Transcriptional control by two interacting regulatory proteins: identification of the PtxS binding site at PtxR

DNA-binding proteins control many fundamental biological processes such as transcription, recombination and replication. A major goal is to decipher the role that DNA sequence plays in orchestrating the binding and activity of such regulatory proteins. To address this goal, it is useful to rationally design DNA sequences with desired numbers, affinities and arrangements of protein binding sites. However, removing binding sites from DNA is computationally non-trivial since one risks creating new sites in the process of deleting or moving others. Here we present an online binding site removal tool, SiteOut, that enables users to design arbitrary DNA sequences that entirely lack binding sites for factors of interest. SiteOut can also be used to delete sites from a specific sequence, or to introduce site-free spacers between functional sequences without creating new sites at the junctions. In combination with commercial DNA synthesis services, SiteOut provides a powerful and flexible platform for synthetic projects that interrogate regulatory DNA. Here we describe the algorithm and illustrate the ways in which SiteOut can be used; it is publicly available at https://depace.med.harvard.edu/siteout/

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Structural basis for the allosteric regulation of the SbtA bicarbonate transporter by the PII-like protein, SbtB, from Cyanobium sp. PCC7001

10.1101/762807 ◽

2019 ◽

Author(s):

Joe A. Kaczmarski ◽

Nan-Sook Hong ◽

Bratati Mukherjee ◽

Laura T. Wey ◽

Loraine Rourke ◽

...

Keyword(s):

Metal Ion ◽

Transcriptional Control ◽

Allosteric Regulation ◽

Inhibitory Effect ◽

Photosynthetic Performance ◽

Regulatory Proteins ◽

Structural Basis ◽

Titration Calorimetry ◽

X Ray ◽

Bicarbonate Transporter

ABSTRACTCyanobacteria have evolved a suite of enzymes and inorganic carbon (Ci) transporters that improve photosynthetic performance by increasing the localized concentration of CO2 around the primary CO2-fixating enzyme, Rubisco. This CO2-concentrating mechanism (CCM) is highly regulated, responds to illumination/darkness cycles and allows cyanobacteria to thrive under limiting Ci conditions. While the transcriptional control of CCM activity is well understood, less is known about how regulatory proteins might allosterically regulate Ci transporters in response to changing conditions. Cyanobacterial sodium-dependent bicarbonate transporters (SbtAs) are inhibited by PII-like regulatory proteins (SbtBs), with the inhibitory effect being modulated by adenylnucleotides. Here, we used isothermal titration calorimetry to show that SbtB from Cyanobium sp. PCC7001 (SbtB7001) binds AMP, ADP, cAMP and ATP with micromolar-range affinities. X-ray crystal structures of apo- and nucleotide-bound SbtB7001 revealed that while AMP, ADP and cAMP have little effect on the SbtB7001 structure, binding of ATP stabilizes the otherwise flexible T-loop and that the flexible C-terminal C-loop adopts several distinct conformations. We also show that ATP binding affinity is increased ten-fold in the presence of Ca2+ and we present an X-ray crystal structure of Ca2+ATP:SbtB7001 that shows how this metal ion facilitates additional stabilizing interactions with the apex of the T-loop. We propose that the Ca2+ATP-induced conformational change observed in SbtB7001 is important for allosteric regulation of SbtA activity by SbtB and is consistent with changing adenylnucleotide levels in illumination/darkness cycles.GRAPHICAL ABSTRACT

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Joint Transcriptional Control of xpsR, the Unusual Signal Integrator of the Ralstonia solanacearum Virulence Gene Regulatory Network, by a Response Regulator and a LysR-Type Transcriptional Activator

Journal of Bacteriology ◽

10.1128/jb.180.10.2736-2743.1998 ◽

1998 ◽

Vol 180 (10) ◽

pp. 2736-2743 ◽

Cited By ~ 35

Author(s):

Jianzhong Huang ◽

Wandee Yindeeyoungyeon ◽

Ram P. Garg ◽

Timothy P. Denny ◽

Mark A. Schell

Keyword(s):

