. Here we show that simultaneous binding of the polymerase at the canonical fis promoter and an upstream transcriptionally inactive site stabilizes a RNAP oligomeric complex in vitro. We further show that modulation of the upstream binding of RNA polymerase affects the fis promoter activity both in vivo and in vitro. The effect of the upstream RNA polymerase binding on the fis promoter activity depends on the spatial arrangement of polymerase binding sites and DNA supercoiling. Our data suggest that a specific DNA geometry of the nucleoprotein complex stabilized on concomitant binding of RNA polymerase molecules at the fis promoter and the upstream region acts as a topological device regulating the fis transcription. We propose that transcriptionally inactive RNA polymerase molecules can act as accessory factors regulating the transcription initiation from a nearby promoter.

The influence of promoter architectures and regulatory motifs on gene expression in Escherichia coli.

The ability to regulate gene expression is of central importance for the adaptability of living organisms to changes in their external and internal environment. At the transcriptional level, binding of transcription factors (TFs) in the promoter region can modulate the transcription rate, hence making TFs central players in gene regulation. For some model organisms, information about the locations and identities of discovered TF binding sites have been collected in continually updated databases, such as RegulonDB for the well-studied case of E. coli. In order to reveal the general principles behind the binding-site arrangement and function of these regulatory architectures we propose a random promoter architecture model that preserves the overall abundance of binding sites to identify overrepresented binding site configurations. This model is analogous to the random network model used in the study of genetic network motifs, where regulatory motifs are identified through their overrepresentation with respect to a “randomly connected” genetic network. Using our model we identify TF pairs which coregulate operons in an overrepresented fashion, or individual TFs which act at multiple binding sites per promoter by, for example, cooperative binding, DNA looping, or through multiple binding domains. We furthermore explore the relationship between promoter architecture and gene expression, using three different genome-wide protein copy number censuses. Perhaps surprisingly, we find no systematic correlation between the number of activator and repressor binding sites regulating a gene and the level of gene expression. A position-weight-matrix model used to estimate the binding affinity of RNA polymerase (RNAP) to the promoters of activated and repressed genes suggests that this lack of correlation might in part be due to differences in basal transcription levels, with repressed genes having a higher basal activity level. This quantitative catalogue relating promoter architecture and function provides a first step towards genome-wide predictive models of regulatory function.

Expanding the Logic of Bacterial Promoters Using Engineered Overlapping Operators for Global Regulators

The understanding of how the architecture of cis-regulatory elements at bacterial promoters determines their final output is of central interest in modern biology. In this work, we attempt to gain insight into this process by analyzing complex promoter architectures in the model organism Escherichia coli. By focusing on the relationship between different TFs at the genomic scale in terms of their binding site arrangement and their effect on the target promoters, we found no strong constraint limiting the combinatorial assembly of TF pairs in E. coli. More strikingly, overlapping binding sites were found equally associated with both equivalent (both TFs have the same effect on the promoter) and opposite (one TF activates while the other repress the promoter) effects on gene expression. With this information on hand, we set an in silico approach to design overlapping sites for three global regulators (GRs) of E. coli, specifically CRP, Fis, and IHF. Using random sequence assembly and an evolutionary algorithm, we were able to identify potential overlapping operators for all TF pairs. In order to validate our prediction, we constructed two lac promoter variants containing overlapping sites for CRP and IHF designed in silico. By assaying the synthetic promoters using a GFP reporter system, we demonstrated that these variants were functional and activated by CRP and IHF in vivo. Taken together, presented results add new information on the mechanisms of signal integration in bacterial promoters and provide new strategies for the engineering of synthetic regulatory circuits in bacteria.

Common features in arrangements of ribosomal protein S26e binding sites on its pre-mRNA and 18S rRNA

It is known that human ribosomal protein (rp) S26e can bind to the first intron of its own pre-mRNA and thereby inhibit its splicing. In this work, hydroxyl radical footprinting was applied for detailed mapping of the rpS26e binding site on an RNA transcript corresponding to the rpS26e pre-mRNA fragment containing the first intron flanked by the first exon and a part of the second exon sequences. Nucleotides of this RNA protected from hydroxyl radical attack in the presence of rpS26e were identified. Most of them are found in the region of the 3'-splice site of the first intron within a purine-rich sequence, which forms a loop connecting two helices in the predicted secondary structure of the rpS26e pre-mRNA fragment, and the remaining nucleotides are located near the 5'-splice site. Comparison of arrangements of rpS26e binding sites on the pre-mRNA and 18S rRNA secondary structures reveals similar elements in the organization of these sites. It was found that both sites contain a structural motif, represented by an extended purine-rich loop between two helices, which could be recognized by rpS26e upon binding to these RNAs. The data obtained shed light on the structural aspects of RNA-protein interactions underlying autoregulation of human RPS26e gene expression at the splicing step.

