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1.
Mol Cell ; 76(3): 382-394.e6, 2019 11 07.
Artigo em Inglês | MEDLINE | ID: mdl-31492634

RESUMO

DNA double-strand breaks (DSBs) threaten genome stability throughout life and are linked to tumorigenesis in humans. To initiate DSB repair by end joining or homologous recombination, the Mre11-nuclease Rad50-ATPase complex detects and processes diverse and obstructed DNA ends, but a structural mechanism is still lacking. Here we report cryo-EM structures of the E. coli Mre11-Rad50 homolog SbcCD in resting and DNA-bound cutting states. In the resting state, Mre11's nuclease is blocked by ATP-Rad50, and the Rad50 coiled coils appear flexible. Upon DNA binding, the two coiled coils zip up into a rod and, together with the Rad50 nucleotide-binding domains, form a clamp around dsDNA. Mre11 moves to the side of Rad50, binds the DNA end, and assembles a DNA cutting channel for the nuclease reactions. The structures reveal how Mre11-Rad50 can detect and process diverse DNA ends and uncover a clamping and gating function for the coiled coils.


Assuntos
Hidrolases Anidrido Ácido/metabolismo , Quebras de DNA de Cadeia Dupla , Replicação do DNA , DNA Bacteriano/metabolismo , Desoxirribonucleases/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Exonucleases/metabolismo , Proteína Homóloga a MRE11/metabolismo , Hidrolases Anidrido Ácido/genética , Hidrolases Anidrido Ácido/ultraestrutura , Microscopia Crioeletrônica , DNA Bacteriano/genética , DNA Bacteriano/ultraestrutura , Desoxirribonucleases/genética , Desoxirribonucleases/ultraestrutura , Escherichia coli/genética , Escherichia coli/ultraestrutura , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/ultraestrutura , Exonucleases/genética , Exonucleases/ultraestrutura , Proteína Homóloga a MRE11/genética , Proteína Homóloga a MRE11/ultraestrutura , Conformação de Ácido Nucleico , Relação Estrutura-Atividade
2.
Nature ; 565(7739): 382-385, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30626968

RESUMO

A key regulated step of transcription is promoter melting by RNA polymerase (RNAP) to form the open promoter complex1-3. To generate the open complex, the conserved catalytic core of the RNAP combines with initiation factors to locate promoter DNA, unwind 12-14 base pairs of the DNA duplex and load the template-strand DNA into the RNAP active site. Formation of the open complex is a multi-step process during which transient intermediates of unknown structure are formed4-6. Here we present cryo-electron microscopy structures of bacterial RNAP-promoter DNA complexes, including structures of partially melted intermediates. The structures show that late steps of promoter melting occur within the RNAP cleft, delineate key roles for fork-loop 2 and switch 2-universal structural features of RNAP-in restricting access of DNA to the RNAP active site, and explain why clamp opening is required to allow entry of single-stranded template DNA into the active site. The key roles of fork-loop 2 and switch 2 suggest a common mechanism for late steps in promoter DNA opening to enable gene expression across all domains of life.


Assuntos
Microscopia Crioeletrônica , DNA Bacteriano/química , DNA Bacteriano/ultraestrutura , RNA Polimerases Dirigidas por DNA/metabolismo , Mycobacterium tuberculosis/enzimologia , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas , Proteínas de Bactérias/metabolismo , Sequência de Bases , Domínio Catalítico , DNA Bacteriano/metabolismo , Estabilidade Enzimática/efeitos dos fármacos , Escherichia coli/enzimologia , Lactonas/farmacologia , Modelos Moleculares , Mycobacterium tuberculosis/metabolismo , Desnaturação de Ácido Nucleico , Ligação Proteica , Termodinâmica , Iniciação da Transcrição Genética/efeitos dos fármacos
3.
Infect Immun ; 87(2)2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30510104

