scholarly journals Facilitated Dissociation of Transcription Factors from Single DNA Binding Sites

2017 ◽  
Author(s):  
Ramsey I. Kamar ◽  
Edward J. Banigan ◽  
Aykut Erbas ◽  
Rebecca D. Giuntoli ◽  
Monica Olvera de la Cruz ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Binding Site ◽  
Single Molecule ◽  
Binding Sites ◽  
Binding Kinetics ◽  
General Mechanism ◽  
Dissociation Kinetics ◽  
Dna Binding Sites ◽  
Standard Picture ◽  
Dynamics Simulations

ABSTRACTThe binding of transcription factors (TFs) to DNA controls most aspects of cellular function, making the understanding of their binding kinetics imperative. The standard description of bimolecular interactions posits TF off-rates are independent of TF concentration in solution. However, recent observations have revealed that proteins in solution can accelerate the dissociation of DNA-bound proteins. To study the molecular basis of facilitated dissociation (FD), we have used single-molecule imaging to measure dissociation kinetics of Fis, a key E. coli TF and major bacterial nucleoid protein, from single dsDNA binding sites. We observe a strong FD effect characterized by an exchange rate ∽1 × 104 M-1s-1, establishing that FD of Fis occurs at the single-binding-site level, and we find that the off-rate saturates at large Fis concentrations in solution. While spontaneous (i.e., competitor-free) dissociation shows a strong salt dependence, we find that facilitated dissociation depends only weakly on salt. These results are quantitatively explained by a model in which partially dissociated bound proteins are susceptible to invasion by competitor proteins in solution. We also report FD of NHP6A, a yeast TF whose structure differs significantly from Fis. We further perform molecular dynamics simulations, which indicate that FD can occur for molecules that interact far more weakly than those we have studied. Taken together, our results indicate that FD is a general mechanism assisting in the local removal of TFs from their binding sites and does not necessarily require cooperativity, clustering, or binding site overlap.SIGNIFICANCE STATEMENTTranscription factors (TFs) control biological processes by binding and unbinding to DNA. Therefore it is crucial to understand the mechanisms that affect TF binding kinetics. Recent studies challenge the standard picture of TF binding kinetics by demonstrating cases of proteins in solution accelerating TF dissociation rates through a facilitated dissociation (FD) process. Our study shows that FD can occur at the level of single binding sites, without the action of large protein clusters or long DNA segments. Our results quantitatively support a model of FD in which competitor proteins invade partially dissociated states of DNA-bound TFs. FD is expected to be a general mechanism for modulating gene expression by altering the occupancy of TFs on the genome.Author ContributionsRamsey I. Kamardesigned research, performed research, contributed new reagents/analytic tools, analyzed data, wrote the paperEdward J. Banigandesigned research, performed research, contributed new reagents/analytic tools, analyzed data, wrote the paperAykut Erbasdesigned research, performed research, contributed new reagents/analytic tools, analyzed data, wrote the paperRebecca D. Giuntolidesigned research, performed research, contributed new reagents/analytic tools, analyzed data, wrote the paperMonica Olvera de la Cruzdesigned research, performed research, wrote the paperReid C. Johnsondesigned research, performed research, contributed new reagents/analytic tools, analyzed data, wrote the paperJohn F. Markodesigned research, performed research, contributed new reagents/analytic tools, analyzed data, wrote the paper

scholarly journals Facilitated dissociation of transcription factors from single DNA binding sites

2017 ◽  
Vol 114 (16) ◽  
pp. E3251-E3257 ◽  
Author(s):  
Ramsey I. Kamar ◽  
Edward J. Banigan ◽  
Aykut Erbas ◽  
Rebecca D. Giuntoli ◽  
Monica Olvera de la Cruz ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Binding Site ◽  
Single Molecule ◽  
Binding Sites ◽  
General Mechanism ◽  
Dissociation Kinetics ◽  
Dna Binding Sites ◽  
Bimolecular Interactions ◽  
Dynamics Simulations ◽  
Kinetics Of

