scholarly journals Genetic and Biochemical Analyses of BvgA Interaction with the Secondary Binding Region of the fhaPromoter of Bordetella pertussis

2001 ◽  
Vol 183 (2) ◽  
pp. 536-544 ◽  
Author(s):  
Philip E. Boucher ◽  
Mei-Shin Yang ◽  
Deanna M. Schmidt ◽  
Scott Stibitz
Keyword(s):  
Bordetella Pertussis ◽  
Response Regulator ◽  
Sequence Specificity ◽  
Binding Region ◽  
Dna Sequence Specificity ◽  
Genes Encoding ◽  
Two Component Signal Transduction ◽  
Biochemical Analyses

ABSTRACT The BvgA-BvgS two-component signal transduction system regulates expression of virulence factors in Bordetella pertussis. The BvgA response regulator activates transcription by binding to target promoters, which include those for the genes encoding filamentous hemagglutinin (fha) and pertussis toxin (ptx). We have previously shown that at both promoters the phosphorylated form of BvgA binds multiple high- and low-affinity sites. Specifically, at the fha promoter, we proposed that there may be high- and a low-affinity binding sites for the BvgA dimer. In our present investigation, we used DNA binding analyses and in vitro and in vivo assays of promoters with substitutions and deletions to support and extend this hypothesis. Our observations indicate that (i) binding of BvgA∼P to a primary (high-affinity) site and a secondary binding region (lower affinity) is cooperative, (ii) although both the primary binding site and the secondary binding region are required for full activity of the wild-type (undeleted) promoter, deletion of two helical turns within the secondary binding region can produce a fully active or hyperactive promoter, and (iii) BvgA binding to the secondary binding region shows limited DNA sequence specificity.

scholarly journals Functional reconstitution of the Salmonella typhimurium PhoQ histidine kinase sensor in proteoliposomes

2005 ◽  
Vol 390 (3) ◽  
pp. 769-776 ◽  
Author(s):  
Sarah Sanowar ◽  
Hervé Le Moual
Keyword(s):  
Salmonella Typhimurium ◽  
Histidine Kinase ◽  
Catalytic Domain ◽  
Response Regulator ◽  
Phosphoryl Group ◽  
Two Component ◽  
Two Component Signal Transduction ◽  
High Concentrations

Two-component signal-transduction systems are widespread in bacteria. They are usually composed of a transmembrane histidine kinase sensor and a cytoplasmic response regulator. The PhoP/PhoQ two-component system of Salmonella typhimurium contributes to virulence by co-ordinating the adaptation to low concentrations of environmental Mg2+. Limiting concentrations of extracellular Mg2+ activate the PhoP/PhoQ phosphorylation cascade modulating the transcription of PhoP-regulated genes. In contrast, high concentrations of extracellular Mg2+ stimulate the dephosphorylation of the response regulator PhoP by the PhoQ kinase sensor. In the present study, we report the purification and functional reconstitution of PhoQHis, a PhoQ variant with a C-terminal His tag, into Escherichia coli liposomes. The functionality of PhoQHis was essentially similar to that of PhoQ as shown in vivo and in vitro. Purified PhoQHis was inserted into liposomes in a unidirectional orientation, with the sensory domain facing the lumen and the catalytic domain facing the extraluminal environment. Reconstituted PhoQHis exhibited all the catalytic activities that have been described for histidine kinase sensors. Reconstituted PhoQHis was capable of autokinase activity when incubated in the presence of Mg2+-ATP. The phosphoryl group could be transferred from reconstituted PhoQHis to PhoP. Reconstituted PhoQHis catalysed the dephosphorylation of phospho-PhoP and this activity was stimulated by the addition of extraluminal ADP.

scholarly journals CovR Activation of the Dipeptide Permease Promoter (PdppA) in Group A Streptococcus