Binding Site ◽

Transcriptional Control ◽

Response Regulator ◽

Consensus Sequence ◽

Virulence Gene ◽

Transcriptional Activator ◽

Binding Assays ◽

Environmental Signals

ABSTRACT Ralstonia (Pseudomonas)solanacearum is a soil-borne phytopathogen that causes a wilting disease of many important crops. It makes large amounts of the exopolysaccharide EPS I, which it requires for efficient colonization, wilting, and killing of plants. Transcription of the epsoperon, encoding biosynthetic enzymes for EPS I, is controlled by a unique and complex sensory network that responds to multiple environmental signals. This network is comprised of the novel transcriptional activator XpsR, three distinct two-component regulatory systems (VsrAD, VsrBC, and PhcSR), and the LysR-type regulator PhcA, which is under the control of PhcSR. Here we show that thexpsR promoter (P xpsR ) is simultaneously controlled by PhcA and VsrD, permitting XpsR to act like a signal integrator, simultaneously coordinating signal input into theeps promoter from both VsrAD and PhcSR. Additionally, we used in vivo expression analysis and in vitro DNA binding assays with substitution and deletion mutants of P xpsR to show the following. (i) PhcA primarily interacts with a typical 14-bp LysR-type consensus sequence around position −77, causing a sixfold activation of P xpsR ; a weaker, less-defined binding site between −183 and −239 likely enhances PhcA binding and activation via the −77 site another twofold. (ii) Full 70-fold activation of P xpsR requires the additional interaction of the VsrD response regulator (or its surrogate) with a 14-bp dyadic sequence centered around −315 where it enhances activation (and possibly binding) by PhcA; however, VsrD alone cannot activate P xpsR . (iii) Increasing the distance between the putative VsrD binding site from that of PhcA by up to 232 bp did not dramatically affect P xpsR activation or regulation.

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Localization and Interaction of the Proteins Constituting the GAL Genetic Switch in Saccharomyces cerevisiae

Eukaryotic Cell ◽

10.1128/ec.00261-08 ◽

2008 ◽

Vol 7 (12) ◽

pp. 2061-2068 ◽

Cited By ~ 22

Author(s):

Raymond Wightman ◽

Rachel Bell ◽

Richard J. Reece

Keyword(s):

Gene Expression ◽

Saccharomyces Cerevisiae ◽

Cellular Localization ◽

Transcriptional Control ◽

Resonance Energy ◽

Regulatory Proteins ◽

Yeast Cells ◽

Galactose Metabolism ◽

Genetic Switch ◽

Metabolism Regulation

ABSTRACT In Saccharomyces cerevisiae, the GAL genes encode the enzymes required for galactose metabolism. Regulation of these genes has served as the paradigm for eukaryotic transcriptional control over the last 50 years. The switch between inert and active gene expression is dependent upon three proteins—the transcriptional activator Gal4p, the inhibitor Gal80p, and the ligand sensor Gal3p. Here, we present a detailed spatial analysis of the three GAL regulatory proteins produced from their native genomic loci. Using a novel application of photobleaching, we demonstrate, for the first time, that the Gal3p ligand sensor enters the nucleus of yeast cells in the presence of galactose. Additionally, using Förster resonance energy transfer, we show that the interaction between Gal3p and Gal80p occurs throughout the yeast cell. Taken together, these data challenge existing models for the cellular localization of the regulatory proteins during the induction of GAL gene expression by galactose and suggest a mechanism for the induction of the GAL genes in which galactose-bound Gal3p moves from the cytoplasm to the nucleus to interact with the transcriptional inhibitor Gal80p.

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The role of TFIID, the initiator element and a novel 5′ TFIID binding site in the transcriptional control of the TATA-less human cytosolic phospholipase A2-α promoter

Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression ◽

10.1016/j.bbaexp.2004.09.006 ◽

2004 ◽

Vol 1680 (3) ◽

pp. 145-157 ◽

Cited By ~ 11

Author(s):

Mark J. Cowan ◽

Xiang-Lan Yao ◽

Rafal Pawliczak ◽

Xiuli Huang ◽

Carolea Logun ◽

...