The binding of transcription factors (TFs) to DNA controls most aspects of cellular function, making the understanding of their binding kinetics imperative. The standard description of bimolecular interactions posits that TF off rates are independent of TF concentration in solution. However, recent observations have revealed that proteins in solution can accelerate the dissociation of DNA-bound proteins. To study the molecular basis of facilitated dissociation (FD), we have used single-molecule imaging to measure dissociation kinetics of Fis, a key Escherichia coli TF and major bacterial nucleoid protein, from single dsDNA binding sites. We observe a strong FD effect characterized by an exchange rate ∼1×104 M−1s−1, establishing that FD of Fis occurs at the single-binding site level, and we find that the off rate saturates at large Fis concentrations in solution. Although spontaneous (i.e., competitor-free) dissociation shows a strong salt dependence, we find that FD depends only weakly on salt. These results are quantitatively explained by a model in which partially dissociated bound proteins are susceptible to invasion by competitor proteins in solution. We also report FD of NHP6A, a yeast TF with structure that differs significantly from Fis. We further perform molecular dynamics simulations, which indicate that FD can occur for molecules that interact far more weakly than those that we have studied. Taken together, our results indicate that FD is a general mechanism assisting in the local removal of TFs from their binding sites and does not necessarily require cooperativity, clustering, or binding site overlap.

Selection of new HSF1 and HSF2 DNA-binding sites reveals difference in trimer cooperativity

1994 ◽  
Vol 14 (11) ◽  
pp. 7592-7603
Author(s):  
P E Kroeger ◽  
R I Morimoto
Keyword(s):  
Transcription Factors ◽  
Heat Shock ◽  
Dna Binding ◽  
Binding Site ◽  
Binding Sites ◽  
Cellular Stress Response ◽  
Specific Factor ◽  
Heat Shock Element ◽  
Cooperative Interactions ◽  
Dna Binding Sites

Multiple heat shock transcription factors (HSFs) have been discovered in several higher eukaryotes, raising questions about their respective functions in the cellular stress response. Previously, we had demonstrated that the two mouse HSFs (mHSF1 and mHSF2) interacted differently with the HSP70 heat shock element (HSE). To further address the issues of cooperativity and the interaction of multiple HSFs with the HSE, we selected new mHSF1 and mHSF2 DNA-binding sites through protein binding and PCR amplification. The selected sequences, isolated from a random population, were composed primarily of alternating inverted arrays of the pentameric consensus 5'-nGAAn-3', and the nucleotides flanking the core GAA motif were nonrandom. The average number of pentamers selected in each binding site was four to five for mHSF1 and two to three for mHSF2, suggesting differences in the potential for cooperative interactions between adjacent trimers. Our comparison of mHSF1 and mHSF2 binding to selected sequences further substantiated these differences in cooperativity as mHSF1, unlike mHSF2, was able to bind to extended HSE sequences, confirming previous observations on the HSP70 HSE. Certain selected sequences that exhibited preferential binding of mHSF1 or mHSF2 were mutagenized, and these studies demonstrated that the affinity of an HSE for a particular HSF and the extent of HSF interaction could be altered by single base substitutions. The domain of mHSF1 utilized for cooperative interactions was transferable, as chimeric mHSF1/mHSF2 proteins demonstrated that sequences within or adjacent to the mHSF1 DNA-binding domain were responsible. We have demonstrated that HSEs can have a greater affinity for a specific HSF and that in mice, mHSF1 utilizes a higher degree of cooperativity in DNA binding. This suggests two ways in which cells have developed to regulate the activity of closely related transcription factors: developing the ability to fully occupy the target binding site and alteration of the target site to favor interaction with a specific factor.

scholarly journals Selection of new HSF1 and HSF2 DNA-binding sites reveals difference in trimer cooperativity.

