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Molecular switching of a DNA-sliding clamp to a repressor mediates long-range gene silencing

ABSTRACT: Long-range gene regulation is rare in bacteria and is confined to the classical DNA looping model. Here, we use a combination of biophysical approaches, including X-ray crystallography and single-molecule analysis, to show that long-range gene silencing on the plasmid RK2, a source of multidrug resistance across diverse Gram-negative bacteria, is achieved cooperatively by a DNA-sliding clamp, KorB, and a clamp-locking protein, KorA. We find that KorB is a CTPase clamp that can entrap and slide along DNA to reach distal target promoters. We resolved the tripartite crystal structure of a KorB-KorA-DNA co-complex, revealing that KorA latches KorB into a closed-clamp state. KorA thus stimulates repression by stalling KorB sliding at target promoters to occlude RNA polymerase holoenzymes. Altogether, our findings explain the mechanistic basis for KorB role-switching from a DNA-sliding clamp to a co-repressor, and provide a new paradigm for the long-range regulation of gene expression.

ORGANISM(S): Escherichia coli

PROVIDER: GSE274567 | GEO | 2024/09/01

REPOSITORIES: GEO

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The virulence regulator VirB from Shigella flexneri uses a CTPdependent switch mechanism to activate gene expression

Project description:The transcriptional antisilencer VirB acts as a master regulator of virulence gene expression in the human pathogen Shigella flexneri. It binds defined sequences (virS) upstream of VirB-dependent promoters and counteracts their silencing by the nucleoid-organizing protein H-NS. However, its precise mode of action remains unclear. Notably, VirB is not a classical transcription factor but related to DNA partitioning pro- teins of the ParB family, which have recently been recognized as DNA-sliding clamps using CTP binding and hydrolysis to control their DNA entry gate. Here, we show that VirB binds CTP, embraces DNA in a clamp-like fashion upon its CTP-dependent loading at virS sites and slides laterally on DNA after clamp closure. Mutations that prevent CTP binding block the loading of VirB clamps in vitro and the formation of VirB nucleoprotein complexes in vivo. Thus, VirB represents a CTP-dependent molecular switch that uses a loading-and-sliding mechanism to control transcription during bacterial pathogenesis.

2023-12-06 | PXD046358 | Pride

Sliding of Bacillus subtilis Natto

Project description:Transcriptome comparison of Bacillus subtilis Natto under sliding permissive (0.7% agar) and restrictive (1.5% agar or spo0A mutant strain) conditions.

2015-08-04 | GSE43840 | GEO

Peptide-Based Covalent Inhibitors Bearing Mild Electrophiles to Target a Conserved His Residue of the Bacterial Sliding Clamp

Project description:Bacterial infections caused by drug-resistant pathogens represent a major threat against which effective antibiotics acting via new mechanisms and engaging new biological targets are highly needed. The sliding clamp (SC), an essential component of the bacterial DNA replication machinery, has been identified as a new target. This processivity factor operates through multiple protein-protein interactions with conserved peptide motifs in multiple proteins involved in DNA replication and repair. Herein, we report the structure-guided design of targeted covalent inhibitors (TCIs) able to bind covalently and selectively to the E. coli SC (EcSC). The inhibitors target His175, a well-conserved SC residue located on the edge of the peptide-binding pocket. Thermodynamic and kinetic analyses confirmed the higher efficiency of the chloroacetamide warhead compared to Michael acceptors. High-resolution X-ray structures of two different covalent inhibitor–SC adducts revealed the canonical ligand orientation and confirmed the formation of the covalent bond with His175. Proteomic studies were consistent with a selective EcSC engagement by the chloroacetamide-based TCI. Finally, the TCI of SC was substantially more active than the parent non-covalent inhibitor in an in vitro SC-dependent replicative assay, suggesting the potential of the approach to target the SC of priority pathogens.

2024-01-29 | PXD042384 | Pride

A CTP-dependent gating mechanism enables ParB spreading on DNA

Project description:Proper chromosome segregation is essential in all living organisms. The ParA-ParB-parS system is widely employed for chromosome segregation in bacteria. Previously, we showed that Caulobacter crescentus ParB requires cytidine triphosphate to escape the nucleation site parS to spread by sliding to the neighboring DNA. Here, we provide the structural basis for this transition from nucleation to spreading by solving co-crystal structures of a C-terminal domain truncated C. crescentus ParB with parS and with a CTP analog. Nucleating ParB is an open clamp, in which parS is captured at the DNA-binding domain (the DNA-gate). Upon binding CTP, the N-terminal domain (NTD) self-dimerizes to close the NTD-gate of the clamp. The DNA-gate also closes, thus driving parS into a compartment between the DNA-gate and the C-terminal domain. CTP hydrolysis and/or the release of hydrolytic products may re-open the gates. Overall, we suggest a CTP-operated gating mechanism that regulates ParB nucleation and spreading.

2021-05-01 | GSE168968 | GEO

Sliding of Bacillus subtilis Natto

Project description:Transcriptome comparison of Bacillus subtilis Natto under sliding permissive (0.7% agar) and restrictive (1.5% agar or spo0A mutant strain) conditions. B subtilis Natto wild type cells were grown on the top of LB solid medium with 1.5% and 0.7% agar concentration (samples 1-4). B subtilis Natto wild type and spo0A derivative were grown on top of LB solid medium with 0.7% agar concentration (Sample 5-7). In first experiment, 4 biological replicates were used, while in the second experiment 3 biological replicates included. Dye swaps are included in both experiments.