Structural Basis for the Specific Recognition of Dual Receptors by the Homopolymeric Psa Fimbriae of Yersinia Pestis

The pH 6 antigen or Psa fimbriae of Yersinia pestis bind to two receptors, β1-linked galactosyl residues in glycosphingolipids and phosphocholine group in phospholipids. Despite the ubiquitous presence of either moiety on the surface of many mammalian cells, Y. pestis appears to prefer interacting with certain types of human cells such as macrophages and alveolar epithelial cells of the lung. The molecular mechanism of this apparent selectivity is not clear. Site-directed mutagenesis of the consensus choline-binding motif in the sequence of PsaA, the subunit of the Psa fimbrial homopolymer, identified residues that abolish either galactosylceramide or phosphatidylcholine binding or both. The crystal structure of the ternary complex of an in cis donor-strand complemented PsaA, galactose and phosphocholine reveals separate galactose and phosphocholine binding sites that share a common structural motif, thus suggesting potential interaction between the two sites. Mutagenesis of this shared structural motif identified Tyr126, which is part of the choline-binding consensus sequence but is found in direct contact with the galactose in the structure of PsaA, important for both receptor binding. This is the first structural resolution of a fimbrial subunit that forms a polymeric polyadhesin describing a unique arrangement of dual receptor binding sites. These findings move the field forward by providing insights into new types of multiple receptor-ligand interactions and should steer research into the synthesis of dual receptor inhibitor molecules to slow down the rapid progression of plague.

Structural basis for the specific recognition of dual receptors by the homopolymeric pH 6 antigen (Psa) fimbriae of Yersinia pestis

The pH 6 antigen (Psa) of Yersinia pestis consists of fimbriae that bind to two receptors: β1-linked galactosyl residues in glycosphingolipids and the phosphocholine group in phospholipids. Despite the ubiquitous presence of either moiety on the surface of many mammalian cells, Y. pestis appears to prefer interacting with certain types of human cells, such as macrophages and alveolar epithelial cells of the lung. The molecular mechanism of this apparent selectivity is not clear. Site-directed mutagenesis of the consensus choline-binding motif in the sequence of PsaA, the subunit of the Psa fimbrial homopolymer, identified residues that abolish galactosylceramide binding, phosphatidylcholine binding, or both. The crystal structure of PsaA in complex with both galactose and phosphocholine reveals separate receptor binding sites that share a common structural motif, thus suggesting a potential interaction between the two sites. Mutagenesis of this shared structural motif identified Tyr126, which is part of the choline-binding consensus sequence but is found in direct contact with the galactose in the structure of PsaA, important for both receptor binding. Thus, this structure depicts a fimbrial subunit that forms a polymeric adhesin with a unique arrangement of dual receptor binding sites. These findings move the field forward by providing insights into unique types of multiple receptor-ligand interactions and should steer research into the synthesis of dual receptor inhibitor molecules to slow down the rapid progression of plague.