RESUMO

Helicobacter pylori CagA is a secreted effector protein that contributes to gastric carcinogenesis. Previous studies showed that there is variation among H. pylori strains in the steady-state levels of CagA and that a strain-specific motif downstream of the cagA transcriptional start site (the +59 motif) is associated with both high levels of CagA and premalignant gastric histology. The cagA 5' untranslated region contains a predicted stem-loop-forming structure adjacent to the +59 motif. In the current study, we investigated the effect of the +59 motif and the adjacent stem-loop on cagA transcript levels and cagA mRNA stability. Using site-directed mutagenesis, we found that mutations predicted to disrupt the stem-loop structure resulted in decreased steady-state levels of both the cagA transcript and the CagA protein. Additionally, these mutations resulted in a decreased cagA mRNA half-life. Mutagenesis of the +59 motif without altering the stem-loop structure resulted in reduced steady-state cagA transcript and CagA protein levels but did not affect cagA transcript stability. cagA transcript stability was not affected by increased sodium chloride concentrations, an environmental factor known to augment cagA transcript levels and CagA protein levels. These results indicate that both a predicted stem-loop structure and a strain-specific +59 motif in the cagA 5' untranslated region influence the levels of cagA expression.


Assuntos
Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , DNA Bacteriano/ultraestrutura , Infecções por Helicobacter/genética , Helicobacter pylori/genética , Estabilidade de RNA/genética , RNA Mensageiro/ultraestrutura , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Humanos , Mutagênese Sítio-Dirigida
4.
Vet Parasitol ; 258: 1-7, 2018 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-30105969

RESUMO

Macrophages are multipurpose phagocytes and are considered to be irreplaceable during the early host innate immune response against microbial and parasitic pathogens. However, no report has investigated the novel anti-parasitic mechanism of macrophage-derived extracellular traps (ETs) against the abortive apicomplexan parasite Neospora caninum (N. caninum) in cattle. Scanning electron microscopy (SEM) was used to visualize and characterize N. caninum tachyzoite-induced macrophage-triggered ETs in exposed bovine macrophages. Fluorescence confocal microscopy was used to confirm the classical backbone structure of DNA embedded with histone 3 (H3) and myeloperoxidase (MPO) in N. caninum tachyzoite-induced macrophage-derived ETs. Furthermore, the lactate dehydrogenase (LDH) levels in the supernatants of parasite-exposed macrophages were detected by a LDH Cytotoxicity Assay® kit. The results clearly demonstrated that N. caninum tachyzoites triggered bovine macrophage-derived ET-like structures. Inhibiting assays revealed that N. caninum tachyzoite-induced macrophage-mediated ET formation may be an ERK 1/2- and p38 MAPK-dependent cell death process. In conclusion, the present study is the first report on the formation of ETs in bovine macrophages against N. caninum tachyzoites and adds new data on the possible role of macrophages in vivo infection by capturing invasive stages and exposing them to other leukocytes.


Assuntos
Coccidiose/veterinária , Armadilhas Extracelulares/imunologia , Interações Hospedeiro-Parasita , Macrófagos/imunologia , Neospora/imunologia , Animais , Bovinos , Coccidiose/imunologia , Coccidiose/parasitologia , Meios de Cultura/análise , DNA Bacteriano/ultraestrutura , DNA de Protozoário/ultraestrutura , MAP Quinases Reguladas por Sinal Extracelular , Armadilhas Extracelulares/parasitologia , Imunidade Inata , L-Lactato Desidrogenase/análise , Macrófagos/parasitologia , Macrófagos/ultraestrutura , Microscopia Eletrônica de Varredura , Neospora/genética
5.
Nucleic Acids Res ; 46(8): 3937-3952, 2018 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-29529244

RESUMO

High-resolution three-dimensional models of Caulobacter crescentus nucleoid structures were generated via a multi-scale modeling protocol. Models were built as a plectonemically supercoiled circular DNA and by incorporating chromosome conformation capture based data to generate an ensemble of base pair resolution models consistent with the experimental data. Significant structural variability was found with different degrees of bending and twisting but with overall similar topologies and shapes that are consistent with C. crescentus cell dimensions. The models allowed a direct mapping of the genomic sequence onto the three-dimensional nucleoid structures. Distinct spatial distributions were found for several genomic elements such as AT-rich sequence elements where nucleoid associated proteins (NAPs) are likely to bind, promoter sites, and some genes with common cellular functions. These findings shed light on the correlation between the spatial organization of the genome and biological functions.