2006 ◽  
Vol 189 (4) ◽  
pp. 1407-1416 ◽  
Author(s):  
Asiya A. Gusa ◽  
Barbara J. Froehlich ◽  
Devak Desai ◽  
Virginia Stringer ◽  
June R. Scott
Keyword(s):  
Group A Streptococcus ◽  
Response Regulator ◽  
Activation Of Transcription ◽  
Genes Encoding ◽  
Group A ◽  
Transcription In Vitro ◽  
Dna Template ◽  
Regulated Promoters

ABSTRACT CovR, the two-component response regulator of Streptococcus pyogenes (group A streptococcus [GAS]) directly or indirectly represses about 15% of the genome, including genes encoding many virulence factors and itself. Transcriptome analyses also showed that some genes are activated by CovR. We asked whether the regulation by CovR of one of these genes, dppA, the first gene in an operon encoding a dipeptide permease, is direct or indirect. Direct regulation by CovR was suggested by the presence of five CovR consensus binding sequences (CBs) near the putative promoter. In this study, we identified the 5′ end of the dppA transcript synthesized in vivo and showed that the start of dppA transcription in vitro is the same. We found that CovR binds specifically to the dppA promoter region (PdppA) in vitro with an affinity similar to that at which it binds to other CovR-regulated promoters. Disruption of any of the five CBs by a substitution of GG for TT inhibited CovR binding to that site in vitro, and binding at two of the CBs appeared cooperative. In vivo, CovR activation of transcription was not affected by individual mutations of any of the four CBs that we could study. This suggests that the binding sites are redundant in vivo. In vitro, CovR did not activate transcription from PdppA in experiments using purified GAS RNA polymerase and either linear or supercoiled DNA template. Therefore, we propose that in vivo, CovR may interfere with the binding of a repressor of PdppA.

scholarly journals The Plant NF-Y DNA Matrix In Vitro and In Vivo

Plants ◽  
2019 ◽  
Vol 8 (10) ◽  
pp. 406 ◽  
Author(s):  
Nerina Gnesutta ◽  
Matteo Chiara ◽  
Andrea Bernardini ◽  
Matteo Balestra ◽  
David S. Horner ◽  
...  
Keyword(s):  
Saturation Mutagenesis ◽  
Sequence Specificity ◽  
Histone Fold ◽  
Nuclear Factor Y ◽  
Histone Fold Domain ◽  
Genes Encoding ◽  
Core Histones ◽  
Ccaat Box

Nuclear Factor Y (NF-Y) is an evolutionarily conserved trimer formed by a Histone-Fold Domain (HFD) heterodimeric module shared by core histones, and the sequence-specific NF-YA subunit. In plants, the genes encoding each of the three subunits have expanded in number, giving rise to hundreds of potential trimers. While in mammals NF-Y binds a well-characterized motif, with a defined matrix centered on the CCAAT box, the specificity of the plant trimers has yet to be determined. Here we report that Arabidopsis thaliana NF-Y trimeric complexes, containing two different NF-YA subunits, bind DNA in vitro with similar affinities. We assayed precisely sequence-specificity by saturation mutagenesis, and analyzed genomic DNA sites bound in vivo by selected HFDs. The plant NF-Y CCAAT matrix is different in nucleotides flanking CCAAT with respect to the mammalian matrix, in vitro and in vivo. Our data point to flexible DNA-binding rules by plant NF-Ys, serving the scope of adapting to a diverse audience of genomic motifs.

scholarly journals Single position substitution of hairpin pyrrole-imidazole polyamides imparts distinct DNA-binding profiles across the human genome

PLoS ONE ◽  
2020 ◽  
Vol 15 (12) ◽  
pp. e0243905
Author(s):  
Paul B. Finn ◽  
Devesh Bhimsaria ◽  
Asfa Ali ◽  
Asuka Eguchi ◽  
Aseem Z. Ansari ◽  
...  
Keyword(s):  
Dna Binding ◽  
Dna Sequences ◽  
Dna Interaction ◽  
Sequence Specificity ◽  
Entire Genome ◽  
Dna Sequence Specificity ◽  
Interaction Sites ◽  
The Impact