Estimation of Pore Geometry of RyR1 using Lanthanide Ruler

It was shown previously that the Ca analogue Gd inhibits RyR1 gating symmetrically with a Kd about 5.5 microM and Hill coefficient (nH) of 4 both on cis and trans side using single channel electrophysiology. We further tested the RyR1-lanthanide interaction using two lanthanides - having an ionic radii between Ca2+ and Gd3+ - by bilayer measurements and ryanodine binding experiments. Cis inhibition of RyR1 by Eu was characterized by a binding constant of Kd=167±5 nM and an nH of 2±0.1 while trans inhibition exhibits Kd=4.8±0.2 microM and nN of 5.2±1.2. The inhibition constants for Sm on the cis side are Kd=64.3±2.5 nM and nH=2.2±0.2 while on the trans side Kd=6.15±0.13 microM and nH=4.68±0.45. Inhibition by Eu and Sm are potential and polarity dependent in contrast to Gd due to the differences in ionic radii of these lanthanides. Increasing the ionic radius from 0.938 (Gd) to 0.964 (Sm) increased the binding affinity from 5.6 microM to 64.3 nM revealing that the size of Ca binding pocket is only slightly higher than the ionic radius of Sm. Ryanodine (Ry) binding experiments revealed that lanthanides bind - at least partially - to the regulatory Ca binding site because the dose response curve of 3H Ry binding starts with an increase of Ry binding, which amounts to about 40% for Eu and 70% for Sm of basic Ry binding. A model has been proposed for one possible spatial arrangement of lanthanide and calcium binding sites of the RyR1 pore based on the ionic radii of Ca and the tested lanthanides. Supported by OTKA 81923.

Transcriptome-wide Analysis of Regulatory Interactions of the RNA-Binding Protein HuR

Posttranscriptional gene regulation relies on hundreds of RNA binding proteins (RBPs) but the function of most RBPs is unknown. The human RBP HuR/ELAVL1 is a conserved mRNA stability regulator. We used PAR-CLIP, a recently developed method based on RNA-protein crosslinking, to identify transcriptome-wide ∼26,000 HuR binding sites. These sites were on average highly conserved, enriched for HuR binding motifs and mainly located in 3' untranslated regions. Surprisingly, many sites were intronic, implicating HuR in mRNA processing. Upon HuR knockdown, mRNA levels and protein synthesis of thousands of target genes were downregulated, validating functionality. HuR and miRNA binding sites tended to reside nearby but generally did not overlap. Additionally, HuR knockdown triggered strong and specific upregulation of miR-7. In summary, we identified thousands of direct and functional HuR targets, found a human miRNA controlled by HuR, and propose a role for HuR in splicing.

Structures of Human Exonuclease 1 DNA Complexes Suggest a Unified Mechanism for Nuclease Family

Human exonuclease 1 (hExo1) plays important roles in DNA repair and recombination processes that maintain genomic integrity. It is a member of the 5' structure-specific nuclease family of exonucleases and endonucleases that includes FEN-1, XPG, and GEN1. We present structures of hExo1 in complex with a DNA substrate, followed by mutagenesis studies, and propose a common mechanism by which this nuclease family recognizes and processes diverse DNA structures. hExo1 induces a sharp bend in the DNA at nicks or gaps. Frayed 5' ends of nicked duplexes resemble flap junctions, unifying the mechanisms of endo- and exonucleolytic processing. Conformational control of a mobile region in the catalytic site suggests a mechanism for allosteric regulation by binding to protein partners. The relative arrangement of substrate binding sites in these enzymes provides an elegant solution to a complex geometrical puzzle of substrate recognition and processing.

Allosteric conformational spread: Exact results using a simple transfer matrix method

A transfer matrix method is described for the conformational spread (CS) model of allosteric cooperativity within a one-dimensional arrangement of four-state binding sites. Each such binding site can realize one of two possible conformational states. Each of these states can either bind ligand or not bind ligand. Thus, analytical expressions that are exact within the context of the CS model are derived for the grand partition function, for the mean fraction of binding sites occupied by ligand versus ligand concentration, and for the mean fraction of binding sites in a given allosteric state versus ligand concentration. The utility of our analytical results is demonstrated by least-mean-square fitting of prior experimental results obtained on the bacterial flagellar motor for the fraction of FliM/FliG/FliN complexes with CheY-P bound [V. Sourjik and H. C. Berg, Proc. Natl. Acad. Sci. U.S.A. 99, 12669 (2002)] and for the cw bias [P. Cluzel, Science 287, 1652 (2000)], which plausibly may be identified as the fraction of protomers realizing state 2. Finally, the relationships between our analytical results and the classical Monod-Wyman-Changeaux, Koshland-Nemethy-Filmer, and McGhee-Von Hippel treatments of allosteric cooperativity are elucidated, as is the connection to an earlier approximate analytical treatment of the CS model.