Assuntos
Caulobacter crescentus/genética , Caulobacter crescentus/ultraestrutura , Cromossomos Bacterianos/genética , Cromossomos Bacterianos/ultraestrutura , Sequência Rica em At , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Caulobacter crescentus/metabolismo , Mapeamento Cromossômico , Cromossomos Bacterianos/química , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Bacteriano/ultraestrutura , DNA Super-Helicoidal/química , DNA Super-Helicoidal/genética , DNA Super-Helicoidal/ultraestrutura , Genoma Bacteriano , Imagem Tridimensional , Modelos Genéticos , Modelos Moleculares , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas
6.
Biochemistry ; 57(13): 1967-1976, 2018 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-29432678

RESUMO

As a guardian of the bacterial genome, the RecG DNA helicase repairs DNA replication and rescues stalled replication. We applied atomic force microscopy (AFM) to directly visualize dynamics of RecG upon the interaction with replication fork substrates in the presence and absence of SSB using high-speed AFM. We directly visualized that RecG moves back and forth over dozens of base pairs in the presence of SSB. There is no RecG translocation in the absence of SSB. Computational modeling was performed to build models of Escherichia coli RecG in a free state and in complex with the fork. The simulations revealed the formation of complexes of RecG with the fork and identified conformational transitions that may be responsible for RecG remodeling that can facilitate RecG translocation along the DNA duplex. Such complexes do not form with the DNA duplex, which is in line with experimental data. Overall, our results provide mechanistic insights into the modes of interaction of RecG with the replication fork, suggesting a novel role of RecG in the repair of stalled DNA replication forks.


Assuntos
Replicação do DNA , DNA Bacteriano/química , Proteínas de Ligação a DNA/química , Proteínas de Escherichia coli/química , Escherichia coli/química , Modelos Moleculares , Complexos Multiproteicos/química , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , DNA Bacteriano/ultraestrutura , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Microscopia de Força Atômica , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo
7.
Curr Opin Microbiol ; 43: 38-45, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29197672

RESUMO

Although DNA replication and repair in bacteria have been extensively studied for many decades, in recent years the development of single-molecule microscopy has provided a new perspective on these fundamental processes. Because single-molecule imaging super-resolves the nanometer-scale dynamics of molecules, and because single-molecule imaging is sensitive to heterogeneities within a sample, this nanoscopic microscopy technique measures the motions, localizations, and interactions of proteins in real time without averaging ensemble observations, both in vitro and in vivo. In this Review, we provide an overview of several recent single-molecule fluorescence microscopy studies on DNA replication and repair. These experiments have shown that, in both Escherichia coli and Bacillus subtilis the DNA replication proteins are highly dynamic. In particular, even highly processive replicative DNA polymerases exchange to and from the replication fork on the scale of a few seconds. Furthermore, single-molecule investigations of the DNA mismatch repair (MMR) pathway have measured the complex interactions between MMR proteins, replication proteins, and DNA. Single-molecule imaging will continue to improve our understanding of fundamental processes in bacteria including DNA replication and repair.


