scholarly journals Molecular architecture of the DNA‐binding sites of the P‐loop ATPases MipZ and ParA from Caulobacter crescentus

2019 ◽  
Author(s):  
Yacine Refes ◽  
Binbin He ◽  
Laura Corrales-Guerrero ◽  
Wieland Steinchen ◽  
Gaël Panis ◽  
...  
Keyword(s):  
Dna Binding ◽  
Binding Sites ◽  
Electrostatic Interactions ◽  
Caulobacter Crescentus ◽  
Bioinformatic Analysis ◽  
Proper Function ◽  
Molecular Architecture ◽  
Independent Manner ◽  
Dna Binding Sites

ABSTRACTTwo related P-loop ATPases, ParA and MipZ, mediate the spatiotemporal regulation of chromosome segregation and cell division in Caulobacter crescentus. Both of these proteins share the ability to form dynamic concentration gradients that control the positioning of regulatory targets within the cell. Their proper localization relies on their nucleotide-dependent cycling between a monomeric and a dimeric state, driven by interaction with the chromosome partitioning protein ParB, and on the ability of the dimeric species to associate non-specifically with the nucleoid. In this study, we use a combination of genetic screening, biochemical analysis and hydrogen/deuterium exchange mass spectrometry to identify the residues mediating the interaction of MipZ with DNA. Our results show that the DNA-binding activity of MipZ relies on a series of positively charged and hydrophobic residues lining both sides of the dimer interface. MipZ thus appears to associate with DNA in a sequence-independent manner through electrostatic interactions with the DNA phosphate backbone. In support of this hypothesis, chromatin immunoprecipitation analyses did not reveal any specific target sites in vivo. When extending our analysis to ParA, we found that the architectures of the MipZ and ParA DNA-binding sites are markedly different, although their relative positions on the dimer surface and their mode of DNA binding are conserved. Importantly, bioinformatic analysis suggests that the same principles apply to other members of the P-loop ATPase family. ParA-like ATPases thus share common mechanistic features, although their modes of action have diverged considerably during the course of evolution.SIGNIFICANCEParA-like P-loop ATPases are involved in a variety of cellular processes in bacteria, including chromosome and plasmid segregation, chemoreceptor and carboxysome positioning, and division site placement. Many members of this large protein family depend on the ability to bind non-specific DNA for proper function. Although previous studies have yielded insights in the DNA-binding properties of some ParA-like ATPases, a comprehensive view of the underlying mechanisms is still lacking. Here, we combine state-of-the-art cell biological, biochemical and biophysical approaches to localize the DNA-binding regions of the ParA-like ATPases MipZ and ParA from Caulobacter crescentus. We show that the two proteins use the same interface and mode of action to associate with DNA, suggesting that the mechanistic basis of DNA binding may be conserved in the ParA-like ATPase family.

scholarly journals Molecular architecture of the DNA-binding sites of the P-loop ATPases MipZ and ParA from Caulobacter crescentus

2020 ◽  
Vol 48 (9) ◽  
pp. 4769-4779 ◽  
Author(s):  
Laura Corrales-Guerrero ◽  
Binbin He ◽  
Yacine Refes ◽  
Gaël Panis ◽  
Gert Bange ◽  
...  
Keyword(s):  
Dna Binding ◽  
Binding Sites ◽  
Caulobacter Crescentus ◽  
Bioinformatic Analysis ◽  
Binding Activity ◽  
Molecular Architecture ◽  
Exchange Mass Spectrometry ◽  
Dna Binding Activity ◽  
Spatiotemporal Regulation ◽  
Dna Binding Sites

Abstract The spatiotemporal regulation of chromosome segregation and cell division in Caulobacter crescentus is mediated by two different P-loop ATPases, ParA and MipZ. Both of these proteins form dynamic concentration gradients that control the positioning of regulatory targets within the cell. Their proper localization depends on their nucleotide-dependent cycling between a monomeric and a dimeric state and on the ability of the dimeric species to associate with the nucleoid. In this study, we use a combination of genetic screening, biochemical analysis and hydrogen/deuterium exchange mass spectrometry to comprehensively map the residues mediating the interactions of MipZ and ParA with DNA. We show that MipZ has non-specific DNA-binding activity that relies on an array of positively charged and hydrophobic residues lining both sides of the dimer interface. Extending our analysis to ParA, we find that the MipZ and ParA DNA-binding sites differ markedly in composition, although their relative positions on the dimer surface and their mode of DNA binding are conserved. In line with previous experimental work, bioinformatic analysis suggests that the same principles may apply to other members of the P-loop ATPase family. P-loop ATPases thus share common mechanistic features, although their functions have diverged considerably during the course of evolution.