Pyrrole–imidazole (Py–Im) polyamides are synthetic molecules that can be rationally designed to target specific DNA sequences to both disrupt and recruit transcriptional machinery. While in vitro binding has been extensively studied, in vivo effects are often difficult to predict using current models of DNA binding. Determining the impact of genomic architecture and the local chromatin landscape on polyamide-DNA sequence specificity remains an unresolved question that impedes their effective deployment in vivo. In this report we identified polyamide–DNA interaction sites across the entire genome, by covalently crosslinking and capturing these events in the nuclei of human LNCaP cells. This technique confirms the ability of two eight ring hairpin-polyamides, with similar architectures but differing at a single ring position (Py to Im), to retain in vitro specificities and display distinct genome-wide binding profiles.

scholarly journals Campylobacter jejuniBumSR directs a response to butyrate via sensor phosphatase activity to impact transcription and colonization

2020 ◽  
Vol 117 (21) ◽  
pp. 11715-11726 ◽  
Author(s):  
Kyle N. Goodman ◽  
Matthew J. Powers ◽  
Alexander A. Crofts ◽  
M. Stephen Trent ◽  
David R. Hendrixson
Keyword(s):  
Signal Transduction ◽  
Target Genes ◽  
Diarrheal Disease ◽  
Response Regulator ◽  
Short Chain Fatty Acids ◽  
Signal Transduction System ◽  
Genes Encoding ◽  
In Vivo Growth

Campylobacter jejunimonitors intestinal metabolites produced by the host and microbiota to initiate intestinal colonization of avian and animal hosts for commensalism and infection of humans for diarrheal disease. We previously discovered thatC. jejunihas the capacity to spatially discern different intestinal regions by sensing lactate and the short-chain fatty acids acetate and butyrate and then alter transcription of colonization factors appropriately for in vivo growth. In this study, we identified theC. jejunibutyrate-modulated regulon and discovered that the BumSR two-component signal transduction system (TCS) directs a response to butyrate by identifying mutants in a genetic screen defective for butyrate-modulated transcription. The BumSR TCS, which is important for infection of humans and optimal colonization of avian hosts, senses butyrate likely by indirect means to alter transcription of genes encoding important colonization determinants. Unlike many canonical TCSs, the predicted cytoplasmic sensor kinase BumS lacked in vitro autokinase activity, which would normally lead to phosphorylation of the cognate BumR response regulator. Instead, BumS has likely evolved mutations to naturally function as a phosphatase whose activity is influenced by exogenous butyrate to control the level of endogenous phosphorylation of BumR and its ability to alter transcription of target genes. To our knowledge, the BumSR TCS is the only bacterial signal transduction system identified so far that mediates responses to the microbiota-generated intestinal metabolite butyrate, an important factor for host intestinal health and homeostasis. Our findings suggest that butyrate sensing by this system is vital forC. jejunicolonization of multiple hosts.

scholarly journals Single position substitution of hairpin pyrrole-imidazole polyamides imparts distinct DNA-binding profiles across the human genome

2020 ◽  
Author(s):  
Paul B. Finn ◽  
Devesh Bhimsaria ◽  
Asfa Ali ◽  
Asuka Eguchi ◽  
Aseem Z. Ansari ◽  
...  
Keyword(s):  
Dna Binding ◽  
Dna Sequences ◽  
Dna Interaction ◽  
Sequence Specificity ◽  
Entire Genome ◽  
Dna Sequence Specificity ◽  
Interaction Sites ◽  
The Impact

ABSTRACTRegulating desired loci in the genome with sequence-specific DNA-binding molecules is a major goal for the development of precision medicine. Pyrrole–imidazole (Py–Im) polyamides are synthetic molecules that can be rationally designed to target specific DNA sequences to both disrupt and recruit transcriptional machinery. While in vitro binding has been extensively studied, in vivo effects are often difficult to predict using current models of DNA binding. Determining the impact of genomic architecture and the local chromatin landscape on polyamide-DNA sequence specificity remains an unresolved question that impedes their effective deployment in vivo. In this report we identified polyamide–DNA interaction sites across the entire genome, by covalently crosslinking and capturing these events in the nuclei of human LNCaP cells. This method, termed COSMIC-seq, confirms the ability of hairpin-polyamides, with similar architectures but differing at a single ring position, to retain in vitro specificities and display distinct genome-wide binding profiles. These results underpin the development of Py-Im polyamides as DNA-targeting molecules that mediate their regulatory or remedial functions at desired genomic loci.