Assuntos
Reparo do DNA , Replicação do DNA , Microscopia/instrumentação , Microscopia/métodos , Proteínas/ultraestrutura , Bacillus subtilis/genética , Bacillus subtilis/ultraestrutura , DNA Bacteriano/ultraestrutura , Escherichia coli/genética , Escherichia coli/ultraestrutura , Proteínas/genética
8.
Science ; 358(6365): 947-951, 2017 11 17.
Artigo em Inglês | MEDLINE | ID: mdl-29146813

RESUMO

In bacteria, the activation of gene transcription at many promoters is simple and only involves a single activator. The cyclic adenosine 3',5'-monophosphate receptor protein (CAP), a classic activator, is able to activate transcription independently through two different mechanisms. Understanding the class I mechanism requires an intact transcription activation complex (TAC) structure at a high resolution. Here we report a high-resolution cryo-electron microscopy structure of an intact Escherichia coli class I TAC containing a CAP dimer, a σ70-RNA polymerase (RNAP) holoenzyme, a complete class I CAP-dependent promoter DNA, and a de novo synthesized RNA oligonucleotide. The structure shows how CAP wraps the upstream DNA and how the interactions recruit RNAP. Our study provides a structural basis for understanding how activators activate transcription through the class I recruitment mechanism.


Assuntos
Proteína Receptora de AMP Cíclico/química , RNA Polimerases Dirigidas por DNA/química , Proteínas de Escherichia coli/química , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Fator sigma/química , Ativação Transcricional , Microscopia Crioeletrônica , Proteína Receptora de AMP Cíclico/ultraestrutura , DNA Bacteriano/química , DNA Bacteriano/ultraestrutura , RNA Polimerases Dirigidas por DNA/ultraestrutura , Proteínas de Escherichia coli/ultraestrutura , Regiões Promotoras Genéticas , Fator sigma/ultraestrutura
9.
Sci Rep ; 7(1): 15275, 2017 11 10.
Artigo em Inglês | MEDLINE | ID: mdl-29127381

RESUMO

Atomic force microscopy (AFM) has proven to be a powerful tool for the study of DNA-protein interactions due to its ability to image single molecules at the nanoscale. However, the use of AFM in force spectroscopy to study DNA-protein interactions has been limited. Here we developed a high throughput, AFM based, pulling assay to measure the strength and kinetics of protein bridging of DNA molecules. As a model system, we investigated the interactions between DNA and the Histone-like Nucleoid-Structuring protein (H-NS). We confirmed that H-NS both changes DNA rigidity and forms bridges between DNA molecules. This straightforward methodology provides a high-throughput approach with single-molecule resolution which is widely applicable to study cross-substrate interactions such as DNA-bridging proteins.


Assuntos
DNA Bacteriano/química , Proteínas de Ligação a DNA/química , Proteínas de Escherichia coli/química , Escherichia coli/química , Proteínas de Fímbrias/química , Microscopia de Força Atômica , DNA Bacteriano/ultraestrutura
10.
Cell ; 169(4): 708-721.e12, 2017 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-28457609

RESUMO

Relaxases play essential roles in conjugation, the main process by which bacteria exchange genetic material, notably antibiotic resistance genes. They are bifunctional enzymes containing a trans-esterase activity, which is responsible for nicking the DNA strand to be transferred and for covalent attachment to the resulting 5'-phosphate end, and a helicase activity, which is responsible for unwinding the DNA while it is being transported to a recipient cell. Here we show that these two activities are carried out by two conformers that can both load simultaneously on the origin of transfer DNA. We solve the structure of one of these conformers by cryo electron microscopy to near-atomic resolution, elucidating the molecular basis of helicase function by relaxases and revealing insights into the mechanistic events taking place in the cell prior to substrate transport during conjugation.


Assuntos
Conjugação Genética , DNA Helicases/metabolismo , DNA Helicases/ultraestrutura , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/ultraestrutura , Escherichia coli/genética , Microscopia Crioeletrônica , DNA Helicases/química , DNA Bacteriano/química , DNA Bacteriano/ultraestrutura , DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismo , Escherichia coli/enzimologia , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Modelos Moleculares
11.
Nucleic Acids Res ; 45(12): 7299-7308, 2017 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-28521053