Keyword(s):

Phospholipase A2 ◽

Binding Site ◽

Transcriptional Control ◽

Cytosolic Phospholipase A2 ◽

Cytosolic Phospholipase ◽

Initiator Element

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Putative Coiled-Coil Structural Elements of the BBA68 Protein of Lyme Disease Spirochetes Are Required for Formation of Its Factor H Binding Site

Journal of Bacteriology ◽

10.1128/jb.187.4.1317-1323.2005 ◽

2005 ◽

Vol 187 (4) ◽

pp. 1317-1323 ◽

Cited By ~ 37

Author(s):

John V. McDowell ◽

Matthew E. Harlin ◽

Elizabeth A. Rogers ◽

Richard T. Marconi

Keyword(s):

Lyme Disease ◽

Binding Site ◽

Immune Evasion ◽

Coiled Coil ◽

Factor H ◽

Regulatory Proteins ◽

Structural Elements ◽

Binding Ability ◽

Complement Regulatory Proteins ◽

Factor I

ABSTRACT Factor H and factor H like-protein 1 (FHL-1) are complement regulatory proteins that serve as cofactors for the factor I-mediated cleavage of C3b. Some Lyme disease and relapsing fever spirochete species bind factor H to their surface to facilitate immune evasion. The Lyme disease spirochetes produce several factor H binding proteins (FHBPs) that form two distinct classes. Class I FHBPs (OspE orthologs and paralogs) bind only factor H, while class II FHBPs (BBA68) bind both factor H and FHL-1. BBA68 belongs to a large paralogous protein family, and of these paralogs, BBA69 is the member most closely related to BBA68. To determine if BBA69 can also bind factor H, recombinant protein was generated and tested for factor H binding. BBA69 did not exhibit factor H binding ability, suggesting that among family 54 paralogs, factor H binding is unique to BBA68. To identify the determinants of BBA68 that are involved in factor H binding, truncation and site-directed mutational analyses were performed. These analyses revealed that the factor H binding site is discontinuous and provide strong evidence that coiled-coil structural elements are involved in the formation of the binding site.

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Enhanced TF binding site maps improve regulatory networks learned from accessible chromatin data

10.1101/545780 ◽

2019 ◽

Author(s):

Shubhada R. Kulkarni ◽

D. Marc Jones ◽

Klaas Vandepoele

Keyword(s):

Binding Site ◽

Dna Sequences ◽

Regulatory Networks ◽

Transcriptional Control ◽

Chromatin Accessibility ◽

Systematic Evaluation ◽

Open Chromatin ◽

Ensemble Approach ◽

Complete Cell ◽

Regulatory Dna

ABSTRACTDetermining where transcription factors (TF) bind in genomes provides insights into which transcriptional programs are active across organs, tissue types, and environmental conditions. Recent advances in high-throughput profiling of regulatory DNA have yielded large amounts of information about chromatin accessibility. Interpreting the functional significance of these datasets requires knowledge of which regulators are likely to bind these regions. This can be achieved by using information about TF binding preferences, or motifs, to identify TF binding events that are likely to be functional. Although different approaches exist to map motifs to DNA sequences, a systematic evaluation of these tools in plants is missing. Here we compare four motif mapping tools widely used in the Arabidopsis research community and evaluate their performance using chromatin immunoprecipitation datasets for 40 TFs. Downstream gene regulatory network (GRN) reconstruction was found to be sensitive to the motif mapper used. We further show that the low recall of FIMO, one of the most frequently used motif mapping tools, can be overcome by using an Ensemble approach, which combines results from different mapping tools. Several examples are provided demonstrating how the Ensemble approach extends our view on transcriptional control for TFs active in different biological processes. Finally, a new protocol is presented to efficiently derive more complete cell type-specific GRNs through the integrative analysis of open chromatin regions, known binding site information, and expression datasets.

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GCUNC-45 Is a Novel Regulator for the Progesterone Receptor/hsp90 Chaperoning Pathway

Molecular and Cellular Biology ◽

10.1128/mcb.26.5.1722-1730.2006 ◽

2006 ◽

Vol 26 (5) ◽

pp. 1722-1730 ◽

Cited By ~ 44

Author(s):

Ahmed Chadli ◽

J. Dinny Graham ◽

M. Greg Abel ◽

Twila A. Jackson ◽

David F. Gordon ◽

...