1994 ◽  
Vol 14 (11) ◽  
pp. 7592-7603 ◽  
Author(s):  
P E Kroeger ◽  
R I Morimoto
Keyword(s):  
Transcription Factors ◽  
Heat Shock ◽  
Dna Binding ◽  
Binding Site ◽  
Binding Sites ◽  
Cellular Stress Response ◽  
Specific Factor ◽  
Heat Shock Element ◽  
Cooperative Interactions ◽  
Dna Binding Sites

Multiple heat shock transcription factors (HSFs) have been discovered in several higher eukaryotes, raising questions about their respective functions in the cellular stress response. Previously, we had demonstrated that the two mouse HSFs (mHSF1 and mHSF2) interacted differently with the HSP70 heat shock element (HSE). To further address the issues of cooperativity and the interaction of multiple HSFs with the HSE, we selected new mHSF1 and mHSF2 DNA-binding sites through protein binding and PCR amplification. The selected sequences, isolated from a random population, were composed primarily of alternating inverted arrays of the pentameric consensus 5'-nGAAn-3', and the nucleotides flanking the core GAA motif were nonrandom. The average number of pentamers selected in each binding site was four to five for mHSF1 and two to three for mHSF2, suggesting differences in the potential for cooperative interactions between adjacent trimers. Our comparison of mHSF1 and mHSF2 binding to selected sequences further substantiated these differences in cooperativity as mHSF1, unlike mHSF2, was able to bind to extended HSE sequences, confirming previous observations on the HSP70 HSE. Certain selected sequences that exhibited preferential binding of mHSF1 or mHSF2 were mutagenized, and these studies demonstrated that the affinity of an HSE for a particular HSF and the extent of HSF interaction could be altered by single base substitutions. The domain of mHSF1 utilized for cooperative interactions was transferable, as chimeric mHSF1/mHSF2 proteins demonstrated that sequences within or adjacent to the mHSF1 DNA-binding domain were responsible. We have demonstrated that HSEs can have a greater affinity for a specific HSF and that in mice, mHSF1 utilizes a higher degree of cooperativity in DNA binding. This suggests two ways in which cells have developed to regulate the activity of closely related transcription factors: developing the ability to fully occupy the target binding site and alteration of the target site to favor interaction with a specific factor.

scholarly journals Specificity landscapes unmask submaximal binding site preferences of transcription factors

2018 ◽  
Vol 115 (45) ◽  
pp. E10586-E10595 ◽  
Author(s):  
Devesh Bhimsaria ◽  
José A. Rodríguez-Martínez ◽  
Junkun Pan ◽  
Daniel Roston ◽  
Elif Nihal Korkmaz ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Binding Site ◽  
Binding Sites ◽  
Energy Landscape ◽  
Side Chains ◽  
Site Preferences ◽  
Dna Binding Sites ◽  
Distinct Sequence ◽  
Allele Specific ◽  
Direct Readout

We have developed Differential Specificity and Energy Landscape (DiSEL) analysis to comprehensively compare DNA–protein interactomes (DPIs) obtained by high-throughput experimental platforms and cutting edge computational methods. While high-affinity DNA binding sites are identified by most methods, DiSEL uncovered nuanced sequence preferences displayed by homologous transcription factors. Pairwise analysis of 726 DPIs uncovered homolog-specific differences at moderate- to low-affinity binding sites (submaximal sites). DiSEL analysis of variants of 41 transcription factors revealed that many disease-causing mutations result in allele-specific changes in binding site preferences. We focused on a set of highly homologous factors that have different biological roles but “read” DNA using identical amino acid side chains. Rather than direct readout, our results indicate that DNA noncontacting side chains allosterically contribute to sculpt distinct sequence preferences among closely related members of transcription factor families.

scholarly journals Participation of Ets transcription factors in the glucocorticoid response of the rat tyrosine aminotransferase gene.