RESUMO

Hfq is a bacterial protein that is involved in several aspects of nucleic acids metabolism. It has been described as one of the nucleoid associated proteins shaping the bacterial chromosome, although it is better known to influence translation and turnover of cellular RNAs. Here, we explore the role of Escherichia coli Hfq's C-terminal domain in the compaction of double stranded DNA. Various experimental methodologies, including fluorescence microscopy imaging of single DNA molecules confined inside nanofluidic channels, atomic force microscopy, isothermal titration microcalorimetry and electrophoretic mobility assays have been used to follow the assembly of the C-terminal and N-terminal regions of Hfq on DNA. Results highlight the role of Hfq's C-terminal arms in DNA binding, change in mechanical properties of the double helix and compaction of DNA into a condensed form. The propensity for bridging and compaction of DNA by the C-terminal domain might be related to aggregation of bound protein and may have implications for protein binding related gene regulation.


Assuntos
DNA Bacteriano/ultraestrutura , DNA/ultraestrutura , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Fator Proteico 1 do Hospedeiro/genética , Sítios de Ligação , Cromossomos Bacterianos/química , Cromossomos Bacterianos/metabolismo , DNA/genética , DNA/metabolismo , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Fator Proteico 1 do Hospedeiro/metabolismo , Cinética , Agregados Proteicos , Ligação Proteica , Domínios Proteicos , Termodinâmica
12.
Methods ; 120: 91-102, 2017 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-28434996

RESUMO

The influence of heparin and heparan sulphate (HepS) on the appearance and analysis of open promoter complex (RPo) formation by E. coli RNA polymerase (RNAP) holoenzyme (σ70RNAP) on linear DNA using ex situ imaging by atomic force microscopy (AFM) has been investigated. Introducing heparin or HepS into the reaction mix significantly reduces non-specific interactions of the σ70RNAP and RNAP after RPo formation allowing for better interpretation of complexes shown within AFM images, particularly on DNA templates containing more than one promoter. Previous expectation was that negatively charged polysaccharides, often used as competitive inhibitors of σRNAP binding and RPo formation, would also inhibit binding of the DNA template to the mica support surface and thereby lower the imaging yield of active RNAP-DNA complexes. We found that the reverse of this was true, and that the yield of RPo formation detected by AFM, for a simple tandem gene model containing two λPR promoters, increased. Moreover and unexpectedly, HepS was more efficient than heparin, with both of them having a dispersive effect on the sample, minimising unwanted RNAP-RNAP interactions as well as non-specific interactions between the RNAP and DNA template. The success of this method relied on the observation that E. coli RNAP has the highest affinity for the mica surface of all the molecular components. For our system, the affinity of the three constituent biopolymers to muscovite mica was RNAP>Heparin or HepS>DNA. While we observed that heparin and HepS can inhibit DNA binding to the mica, the presence of E. coli RNAP overcomes this effect allowing a greater yield of RPos for AFM analysis. This method can be extended to other DNA binding proteins and enzymes, which have an affinity to mica higher than DNA, to improve sample preparation for AFM studies.


Assuntos
DNA Bacteriano/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Heparina/química , Heparitina Sulfato/química , Microscopia de Força Atômica/métodos , Regiões Promotoras Genéticas , Silicatos de Alumínio/química , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Bacteriano/ultraestrutura , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/ultraestrutura , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/ultraestrutura , Holoenzimas/genética , Holoenzimas/metabolismo , Ligação Proteica , Fator sigma/química , Fator sigma/metabolismo , Transcrição Genética
13.
Nat Commun ; 8: 14665, 2017 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-28272414

RESUMO

DNA-binding proteins are central regulators of chromosome organization; however, in genome-reduced bacteria their diversity is largely diminished. Whether the chromosomes of such bacteria adopt defined three-dimensional structures remains unexplored. Here we combine Hi-C and super-resolution microscopy to determine the structure of the Mycoplasma pneumoniae chromosome at a 10 kb resolution. We find a defined structure, with a global symmetry between two arms that connect opposite poles, one bearing the chromosomal Ori and the other the midpoint. Analysis of local structures at a 3 kb resolution indicates that the chromosome is organized into domains ranging from 15 to 33 kb. We provide evidence that genes within the same domain tend to be co-regulated, suggesting that chromosome organization influences transcriptional regulation, and that supercoiling regulates local organization. This study extends the current understanding of bacterial genome organization and demonstrates that a defined chromosomal structure is a universal feature of living systems.