Keyword(s):

Progesterone Receptor ◽

Atpase Activity ◽

Binding Site ◽

Signaling Pathway ◽

Regulatory Proteins ◽

N Terminus ◽

Positive Factor

ABSTRACT The hsp90 chaperoning pathway is a multiprotein system that is required for the production or activation of many cell regulatory proteins, including the progesterone receptor (PR). We report here the identity of GCUNC-45 as a novel modulator of PR chaperoning by hsp90. GCUNC-45, previously implicated in the activities of myosins, can interact in vivo and in vitro with both PR-A and PR-B and with hsp90. Overexpression and knockdown experiments show GCUNC-45 to be a positive factor in promoting PR function in the cell. GCUNC-45 binds to the ATP-binding domain of hsp90 to prevent the activation of its ATPase activity by the cochaperone Aha1. This effect limits PR chaperoning by hsp90, but this can be reversed by FKBP52, a cochaperone that is thought to act later in the pathway. These findings reveal a new cochaperone binding site near the N terminus of hsp90, add insight on the role of FKBP52, and identify GCUNC-45 as a novel regulator of the PR signaling pathway.

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Transcriptional Repression of the VC2105 Protein by Vibrio FadR Suggests that It Is a New Auxiliary Member of thefadRegulon

Applied and Environmental Microbiology ◽

10.1128/aem.00293-16 ◽

2016 ◽

Vol 82 (9) ◽

pp. 2819-2832 ◽

Cited By ~ 6

Author(s):

Rongsui Gao ◽

Jingxia Lin ◽

Han Zhang ◽

Youjun Feng

Keyword(s):

Transcriptional Regulation ◽

Fatty Acid ◽

Ligand Binding ◽

Binding Site ◽

Binding Sites ◽

Regulatory Proteins ◽

Content Type ◽

Ligand Binding Sites

ABSTRACTRecently, our group along with others reported that theVibrioFadR regulatory protein is unusual in that, unlike the prototypicalfadRproduct ofEscherichia coli, which has only one ligand-binding site,VibrioFadR has two ligand-binding sites and represents a new mechanism for fatty acid sensing. The promoter region of thevc2105gene, encoding a putative thioesterase, was mapped, and a putative FadR-binding site (AA CTG GTA AGA GCA CTT) was proposed. Different versions of the FadR regulatory proteins were prepared and purified to homogeneity. Both electrophoretic mobility shift assay (EMSA) and surface plasmon resonance (SPR) determined the direct interaction of thevc2105gene with FadR proteins of various origins. Further, EMSAs illustrated that the addition of long-chain acyl-coenzyme A (CoA) species efficiently dissociates thevc2105promoter from the FadR regulator. The expression level of theVibrio cholerae vc2105gene was elevated 2- to 3-fold in afadRnull mutant strain, validating that FadR is a repressor for thevc2105gene. The β-galactosidase activity of avc2105-lacZtranscriptional fusion was increased over 2-fold upon supplementation of growth medium with oleic acid. Unlike thefadDgene, a member of theVibrio fadregulon, the VC2105 protein played no role in bacterial growth and virulence-associated gene expression ofctxAB(cholera toxin A/B) andtcpA(toxin coregulated pilus A). Given that the transcriptional regulation ofvc2105fits the criteria for fatty acid degradation (fad) genes, we suggested that it is a new member of theVibrio fadregulon.IMPORTANCETheVibrioFadR regulator is unusual in that it has two ligand-binding sites. Different versions of the FadR regulatory proteins were prepared and characterizedin vitroandin vivo. An auxiliaryfadgene (vc2105) fromVibriowas proposed that encodes a putative thioesterase and has a predicted FadR-binding site (AAC TGG TA A GAG CAC TT). The function of this putative binding site was proved using both EMSA and SPR. Furtherin vitroandin vivoexperiments revealed that theVibrioFadR is a repressor for thevc2105gene. UnlikefadD, a member of theVibrio fadregulon, VC2105 played no role in bacterial growth and expression of the two virulence-associated genes (ctxABandtcpA). Therefore, since transcriptional regulation ofvc2105fits the criteria forfadgenes, it seems likely thatvc2105acts as a new auxiliary member of theVibrio fadregulon.

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