scholarly journals A composite double-/single-stranded RNA-binding region in protein Prp3 supports tri-snRNP stability and splicing

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Sunbin Liu ◽  
Sina Mozaffari-Jovin ◽  
Jan Wollenhaupt ◽  
Karine F Santos ◽  
Matthias Theuser ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Rna Binding ◽  
Phylogenetic Analyses ◽  
Binding Region ◽  
U6 Snrna ◽  
Splicing Defects ◽  
Biochemical Analyses ◽  
Rna Interaction

Prp3 is an essential U4/U6 di-snRNP-associated protein whose functions and molecular mechanisms in pre-mRNA splicing are presently poorly understood. We show by structural and biochemical analyses that Prp3 contains a bipartite U4/U6 di-snRNA-binding region comprising an expanded ferredoxin-like fold, which recognizes a 3′-overhang of U6 snRNA, and a preceding peptide, which binds U4/U6 stem II. Phylogenetic analyses revealed that the single-stranded RNA-binding domain is exclusively found in Prp3 orthologs, thus qualifying as a spliceosome-specific RNA interaction module. The composite double-stranded/single-stranded RNA-binding region assembles cooperatively with Snu13 and Prp31 on U4/U6 di-snRNAs and inhibits Brr2-mediated U4/U6 di-snRNA unwinding in vitro. RNP-disrupting mutations in Prp3 lead to U4/U6•U5 tri-snRNP assembly and splicing defects in vivo. Our results reveal how Prp3 acts as an important bridge between U4/U6 and U5 in the tri-snRNP and comparison with a Prp24-U6 snRNA recycling complex suggests how Prp3 may be involved in U4/U6 reassembly after splicing.

scholarly journals Bordetella pertussis fim3gene regulation by BvgA: Phosphorylation controls the formation of inactive vs. active transcription complexes

2015 ◽  
Vol 112 (6) ◽  
pp. E526-E535 ◽  
Author(s):  
Alice Boulanger ◽  
Kyung Moon ◽  
Kimberly B. Decker ◽  
Qing Chen ◽  
Leslie Knipling ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Bordetella Pertussis ◽  
Transcription Initiation ◽  
Response Regulator ◽  
Gene Activation ◽  
Virulence Gene ◽  
In Vitro Transcription ◽  
Transcription Complexes

Two-component systems [sensor kinase/response regulator (RR)] are major tools used by microorganisms to adapt to environmental conditions. RR phosphorylation is typically required for gene activation, but few studies have addressed how and if phosphorylation affects specific steps during transcription initiation. We characterized transcription complexes made with RNA polymerase and theBordetella pertussisRR, BvgA, in its nonphosphorylated or phosphorylated (BvgA∼P) state at Pfim3, the promoter for the virulence genefim3(fimbrial subunit), using gel retardation, potassium permanganate and DNase I footprinting, cleavage reactions with protein conjugated with iron bromoacetamidobenzyl-EDTA, and in vitro transcription. Previous work has shown that the level of nonphosphorylated BvgA remains high in vivo under conditions in which BvgA is phosphorylated. Our results here indicate that surprisingly both BvgA and BvgA∼P form open and initiating complexes with RNA polymerase at Pfim3. However, phosphorylation of BvgA is needed to generate the correct conformation that can transition to competent elongation. Footprints obtained with the complexes made with nonphosphorylated BvgA are atypical; while the initiating complex with BvgA synthesizes short RNA, it does not generate full-length transcripts. Extended incubation of the BvgA/RNA polymerase initiated complex in the presence of heparin generates a stable, but defective species that depends on the initial transcribed sequence offim3. We suggest that the presence of nonphosphorylated BvgA down-regulates Pfim3activity when phosphorylated BvgA is present and may allow the bacterium to quickly adapt to the loss of inducing conditions by rapidly eliminating Pfim3activation once the signal for BvgA phosphorylation is removed.