scholarly journals An effective approach for identification of in vivo protein-DNA binding sites from paired-end ChIP-Seq data

2010 ◽  
Vol 11 (1) ◽  
pp. 81 ◽  
Author(s):  
Congmao Wang ◽  
Jie Xu ◽  
Dasheng Zhang ◽  
Zoe A Wilson ◽  
Dabing Zhang
Keyword(s):  
Dna Binding ◽  
Binding Sites ◽  
Dna Binding Sites

scholarly journals Accurate and sensitive quantification of protein-DNA binding affinity

2018 ◽  
Vol 115 (16) ◽  
pp. E3692-E3701 ◽  
Author(s):  
Chaitanya Rastogi ◽  
H. Tomas Rube ◽  
Judith F. Kribelbauer ◽  
Justin Crocker ◽  
Ryan E. Loker ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Binding ◽  
Binding Sites ◽  
Biophysical Model ◽  
Regulatory Sequences ◽  
Binding Modes ◽  
Perfect Agreement ◽  
Dna Binding Sites

Transcription factors (TFs) control gene expression by binding to genomic DNA in a sequence-specific manner. Mutations in TF binding sites are increasingly found to be associated with human disease, yet we currently lack robust methods to predict these sites. Here, we developed a versatile maximum likelihood framework named No Read Left Behind (NRLB) that infers a biophysical model of protein-DNA recognition across the full affinity range from a library of in vitro selected DNA binding sites. NRLB predicts human Max homodimer binding in near-perfect agreement with existing low-throughput measurements. It can capture the specificity of the p53 tetramer and distinguish multiple binding modes within a single sample. Additionally, we confirm that newly identified low-affinity enhancer binding sites are functional in vivo, and that their contribution to gene expression matches their predicted affinity. Our results establish a powerful paradigm for identifying protein binding sites and interpreting gene regulatory sequences in eukaryotic genomes.

scholarly journals An A257V Mutation in the Bacillus subtilis Response Regulator Spo0A Prevents Regulated Expression of Promoters with Low-Consensus Binding Sites

2009 ◽  
Vol 191 (17) ◽  
pp. 5489-5498 ◽  
Author(s):  
Steve D. Seredick ◽  
Barbara M. Seredick ◽  
David Baker ◽  
George B. Spiegelman
Keyword(s):  
Dna Binding ◽  
Binding Sites ◽  
Transcription Initiation ◽  
Molecular Basis ◽  
Response Regulator ◽  
Mutant Protein ◽  
Wild Type ◽  
Dna Binding Sites

ABSTRACT In Bacillus species, the master regulator of sporulation is Spo0A. Spo0A functions by both activating and repressing transcription initiation from target promoters that contain 0A boxes, the binding sites for Spo0A. Several classes of spo0A mutants have been isolated, and the molecular basis for their phenotypes has been determined. However, the molecular basis of the Spo0A(A257V) substitution, representative of an unusual phenotypic class, is not understood. Spo0A(A257V) is unusual in that it abolishes sporulation; in vivo, it fails to activate transcription from key stage II promoters yet retains the ability to repress the abrB promoter. To determine how Spo0A(A257V) retains the ability to repress but not stimulate transcription, we performed a series of in vitro and in vivo assays. We found unexpectedly that the mutant protein both stimulated transcription from the spoIIG promoter and repressed transcription from the abrB promoter, albeit twofold less than the wild type. A DNA binding analysis of Spo0A(A257V) showed that the mutant protein was less able to tolerate alterations in the sequence and arrangement of its DNA binding sites than the wild-type protein. In addition, we found that Spo0A(A257V) could stimulate transcription of a mutant spoIIG promoter in vivo in which low-consensus binding sites were replaced by high-consensus binding sites. We conclude that Spo0A(A257V) is able to bind to and regulate the expression of only genes whose promoters contain high-consensus binding sites and that this effect is sufficient to explain the observed sporulation defect.

scholarly journals A micro-evolutionary change in target binding sites as key determinant of Ultrabithorax function in Drosophila.