1994 ◽  
Vol 14 (6) ◽  
pp. 4116-4125 ◽  
Author(s):  
M L Espinás ◽  
J Roux ◽  
J Ghysdael ◽  
R Pictet ◽  
T Grange
Keyword(s):  
Transcription Factors ◽  
Binding Site ◽  
Cell Line ◽  
Binding Sites ◽  
Tyrosine Aminotransferase ◽  
Hierarchical Level ◽  
Detectable Effect ◽  
Independent Manner ◽  
Glucocorticoid Response

We have previously shown that two remote glucocorticoid-responsive units (GRUs) of the rat tyrosine aminotransferase (TAT) gene contain multiple binding sites for several transcription factor families, including the glucocorticoid receptor (GR). We report here the identification of two novel binding sites for members of the Ets family of transcription factors in one of these GRUs. One of these binding sites overlaps the major GR-binding site (GRBS), whereas the other is located in its vicinity. Inactivation of the latter binding site leads to a twofold reduction of the glucocorticoid response, whereas inactivation of the site overlapping the GRBS has no detectable effect. In vivo footprinting analysis reveals that the active site is occupied in a glucocorticoid-independent manner, in a TAT-expressing cell line, even though it is located at a position where there is a glucocorticoid-dependent alteration of the nucleosomal structure. This same site is not occupied in a cell line that does not express TAT but expresses Ets-related DNA-binding activities, suggesting the existence of an inhibitory effect of chromatin structure at a hierarchical level above the nucleosome. The inactive Ets-binding site that overlaps the GRBS is not occupied even in TAT-expressing cells. However, this same overlapping site can confer Ets-dependent stimulation of both basal and glucocorticoid-induced levels when it is isolated from the GRU and duplicated. Ets-1 expression in COS cells mimics the activity of the Ets-related activities present in hepatoma cells. These Ets-binding sites could participate in the integration of the glucocorticoid response of the TAT gene with signal transduction pathways triggered by other nonsteroidal extracellular stimuli.

scholarly journals DNA damage independent inhibition of NF-κB transcription by anthracyclines

2020 ◽  
Author(s):  
Angelo Chora ◽  
Dora Pedroso ◽  
Nadja Pejanovic ◽  
Eleni Kyriakou ◽  
Henrique Colaço ◽  
...  
Keyword(s):  
Dna Damage ◽  
Transcription Factors ◽  
Binding Sites ◽  
Inflammatory Responses ◽  
Inflammatory Diseases ◽  
Anticancer Properties ◽  
Dna Binding Sites ◽  
Immune Mediated ◽  
Life Threatening ◽  
Molecular Patterns

AbstractTranscriptional programs leading to induction of a large number of genes can be rapidly initiated by the activation of only few selected transcription factors. Upon stimulation of macrophages with microbial-associated molecular patterns (MAMPs), the activation of the nuclear factor kappa B (NF-κB) family of transcription factors triggers inflammatory responses that, left uncontrolled, can lead to excessive inflammation with life-threatening consequences for the host. Here we identify and characterize a novel effect of Anthracyclines, a class of drugs currently used as potent anticancer drugs, in the regulation of NF-κB transcriptional activity in BMDMs, in addition to the previously reported DNA damage and histone eviction. Anthracyclines, including Doxorubicin, Daunorubicin and Epirubicin, disturb the complexes formed between the NF-κB subunit RelA and its DNA binding sites, to limit NF-κB-dependent gene transcription during inflammatory responses, including of pivotal pro-inflammatory mediators such as TNF. We observed that suppression of inflammation can also be mediated by Aclarubicin, Doxorubicinone and the newly developed Dimethyl-doxorubicin, which share anticancer properties with the other Anthracyclines, but do not induce DNA damage in the tested concentrations. This novel mechanism of action of Anthracyclines, contributing to the reduction of inflammation, is thus independent of the activation of DNA damage responses and may be relevant for the development of novel strategies targeting immune-mediated inflammatory diseases.

scholarly journals Single Molecule Investigation of Ag+ Interactions with Single Cytosine-, Methylcytosine- and Hydroxymethylcytosine-Cytosine Mismatches in a Nanopore