scholarly journals Functional Analysis of theMycobacterium tuberculosis MprAB Two-Component SignalTransductionSystem

2003 ◽  
Vol 71 (12) ◽  
pp. 6962-6970 ◽  
Author(s):  
Thomas C. Zahrt ◽  
Christopher Wozniak ◽  
Denise Jones ◽  
Andrea Trevett
Keyword(s):  
Signal Transduction ◽  
Functional Analysis ◽  
Persistent Infection ◽  
Response Regulator ◽  
Sensor Kinase ◽  
Two Component System ◽  
Two Component ◽  
Two Component Signal Transduction

ABSTRACT The mechanisms utilized by Mycobacterium tuberculosis to establish, maintain, or reactivate from latent infection in the host are largely unknown but likely include genes that mediate adaptation to conditions encountered during persistence. Previously, a two-component signal transduction system, mprAB, was found to be required in M. tuberculosis for establishment and maintenance of persistent infection in a tissue- and stage-specific fashion. To begin to characterize the role of this system in M. tuberculosis physiology and virulence, a functional analysis of the mprA and mprB gene products was initiated. Here, evidence is presented demonstrating that sensor kinase MprB and response regulator MprA function as an intact signal-transducing pair in vitro and in vivo. Sensor kinase MprB can be autophosphorylated, can donate phosphate to MprA, and can act as a phospho-MprA phosphatase in vitro. Correspondingly, response regulator MprA can accept phosphate from MprB or from small phosphodonors including acetyl phosphate. Mutagenesis of residues His249 in MprB and Asp48 in MprA abolished the ability of these proteins to be phosphorylated in vitro. Introduction of these alleles into Mycobacterium bovis BCGattenuated virulence in macrophages in vivo. Together, these results support a role for the mprAB two-component system in M. tuberculosis physiology and pathogenesis. Characterization of two-component signal transduction systems will enhance our understanding of processes regulated by M. tuberculosis during acute and/or persistent infection in the host.

scholarly journals The Atypical Hybrid Histidine Protein Kinase RodK in Myxococcus xanthus: Spatial Proximity Supersedes Kinetic Preference in Phosphotransfer Reactions

2009 ◽  
Vol 191 (6) ◽  
pp. 1765-1776 ◽  
Author(s):  
Sigrun Wegener-Feldbrügge ◽  
Lotte Søgaard-Andersen
Keyword(s):  
Response Regulator ◽  
Myxococcus Xanthus ◽  
Kinase Domain ◽  
Full Length ◽  
Spatial Proximity ◽  
Fruiting Body Formation ◽  
Domain Structures ◽  
Two Component Signal Transduction

ABSTRACT Many proteins of two-component signal transduction systems (TCS) have domain structures that do not comply with a phosphate flow as observed in linear TCS, phosphorelays, or simple branched pathways. An example is RodK, which is essential for fruiting body formation in Myxococcus xanthus and, in addition to a sensor domain, consists of a kinase domain and three receiver domains (RodK-R1, -R2, and -R3), all of which are functionally important. We identified the RokA response regulator as part of the RodK pathway. In vitro the isolated RodK kinase domain engages in phosphotransfer to RodK-R3 and RokA, with a kinetic preference for RokA. However, in the context of the full-length protein, the RodK kinase domain has a preference for phosphotransfer to RodK-R3 over RokA. We suggest that in full-length RodK, the spatial proximity of the RodK kinase domain and RodK-R3 compensate for the kinetic preference of the isolated kinase domain for RokA. Thus, the kinetic preference observed using an isolated kinase domain of a hybrid kinase does not necessarily reflect the phosphotransfer preference of the full-length protein. We speculate that the phosphorylation status of RodK-R1 and RodK-R2 determines whether RodK engages in phosphotransfer to RodK-R3 or RokA in vivo.

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