2021 ◽  
Author(s):  
Soumen Khan ◽  
Saurabh J. Pradhan ◽  
Guillaume Giraud ◽  
Françoise Bleicher ◽  
Rachel Paul ◽  
...  
Keyword(s):  
Dna Binding ◽  
Binding Sites ◽  
Morphological Changes ◽  
Binding Motif ◽  
Core Sequence ◽  
Dna Binding Sites ◽  
Target Binding ◽  
Hox Protein

All Hox proteins are known to recognize, in vitro, similar DNA-binding sites containing a TAAT core sequence. This poor DNA-binding specificity is in sharp contrast with their specific functions in vivo. Here we report a new binding motif with TAAAT core sequence to which the Hox protein Ultrabithorax (Ubx) binds with higher affinity and specificity. Using transgenic and luciferase assays, we show that this new motif is critical for Ubx-mediated regulation of a target gene in Drosophila melanogaster. Interestingly, this new motif with TAAAT core sequences is not associated with the targets of Ubx in the honeybee, Apis mellifera, wherein hindwings are nearly identical to the forewings. We show that introduction of TAAAT motif in the place of TAAT motif is sufficient to bring an enhancer of a wing-promoting gene of A. mellifera under the regulation of Ubx. Our results, thus, suggest that binding motifs with a TAAAT core sequence may help identify functionally relevant direct targets of Ubx in D. melanogaster and the emergence of these binding sites may be crucial for Hox-mediated morphological changes during insect evolution.

scholarly journals Transcriptional repression by Msx-1 does not require homeodomain DNA-binding sites.

1995 ◽  
Vol 15 (2) ◽  
pp. 861-871 ◽  
Author(s):  
K M Catron ◽  
H Zhang ◽  
S C Marshall ◽  
J A Inostroza ◽  
J M Wilson ◽  
...  
Keyword(s):  
Dna Binding ◽  
Binding Sites ◽  
Transcriptional Repression ◽  
Binding Domain ◽  
Specific Cell ◽  
Homeodomain Protein ◽  
Dna Binding Domain ◽  
Dna Binding Sites

This study investigates the transcriptional properties of Msx-1, a murine homeodomain protein which has been proposed to play a key role in regulating the differentiation and/or proliferation state of specific cell populations during embryogenesis. We show, using basal and activated transcription templates, that Msx-1 is a potent repressor of transcription and can function through both TATA-containing and TATA-less promoters. Moreover, repression in vivo and in vitro occurs in the absence of DNA-binding sites for the Msx-1 homeodomain. Utilizing a series of truncated Msx-1 polypeptides, we show that multiple regions of Msx-1 contribute to repression, and these are rich in alanine, glycine, and proline residues. When fused to a heterologous DNA-binding domain, both N- and C-terminal regions of Msx-1 retain repressor function, which is dependent upon the presence of the heterologous DNA-binding site. Moreover, a polypeptide consisting of the full-length Msx-1 fused to a heterologous DNA-binding domain is a more potent repressor than either the N- or C-terminal regions alone, and this fusion retains the ability to repress transcription in the absence of the heterologous DNA site. We further show that Msx-1 represses transcription in vitro in a purified reconstituted assay system and interacts with protein complexes composed of TBP and TFIIA (DA) and TBP, TFIIA, and TFIIB (DAB) in gel retardation assays, suggesting that the mechanism of repression is mediated through interaction(s) with a component(s) of the core transcription complex. We speculate that the repressor function of Msx-1 is critical for its proposed role in embryogenesis as a regulator of cellular differentiation.

scholarly journals CtrA, a Global Response Regulator, Uses a Distinct Second Category of Weak DNA Binding Sites for Cell Cycle Transcription Control in Caulobacter crescentus

2009 ◽  
Vol 191 (17) ◽  
pp. 5458-5470 ◽  
Author(s):  
William Spencer ◽  
Rania Siam ◽  
Marie-Claude Ouimet ◽  
D. Patrick Bastedo ◽  
Gregory T. Marczynski
Keyword(s):  
Cell Cycle ◽  
Binding Sites ◽  
Caulobacter Crescentus ◽  
Response Regulator ◽  
Cell Cycle Control ◽  
Transcription Control ◽  
Mobility Shift ◽  
Dna Binding Sites ◽  
Electrophoretic Mobility Shift Assays

ABSTRACT CtrA controls cell cycle programs of chromosome replication and genetic transcription. Phosphorylated CtrA∼P exhibits high affinity (dissociation constant [Kd ], <10 nM) for consensus TTAA-N7-TTAA binding sites with “typical” (N = 7) spacing. We show here that ctrA promoters P1 and P2 use low-affinity (Kd , >500 nM) CtrA binding sites with “atypical” (N ≠ 7) spacing. Footprints demonstrated that phosphorylated CtrA∼P does not exhibit increased affinity for “atypical” sites, as it does for sites in the replication origin. Instead, high levels of CtrA (>10 μM) accumulate, which can drive CtrA binding to “atypical” sites. In vivo cross-linking showed that when the stable CtrAΔ3 protein persists during the cell cycle, the “atypical” sites at ctrA and motB are persistently bound. Interestingly, the cell cycle timing of ctrA P1 and P2 transcription is not altered by persistent CtrAΔ3 binding. Therefore, operator DNA occupancy is not sufficient for regulation, and it is the cell cycle variation of CtrA∼P phosphorylation that provides the dominant “activation” signal. Protein dimerization is one potential means of “activation.” The glutathione S-transferase (GST) protein dimerizes, and fusion with CtrA (GST-CtrA) creates a stable dimer with enhanced affinity for TTAA motifs. Electrophoretic mobility shift assays with GST-CtrA revealed cooperative modes of binding that further distinguish the “atypical” sites. GST-CtrA also binds a single TTAA motif in ctrA P1 aided by DNA in the extended TTAACCAT motif. We discuss how “atypical” sites are a common yet distinct category of CtrA regulatory sites and new implications for the working and evolution of cell cycle control networks.