Genetic flexibility of regulatory networks

Gene regulatory networks are based on simple building blocks such as promoters, transcription factors (TFs) and their binding sites on DNA. But how diverse are the functions that can be obtained by different arrangements of promoters and TF binding sites? In this work we constructed synthetic regulatory regions using promoter elements and binding sites of two noninteracting TFs, each sensing a single environmental input signal. We show that simply by combining these three kinds of elements, we can obtain 11 of the 16 Boolean logic gates that integrate two environmental signals in vivo. Further, we demonstrate how combination of logic gates can result in new logic functions. Our results suggest that simple elements of transcription regulation form a highly flexible toolbox that can generate diverse functions under natural selection.

Comparing anterior and posterior Hox complex formation reveals guidelines for predicting cis-regulatory elements

Hox transcription factors specify numerous cell fates along the anterior–posterior axis by regulating the expression of downstream target genes. While expression analysis has uncovered large numbers of de-regulated genes in cells with altered Hox activity, determining which are direct versus indirect targets has remained a significant challenge. Here, we characterize the DNA binding activity of Hox transcription factor complexes on eight experimentally verified cis-regulatory elements. Hox factors regulate the activity of each element by forming protein complexes with two cofactor proteins, Extradenticle (Exd) and Homothorax (Hth). Using comparative DNA binding assays, we found that a number of flexible arrangements of Hox, Exd, and Hth binding sites mediate cooperative transcription factor complexes. Moreover, analysis of a Distal-less regulatory element (DMXR) that is repressed by abdominal Hox factors revealed that suboptimal binding sites can be combined to form high affinity transcription complexes. Lastly, we determined that the anterior Hox factors are more dependent upon Exd and Hth for complex formation than posterior Hox factors. Based upon these findings, we suggest a general set of guidelines to serve as a basis for designing bioinformatics algorithms aimed at identifying Hox regulatory elements using the wealth of recently sequenced genomes.

A Biophysical Model for Analysis of Transcription Factor Interaction and Binding Site Arrangement from Genome-Wide Binding Data

BackgroundHow transcription factors (TFs) interact with cis-regulatory sequences and interact with each other is a fundamental, but not well understood, aspect of gene regulation.Methodology/Principal FindingsWe present a computational method to address this question, relying on the established biophysical principles. This method, STAP (sequence to affinity prediction), takes into account all combinations and configurations of strong and weak binding sites to analyze large scale transcription factor (TF)-DNA binding data to discover cooperative interactions among TFs, infer sequence rules of interaction and predict TF target genes in new conditions with no TF-DNA binding data. The distinctions between STAP and other statistical approaches for analyzing cis-regulatory sequences include the utility of physical principles and the treatment of the DNA binding data as quantitative representation of binding strengths. Applying this method to the ChIP-seq data of 12 TFs in mouse embryonic stem (ES) cells, we found that the strength of TF-DNA binding could be significantly modulated by cooperative interactions among TFs with adjacent binding sites. However, further analysis on five putatively interacting TF pairs suggests that such interactions may be relatively insensitive to the distance and orientation of binding sites. Testing a set of putative Nanog motifs, STAP showed that a novel Nanog motif could better explain the ChIP-seq data than previously published ones. We then experimentally tested and verified the new Nanog motif. A series of comparisons showed that STAP has more predictive power than several state-of-the-art methods for cis-regulatory sequence analysis. We took advantage of this power to study the evolution of TF-target relationship in Drosophila. By learning the TF-DNA interaction models from the ChIP-chip data of D. melanogaster (Mel) and applying them to the genome of D. pseudoobscura (Pse), we found that only about half of the sequences strongly bound by TFs in Mel have high binding affinities in Pse. We show that prediction of functional TF targets from ChIP-chip data can be improved by using the conservation of STAP predicted affinities as an additional filter.Conclusions/SignificanceSTAP is an effective method to analyze binding site arrangements, TF cooperativity, and TF target genes from genome-wide TF-DNA binding data.