Assuntos
Cromossomos Bacterianos/ultraestrutura , DNA Bacteriano/ultraestrutura , DNA Super-Helicoidal/ultraestrutura , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano/genética , Mycoplasma pneumoniae/genética , Estruturas Cromossômicas , Microscopia , Conformação Molecular , Mycoplasma pneumoniae/ultraestrutura , Conformação de Ácido Nucleico
14.
Nano Lett ; 17(3): 1938-1948, 2017 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-28191853

RESUMO

Bacterial chromosome has a compact structure that dynamically changes its shape in response to bacterial growth rate and growth phase. Determining how chromatin remains accessible to DNA binding proteins, and transcription machinery is crucial to understand the link between genetic regulation, DNA structure, and topology. Here, we study very large supercoiled dsDNA using high-resolution characterization, theoretical modeling, and molecular dynamics calculations. We unveil a new type of highly ordered DNA organization forming in the presence of attractive DNA-DNA interactions, which we call hyperplectonemes. We demonstrate that their formation depends on DNA size, supercoiling, and bacterial physiology. We compare structural, nanomechanic, and dynamic properties of hyperplectonemes bound by three highly abundant nucleoid-associated proteins (FIS, H-NS, and HU). In all these cases, the negative supercoiling of DNA determines molecular dynamics, modulating their 3D shape. Overall, our findings provide a mechanistic insight into the critical role of DNA topology in genetic regulation.


Assuntos
DNA Bacteriano/ultraestrutura , DNA Super-Helicoidal/ultraestrutura , Escherichia coli/ultraestrutura , DNA Bacteriano/química , DNA Super-Helicoidal/química , Escherichia coli/química , Microscopia de Força Atômica , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico
15.
Sci Rep ; 6: 34934, 2016 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-27731339

RESUMO

Some cyanobacteria exhibit compaction of DNA in synchrony with their circadian rhythms accompanying cell division. Since the structure is transient, it has not yet been described in detail. Here, we successfully visualize the ultrastructure of compacted DNA in the cyanobacterium Synechococcus elongatus PCC 7942 under rigorous synchronized cultivation by means of high-voltage cryo-electron tomography. In 3D reconstructions of rapidly frozen cells, the compacted DNA appears as an undulating rod resembling a eukaryotic condensed chromosome. The compacted DNA also includes many small and paired polyphosphate bodies (PPBs), some of which seem to maintain contact with DNA that appears to twist away from them, indicating that they may act as interactive suppliers and regulators of phosphate for DNA synthesis. These observations throw light on the duplication and segregation mechanisms of cyanobacterial DNA and point to an important role for PPBs.


Assuntos
Cianobactérias/ultraestrutura , DNA Bacteriano/fisiologia , DNA Bacteriano/ultraestrutura , Cromossomos/ultraestrutura , Microscopia Crioeletrônica , Tomografia com Microscopia Eletrônica , Processamento de Imagem Assistida por Computador , Imagem Tridimensional , Microscopia de Fluorescência , Polifosfatos/química , Synechococcus/metabolismo
16.
Free Radic Biol Med ; 97: 351-361, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27375130