scholarly journals Functional divergence between the half-sites of the DNA-binding sequence for the yeast transcriptional regulator Rap1p

1999 ◽  
Vol 341 (3) ◽  
pp. 477-482 ◽  
Author(s):  
Fátima-Zahra IDRISSI ◽  
Benjamin PIÑA
Keyword(s):  
Dna Binding ◽  
Binding Sites ◽  
Functional Divergence ◽  
Consensus Sequence ◽  
Transcriptional Regulator ◽  
Dna Complexes ◽  
Dna Binding Sites ◽  
Binding Sequence ◽  
Half Site

The yeast transcriptional regulator Rap1p binds to the DNA consensus sequence ACACCCAYACAYYY. We have previously shown that DNA-binding sites in which all four Y (Y = T or C) positions were Ts (UASrpg sequences) synergized more efficiently to activate transcription than sequences in which all Ys were Cs (telomere sequences) [F.-Z. Idrissi, J. Fernández-Larrea and B. Piña (1998) J. Mol. Biol. 284, 925-935]. Here we provide evidence that the DNA consensus sequence for Rap1p behaves as a combination of two ACAYYY half-sites with different functionality, the presence of Ts in the second half-site being the determinant for the transcriptional behaviour of the UASrpg sequences. DNA structure in the different complexes with Rap1p varied from being relatively uniform to appear rather distorted, this also being dependent on the presence of Ts in the second half-site. These distortions did not cause sharp bends or kinks in the DNA molecule. Computer analysis suggests that high-affinity binding of Rap1p to UASrpg sequences requires a rearrangement of the C-terminal Myb domain of the protein. We propose that the structural alterations in Rap1p-DNA complexes, both in the DNA and in the protein, affect the transcription potential of the complex in an allosteric manner. We also propose that the dimeric nature of the Rap1 DNA-binding domain is a key structural feature that explains the disparate functions of its DNA-binding sites in vivo.

scholarly journals DamIP: A novel method to identify DNA binding sites in vivo

2010 ◽  
Vol 8 (1) ◽  
pp. nrs.08003 ◽  
Author(s):  
Rui Xiao ◽  
Ramon Roman-Sanchez ◽  
David D. Moore
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Binding Sites ◽  
Protein Function ◽  
Target Genes ◽  
Estrogen Receptor Α ◽  
Cross Linking ◽  
Mutant Form ◽  
Dna Binding Sites

Identifying binding sites and target genes of transcription factors is a major biologic problem. The most commonly used current technique, chromatin immunoprecipitation (ChIP), is dependent on a high quality antibody for each protein of interest, which is not always available, and is also cumbersome, involving sequential cross-linking and reversal of cross-linking. We have developed a novel strategy to study protein DNA binding sites in vivo, which we term DamIP. By tethering a mutant form of E. coli DNA adenine methyltransferase to the target protein, the fusion protein introduces N-6-adenosine methylation to sequences proximal to the protein binding sites. DNA fragments with this modification, which is absent in eukaryotes, are detected using an antibody directed against methylated adenosine. For an initial test of the method we used human estrogen receptor α (hERα), one of the best studied transcription factors. We found that expression of Dam-hERα fusion proteins in MCF-7 cells introduces adenosine methylation near a series of known direct hERα binding sites. Specific methylation tags are also found at indirect hERα binding sites, including both primary binding sites for the ER interactors AP-1 and SP1, and promoters that are activated by upstream ER bound enhancers. DamIP provides a new tool for the study of DNA interacting protein function in vivo.

scholarly journals Genome-wide identification of in vivo protein-DNA binding sites from ChIP-Seq data

2008 ◽  
Vol 36 (16) ◽  
pp. 5221-5231 ◽  
Author(s):  
R. Jothi ◽  
S. Cuddapah ◽  
A. Barski ◽  
K. Cui ◽  
K. Zhao
Keyword(s):  
Dna Binding ◽  
Binding Sites ◽  
Dna Binding Sites ◽  
Genome Wide
Sign in / Sign up

Export Citation Format

Share Document