Direct activation of a notochord cis-regulatory module by Brachyury and FoxA in the ascidianCiona intestinalis

The notochord is a defining feature of the chordate body plan. Experiments in ascidian, frog and mouse embryos have shown that co-expression of Brachyury and FoxA class transcription factors is required for notochord development. However, studies on the cis-regulatory sequences mediating the synergistic effects of these transcription factors are complicated by the limited knowledge of notochord genes and cis-regulatory modules (CRMs) that are directly targeted by both. We have identified an easily testable model for such investigations in a 155-bp notochord-specific CRM from the ascidian Ciona intestinalis. This CRM contains functional binding sites for both Ciona Brachyury (Ci-Bra) and FoxA (Ci-FoxA-a). By combining point mutation analysis and misexpression experiments, we demonstrate that binding of both transcription factors to this CRM is necessary and sufficient to activate transcription. To gain insights into the cis-regulatory criteria controlling its activity, we investigated the organization of the transcription factor binding sites within the 155-bp CRM. The 155-bp sequence contains two Ci-Bra binding sites with identical core sequences but opposite orientations, only one of which is required for enhancer activity. Changes in both orientation and spacing of these sites substantially affect the activity of the CRM, as clusters of identical sites found in the Ciona genome with different arrangements are unable to activate transcription in notochord cells. This work presents the first evidence of a synergistic interaction between Brachyury and FoxA in the activation of an individual notochord CRM, and highlights the importance of transcription factor binding site arrangement for its function.

Structural and biological characterization of the tubulin interaction with dinitroanilines

Characteristics of the interaction of dinitroaniline compounds with tubulin molecules have an extremely high selectivity: these substances efficiently bind to the tubulins of both plant and protozoan origins and practically do not interact with any animal and fungal tubulins despite a very high similarity between the corresponding sequences. This work summarizes and comprehensively analyzes the specific structural features and mechanisms of these interactions, in particular, the patterns of the structure and arrangement of dinitroaniline binding sites on the surface of different tubulin subunits and tubulins of various origins. Dinitroaniline binding sites are localized to the surface of longitudinal contacts between tubulin subunits and contain diamine amino acid residues (lysine or arginine), which bind the nitrile group of dinitroanilines. The localizations of these sites on the surface of identical subunits of different origins (for example, α-tubulins of plants and protozoans) coincide; however, the location of these binding sites on the surfaces of tubulin α- and β-subunits is different. The characterized sites can also be potential binding sites for other antimicrotubule compounds, in particular, cyanoacrylates.

Counting Electrons Transferred through a Thin Alumina Film into Au Chains

Low-temperature STM measurements combined with density functional theory calculations are employed to study the adsorption of gold on alumina/NiAl(110). The binding of Au monomers involves breaking of an oxide Al-O bond below the adatom and stabilizing the hence undercoordinated O ion by forming a new bond to an Al atom in the NiAl. The adsorption implies negative charging of the adatom. The linear arrangement of favorable binding sites induces the self-organization of Au atoms into chains. For every ad-chain, the number of transfer electrons from the support is determined by analyzing the node structure of the corresponding highest occupied molecular orbital.

When Two Is Better Than One

Gene duplication and divergence has long been considered an important route to adaptation and phenotypic evolution. Reporting in Nature, Hittinger and Carroll (2007) provide the first clear example of adaptations in both regulatory regions and protein-coding regions after gene duplication. This combination of evolutionary changes appears to have resolved an adaptive conflict, leading to increased organismal fitness.

The roles of binding site arrangement and combinatorial targeting in microRNA repression of gene expression

A genome wide analysis of factors affecting repression of mRNAs by microRNAs reveals roles for 3'UTR length, the number of target sites on the mRNA and the distance between pairs of binding sites.

Protonation-mediated structural flexibility in the F conjugation regulatory protein, TraM

TraM is essential for F plasmid-mediated bacterial conjugation, where it binds to the plasmid DNA near the origin of transfer, and recognizes a component of the transmembrane DNA transfer complex, TraD. Here we report the 1.40 A crystal structure of the TraM core tetramer (TraM58-127). TraM58-127 is a compact eight-helical bundle, in which the N-terminal helices from each protomer interact to form a central, parallel four-stranded coiled-coil, whereas each C-terminal helix packs in an antiparallel arrangement around the outside of the structure. Four protonated glutamic acid residues (Glu88) are packed in a hydrogen-bonded arrangement within the central four-helix bundle. Mutational and biophysical analyses indicate that this protonated state is in equilibrium with a deprotonated tetrameric form characterized by a lower helical content at physiological pH and temperature. Comparison of TraM to its Glu88 mutants predicted to stabilize the helical structure suggests that the protonated state is the active form for binding TraD in conjugation.