2014 ◽  
Vol 4 (1) ◽  
Author(s):  
Yong Wang ◽  
Bin-Quan Luan ◽  
Zhiyu Yang ◽  
Xinyue Zhang ◽  
Brandon Ritzo ◽  
...  
Keyword(s):  
Hydrogen Bond ◽  
Binding Site ◽  
Single Molecule ◽  
Metal Ion ◽  
Detection Method ◽  
Direct Detection ◽  
Alpha Hemolysin ◽  
Duplex Stability ◽  
Dynamics Simulations ◽  
Direct Detection Method

Abstract Both cytosine-Ag-cytosine interactions and cytosine modifications in a DNA duplex have attracted great interest for research. Cytosine (C) modifications such as methylcytosine (mC) and hydroxymethylcytosine (hmC) are associated with tumorigenesis. However, a method for directly discriminating C, mC and hmC bases without labeling, modification and amplification is still missing. Additionally, the nature of coordination of Ag+ with cytosine-cytosine (C-C) mismatches is not clearly understood. Utilizing the alpha-hemolysin nanopore, we show that in the presence of Ag+, duplex stability is most increased for the cytosine-cytosine (C-C) pair, followed by the cytosine-methylcytosine (C-mC) pair and the cytosine-hydroxymethylcytosine (C-hmC) pair, which has no observable Ag+ induced stabilization. Molecular dynamics simulations reveal that the hydrogen-bond-mediated paring of a C-C mismatch results in a binding site for Ag+. Cytosine modifications (such as mC and hmC) disrupted the hydrogen bond, resulting in disruption of the Ag+ binding site. Our experimental method provides a novel platform to study the metal ion-DNA interactions and could also serve as a direct detection method for nucleobase modifications.

scholarly journals Detailed studies of the binding mechanism of the Sinorhizobium meliloti transcriptional activator ExpG to DNA

Microbiology ◽  
2005 ◽  
Vol 151 (1) ◽  
pp. 259-268 ◽  
Author(s):  
Birgit Baumgarth ◽  
Frank Wilco Bartels ◽  
Dario Anselmetti ◽  
Anke Becker ◽  
Robert Ros
Keyword(s):  
Binding Site ◽  
Single Molecule ◽  
Sinorhizobium Meliloti ◽  
Specific Binding ◽  
Dna Interaction ◽  
Dynamic Force ◽  
Sequence Motifs ◽  
Promoter Regions ◽  
Dissociation Kinetics ◽  
Direct Binding

The exopolysaccharide galactoglucan promotes the establishment of symbiosis between the nitrogen-fixing Gram-negative soil bacterium Sinorhizobium meliloti 2011 and its host plant alfalfa. The transcriptional regulator ExpG activates expression of galactoglucan biosynthesis genes by direct binding to the expA1, expG/expD1 and expE1 promoter regions. ExpG is a member of the MarR family of regulatory proteins. Analysis of target sequences of an ExpG(His)6 fusion protein in the exp promoter regions resulted in the identification of a binding site composed of a conserved palindromic region and two associated sequence motifs. Association and dissociation kinetics of the specific binding of ExpG(His)6 to this binding site were characterized by standard biochemical methods and by single-molecule spectroscopy based on the atomic force microscope (AFM). Dynamic force spectroscopy indicated a distinct difference in the kinetics between the wild-type binding sequence and two mutated binding sites, leading to a closer understanding of the ExpG–DNA interaction.

scholarly journals Binding of disparate transcriptional activators to nucleosomal DNA is inherently cooperative.

1995 ◽  
Vol 15 (3) ◽  
pp. 1405-1421 ◽  
Author(s):  
C C Adams ◽  
J L Workman
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Binding Sites ◽  
Specific Binding ◽  
General Mechanism ◽  
Nf Kappa B ◽  
Nucleosomal Dna ◽  
Nucleosome Binding