RESUMO

The biology of nickel has been widely studied in mammals because of its carcinogenic properties, whereas few studies have been performed in microorganisms. In the present work, changes accompanying stress caused by nickel were evaluated at the cellular level using RNA-Seq in Escherichia coli K-12. Interestingly, a very large number of genes were found to be deregulated by Ni stress. Iron and oxidative stress homeostasis maintenance were among the most highly enriched functional categories, and genes involved in periplasmic copper efflux were among the most highly upregulated. These results suggest that the deregulation of Fe and Cu homeostatic genes is caused by a release of free Cu and Fe ions in the cell which in turn activate the Cu and Fe homeostatic systems. The content of Cu was not significantly affected upon the addition of Ni to the growth medium, nor were the Cus and CopA Cu-efflux systems important for the survival of bacteria under Ni stress In contrast the addition of Ni slightly decreased the amount of cellular Fe and activated the transcription of Fur regulated genes in a Fur-dependent manner. Cu or Fe imbalance together with oxidative stress might affect the structure of DNA. Further experiments revealed that Ni alters the state of DNA folding by causing a relaxed conformation, a phenomenon that is reversible by addition of the antioxidant Tiron or the Fe chelator Dip. The Tiron-reversible DNA relaxation was also observed for Fe and to a lesser extent with Cu but not with Co. DNA supercoiling is well recognized as an integral aspect of gene regulation. Moreover our results show that Ni modifies the expression of several nucleoid-associated proteins (NAPs), important agents of DNA topology and global gene regulation. This is the first report describing the impact of metal-induced oxidative on global regulatory networks.


Assuntos
DNA Bacteriano/metabolismo , Escherichia coli K12/metabolismo , Ferro/metabolismo , Níquel/farmacologia , Estresse Oxidativo , Empacotamento do DNA/efeitos dos fármacos , DNA Bacteriano/ultraestrutura , Escherichia coli K12/efeitos dos fármacos , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Homeostase , Espécies Reativas de Oxigênio/metabolismo , Transcriptoma/efeitos dos fármacos
17.
Science ; 352(6291): 1330-3, 2016 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-27284196

RESUMO

Class II transcription activators function by binding to a DNA site overlapping a core promoter and stimulating isomerization of an initial RNA polymerase (RNAP)-promoter closed complex into a catalytically competent RNAP-promoter open complex. Here, we report a 4.4 angstrom crystal structure of an intact bacterial class II transcription activation complex. The structure comprises Thermus thermophilus transcription activator protein TTHB099 (TAP) [homolog of Escherichia coli catabolite activator protein (CAP)], T. thermophilus RNAP σ(A) holoenzyme, a class II TAP-dependent promoter, and a ribotetranucleotide primer. The structure reveals the interactions between RNAP holoenzyme and DNA responsible for transcription initiation and reveals the interactions between TAP and RNAP holoenzyme responsible for transcription activation. The structure indicates that TAP stimulates isomerization through simple, adhesive, stabilizing protein-protein interactions with RNAP holoenzyme.


Assuntos
Proteínas de Bactérias/química , Proteína Receptora de AMP Cíclico/química , DNA Bacteriano/química , RNA Polimerases Dirigidas por DNA/química , Regulação Bacteriana da Expressão Gênica , Fator sigma/química , Ativação Transcricional , Proteínas de Bactérias/ultraestrutura , Cristalografia por Raios X , Proteína Receptora de AMP Cíclico/ultraestrutura , DNA Bacteriano/ultraestrutura , RNA Polimerases Dirigidas por DNA/ultraestrutura , Holoenzimas/química , Holoenzimas/ultraestrutura , Regiões Promotoras Genéticas , Conformação Proteica , Fator sigma/ultraestrutura , Thermus thermophilus/enzimologia , Thermus thermophilus/genética
18.
Biochem Biophys Res Commun ; 474(4): 686-690, 2016 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-27150628

RESUMO

Two-component system SaeRS of Staphylococcus regulates virulence factor expression through phosphorylation of the DNA-binding regulator SaeR by the sensor histidine kinase SaeS. Here crystal structures of the DNA-binding domain (DBD) of SaeR from two Staphylococcal species Staphylococcus epidermidis and Staphylococcus aureus were determined and showed similar folds. Analyzing the DNA binding activity of three mutants of SeSaeR, we observed that Thr217 is important in binding to the phosphate group of DNA and Trp219 may interact with the base pairs. Additionally, the tandem arrangement of DBD may represent a possible way for SaeR oligomerization on DNA.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/ultraestrutura , DNA Bacteriano/química , DNA Bacteriano/ultraestrutura , Sítios de Ligação , Simulação por Computador , Cristalografia/métodos , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/ultraestrutura , Modelos Químicos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade , Fatores de Transcrição
19.
Proc Natl Acad Sci U S A ; 113(18): 4988-93, 2016 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-27091999