2015-08-04 | E-GEOD-43840 | biostudies-arrayexpress

Novel inducible filamentous sliding motility identified in pathogenic Bacillus cereus group species

Project description:We isolated an atmospheric contaminant, subsequently identified as a new strain of Bacillus mobilis, which showed a novel, robust, inducible filamentous sliding motility and completely colonized a bacterial culture plate in less than 48 h under some conditions. This flagella-independent sliding motility was characterized by long filamentous cells at the expanding edge, and was induced when cells were inoculated onto lawns of metabolically inactive Campylobacter jejuni cells, heat killed bacterial biomass, and milk or blood dried onto agar plates. Phosphatidylcholine (PC), bacterial membrane components, and sterile human fecal extracts were sufficient to induce filamentous expansion. Screening of eight other Bacillus spp. (five from the B. cereus group and three other Bacillus spp.) showed that filamentous motility was conserved amongst B. cereus group species to varying degrees. RNAseq of filamentously expanding cells collected from PC and milk lawn plates in comparison to rod-shaped cells from control plates revealed that genes related to metabolism, ion and amino acid transport were differently regulated, genes controlling sporulation were reduced, and some virulence genes (e.g., hblA/B/C/D and plcR) were increased. We hypothesize that the robust and conserved nature of filamentous motility in pathogenic B. cereus group species can enhance bacterial colonization during host colonization.

2020-01-01 | GSE136873 | GEO

The ATP-dependent chromatin remodeling enzyme Fun30 represses transcription by sliding promoter proximal nucleosomes [seq]

Project description:The evolutionarily conserved ATP-dependent chromatin remodeling enzyme Fun30 has recently been shown to play important roles in heterochromatin silencing and DNA repair. However, how Fun30 remodels nucleosomes is not clear. Here we report a nucleosome sliding activity of Fun30 and its role in transcriptional repression. We observed that Fun30 repressed the expression of genes involved in amino acid and carbohydrate metabolism, the stress response, and meiosis. In addition, Fun30 was localized at the 5′ and 3′ ends of genes and within the open reading frames of its targets. Consistent with its role in gene repression, we observed that Fun30-target genes lacked histone modifications often associated with gene activation and showed an increased level of ubiquitinated histone H2B. Furthermore, genome-wide nucleosome mapping analysis revealed that the length of the nucleosome-free region at the 5′ end of a subset of genes was changed in Fun30-depleted cells. In addition, the positions of the -1, +2 and +3 nucleosomes at the 5′ end of target genes were significantly shifted, while position of the +1 nucleosome remained largely unchanged in the fun30Δ mutant. Finally, we demonstrated that affinity purified single-component Fun30 exhibited nucleosome sliding activity in an ATP-dependent manner. These results define a role for Fun30 in regulation of transcription and indicate that Fun30 remodels chromatin at the 5′ end of genes by sliding promoter proximal nucleosomes.

2013-07-08 | GSE48562 | GEO

The ATP-dependent chromatin remodeling enzyme Fun30 represses transcription by sliding promoter proximal nucleosomes [array]

2013-07-08 | GSE48570 | GEO

The ATP-dependent chromatin remodeling enzyme Fun30 represses transcription by sliding promoter proximal nucleosomes [seq]

Project description:The evolutionarily conserved ATP-dependent chromatin remodeling enzyme Fun30 has recently been shown to play important roles in heterochromatin silencing and DNA repair. However, how Fun30 remodels nucleosomes is not clear. Here we report a nucleosome sliding activity of Fun30 and its role in transcriptional repression. We observed that Fun30 repressed the expression of genes involved in amino acid and carbohydrate metabolism, the stress response, and meiosis. In addition, Fun30 was localized at the 5M-bM-^@M-2 and 3M-bM-^@M-2 ends of genes and within the open reading frames of its targets. Consistent with its role in gene repression, we observed that Fun30-target genes lacked histone modifications often associated with gene activation and showed an increased level of ubiquitinated histone H2B. Furthermore, genome-wide nucleosome mapping analysis revealed that the length of the nucleosome-free region at the 5M-bM-^@M-2 end of a subset of genes was changed in Fun30-depleted cells. In addition, the positions of the -1, +2 and +3 nucleosomes at the 5M-bM-^@M-2 end of target genes were significantly shifted, while position of the +1 nucleosome remained largely unchanged in the fun30M-NM-^T mutant. Finally, we demonstrated that affinity purified single-component Fun30 exhibited nucleosome sliding activity in an ATP-dependent manner. These results define a role for Fun30 in regulation of transcription and indicate that Fun30 remodels chromatin at the 5M-bM-^@M-2 end of genes by sliding promoter proximal nucleosomes. Micrococcal nuclease digested mononucleosome DNA from wild type and fun30D cells were sequenced by illumina Genome Analyzer. Genome-wide nucleosome positioning and occupancy were analyzed.

2013-07-08 | E-GEOD-48562 | biostudies-arrayexpress

Amara2013 - PCNA ubiquitylation in the activation of PRR pathway

Project description:Mechanistic model of the Post-Replication Repair (PRR), the pathway involved in the bypass of DNA lesions induced by sunlight exposure and UV radiation. PRR acts through two different mechanisms, activated by mono- and poly-ubiquitylation of the DNA sliding clamp, called Proliferating Cell Nuclear Antigen (PCNA). This model has been defined according to the stochastic formulation of chemical kinetics [Gillespie DT, J Phys Chem 1977, 81(25):2340-2361], which requires to specify the set of molecular species occurring in the pathway and their respective interactions, formally described as a set of biochemical reactions. The volume considered for this system is 1.666667e-17L; this value can be used to convert the model into the deterministic formulation.

2024-09-02 | BIOMD0000000475 | BioModels

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