To investigate mechanisms by which multiple transcription factors access complex promoters and enhancers within cellular chromatin, we have analyzed the binding of disparate factors to nucleosome cores. We used a purified in vitro system to analyze binding of four activator proteins, two GAL4 derivatives, USF, and NF-kappa B (KBF1), to reconstituted nucleosome cores containing different combinations of binding sites. Here we show that binding of any two or all three of these factors to nucleosomal DNA is inherently cooperative. Thus, the binuclear Zn clusters of GAL4, the helix-loop-helix/basic domains of USF, and the rel domain of NF-kappa B all participated in cooperative nucleosome binding, illustrating that this effect is not restricted to a particular DNA-binding domain. Simultaneous binding by two factors increased the affinity of individual factors for nucleosomal DNA by up to 2 orders of magnitude. Importantly, cooperative binding resulted in efficient nucleosome binding by factors (USF and NF-kappa B) which independently possess little nucleosome-binding ability. The participation of GAL4 derivatives in cooperative nucleosome binding required only DNA-binding and dimerization domains, indicating that disruption of histone-DNA contacts by factor binding was responsible for the increased affinity of additional factors. Cooperative nucleosome binding required sequence-specific binding of all transcription factors, appeared to have spatial constraints, and was independent of the orientation of the binding sites on the nucleosome. These results indicate that cooperative nucleosome binding is a general mechanism that may play a significant role in loading complex enhancer and promoter elements with multiple diverse factors in chromatin and contribute to the generation of threshold responses and transcriptional synergy by multiple activator sites in vivo.

scholarly journals Molecular regulation of the human IL-3 gene: inducible T cell-restricted expression requires intact AP-1 and Elf-1 nuclear protein binding sites.

1993 ◽  
Vol 178 (5) ◽  
pp. 1681-1692 ◽  
Author(s):  
L R Gottschalk ◽  
D M Giannola ◽  
S G Emerson
Keyword(s):  
T Cells ◽  
Transcription Factors ◽  
T Cell ◽  
Protein Binding ◽  
Binding Site ◽  
Binding Sites ◽  
Nuclear Protein ◽  
Mobility Shift ◽  
Nuclear Extracts ◽  
Mobility Shift Assay

Interleukin 3 (IL-3) is a hematopoietic stem-cell growth and differentiation factor that is expressed solely in activated T and NK cells. Studies to date have identified elements 5' to the IL-3 coding sequences that regulate its transcription, but the sequences that confer T cell-specific expression remain to be clearly defined. We have now identified DNA sequences that are required for T cell-restricted IL-3 gene transcription. A series of transient transfections performed with human IL-3-chloramphenicol acetyltransferase (CAT) reporter plasmids in T and non-T cells revealed that a plasmid containing 319 bp of 5' flanking sequences was active exclusively in T cells. Deletion analysis revealed that T cell specificity was conferred by a 49-bp fragment (bp -319 to -270) that included a potential binding site for AP-1 transcription factors 6 bp upstream of a binding site for Elf-1, a member of the Ets family of transcription factors. DNaseI footprint and electrophoretic mobility shift assay analyses performed with MLA-144 T cell nuclear extracts demonstrated that this 49-bp region contains a nuclear protein binding region that includes consensus AP-1 and Elf-1 binding sites. In addition, extracts prepared from purified human T cells contained proteins that bound to synthetic oligonucleotides corresponding to the AP-1 and Elf-1 binding sites. In vitro-transcribed and -translated Elf-1 protein bound specifically to the Elf-1 site, and Elf-1 antisera competed and super shifted nuclear protein complexes present in MLA-144 nuclear extracts. Moreover, addition of anti-Jun family antiserum in electrophoretic mobility shift assay reactions completely blocked formation of the AP-1-related complexes. Transient transfection studies in MLA-144 T cells revealed that constructs containing mutations in the AP-1 site almost completely abolished CAT activity while mutation of the Elf-1 site or the NF-IL-3 site, a previously described nuclear protein binding site (bp. -155 to -148) in the IL-3 promoter, reduced CAT activity to < 25% of the activity given by wild-type constructs. We conclude that expression of the human IL-3 gene requires the AP-1 and Elf-1 binding sites; however, unlike other previously characterized cytokine genes such as IL-2, the AP-1 and Elf-1 factors can bind independently in the IL-3 gene.(ABSTRACT TRUNCATED AT 400 WORDS)

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