RESUMO

Cell division in most prokaryotes is mediated by FtsZ, which polymerizes to create the cytokinetic Z ring. Multiple FtsZ-binding proteins regulate FtsZ polymerization to ensure the proper spatiotemporal formation of the Z ring at the division site. The DNA-binding protein SlmA binds to FtsZ and prevents Z-ring formation through the nucleoid in a process called "nucleoid occlusion" (NO). As do most FtsZ-accessory proteins, SlmA interacts with the conserved C-terminal domain (CTD) that is connected to the FtsZ core by a long, flexible linker. However, SlmA is distinct from other regulatory factors in that it must be DNA-bound to interact with the FtsZ CTD. Few structures of FtsZ regulator-CTD complexes are available, but all reveal the CTD bound as a helix. To deduce the molecular basis for the unique SlmA-DNA-FtsZ CTD regulatory interaction and provide insight into FtsZ-regulator protein complex formation, we determined structures of Escherichia coli, Vibrio cholera, and Klebsiella pneumonia SlmA-DNA-FtsZ CTD ternary complexes. Strikingly, the FtsZ CTD does not interact with SlmA as a helix but binds as an extended conformation in a narrow, surface-exposed pocket formed only in the DNA-bound state of SlmA and located at the junction between the DNA-binding and C-terminal dimer domains. Binding studies are consistent with the structure and underscore key interactions in complex formation. Combined, these data reveal the molecular basis for the SlmA-DNA-FtsZ interaction with implications for SlmA's NO function and underscore the ability of the FtsZ CTD to adopt a wide range of conformations, explaining its ability to bind diverse regulatory proteins.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/ultraestrutura , Proteínas de Transporte/química , Proteínas de Transporte/ultraestrutura , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/ultraestrutura , DNA Bacteriano/química , DNA Bacteriano/ultraestrutura , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/ultraestrutura , Sítios de Ligação , Segregação de Cromossomos , Cromossomos Bacterianos/química , Cromossomos Bacterianos/ultraestrutura , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/ultraestrutura , Modelos Químicos , Simulação de Acoplamento Molecular , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Subunidades Proteicas
20.
Sci Rep ; 6: 19243, 2016 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-26763930

RESUMO

Escherichia coli lac repressor (LacI) is a paradigmatic transcriptional factor that controls the expression of lacZYA in the lac operon. This tetrameric protein specifically binds to the O1, O2 and O3 operators of the lac operon and forms a DNA loop to repress transcription from the adjacent lac promoter. In this article, we demonstrate that upon binding to the O1 and O2 operators at their native positions LacI constrains three (-) supercoils within the 401-bp DNA loop of the lac promoter and forms a topological barrier. The stability of LacI-mediated DNA topological barriers is directly proportional to its DNA binding affinity. However, we find that DNA supercoiling modulates the basal expression from the lac operon in E. coli. Our results are consistent with the hypothesis that LacI functions as a topological barrier to constrain free, unconstrained (-) supercoils within the 401-bp DNA loop of the lac promoter. These constrained (-) supercoils enhance LacI's DNA-binding affinity and thereby the repression of the promoter. Thus, LacI binding is superhelically modulated to control the expression of lacZYA in the lac operon under varying growth conditions.


Assuntos
DNA Bacteriano , DNA Super-Helicoidal , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Óperon Lac , DNA Bacteriano/ultraestrutura , DNA Super-Helicoidal/ultraestrutura , Escherichia coli/metabolismo , Repressores Lac/química , Repressores Lac/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Multimerização Proteica
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