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1.
Biochim Biophys Acta Gene Regul Mech ; 1863(3): 194501, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32036061

RESUMO

Riboswitches are RNA sensors that have been shown to modulate the expression of downstream genes by altering their structure upon metabolite binding. Riboswitches are unique among cellular regulators in that metabolite detection is strictly performed using RNA interactions with the sensed metabolite and in which no regulatory protein is needed to mediate the interaction. However, recent studies have shed light on riboswitch control mechanisms relying on protein regulators to harness metabolite binding for the mediation of gene expression, thereby increasing the range of cellular factors involved in riboswitch regulation. The interaction between riboswitches and proteins adds another level of evolutionary pressure as riboswitches must maintain key residues for metabolite detection, structural switching and protein binding sites. Here, we review regulatory mechanisms involving Escherichia coli riboswitches that have recently been shown to rely on regulatory proteins. We also discuss the implication of such protein-based riboswitch regulatory mechanisms for genetic regulation.


Assuntos
Regulação da Expressão Gênica , Riboswitch , Endorribonucleases/metabolismo , Escherichia coli/genética , Pequeno RNA não Traduzido/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fator Rho/metabolismo , Terminação da Transcrição Genética
2.
PLoS Genet ; 15(10): e1008425, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31589608

RESUMO

Evolutionarily conserved NusG protein enhances bacterial RNA polymerase processivity but can also promote transcription termination by binding to, and stimulating the activity of, Rho factor. Rho terminates transcription upon anchoring to cytidine-rich motifs, the so-called Rho utilization sites (Rut) in nascent RNA. Both NusG and Rho have been implicated in the silencing of horizontally-acquired A/T-rich DNA by nucleoid structuring protein H-NS. However, the relative roles of the two proteins in H-NS-mediated gene silencing remain incompletely defined. In the present study, a Salmonella strain carrying the nusG gene under the control of an arabinose-inducible repressor was used to assess the genome-wide response to NusG depletion. Results from two complementary approaches, i) screening lacZ protein fusions generated by random transposition and ii) transcriptomic analysis, converged to show that loss of NusG causes massive upregulation of Salmonella pathogenicity islands (SPIs) and other H-NS-silenced loci. A similar, although not identical, SPI-upregulated profile was observed in a strain with a mutation in the rho gene, Rho K130Q. Surprisingly, Rho mutation Y80C, which affects Rho's primary RNA binding domain, had either no effect or made H-NS-mediated silencing of SPIs even tighter. Thus, while corroborating the notion that bound H-NS can trigger Rho-dependent transcription termination in vivo, these data suggest that H-NS-elicited termination occurs entirely through a NusG-dependent pathway and is less dependent on Rut site binding by Rho. We provide evidence that through Rho recruitment, and possibly through other still unidentified mechanisms, NusG prevents pervasive transcripts from elongating into H-NS-silenced regions. Failure to perform this function causes the feedforward activation of the entire Salmonella virulence program. These findings provide further insight into NusG/Rho contribution in H-NS-mediated gene silencing and underscore the importance of this contribution for the proper functioning of a global regulatory response in growing bacteria. The complete set of transcriptomic data is freely available for viewing through a user-friendly genome browser interface.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação Bacteriana da Expressão Gênica , Fatores de Alongamento de Peptídeos/metabolismo , Salmonella typhimurium/genética , Fatores de Transcrição/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Loci Gênicos , Fatores de Alongamento de Peptídeos/genética , RNA Bacteriano/metabolismo , Fator Rho/genética , Fator Rho/metabolismo , Salmonella typhimurium/patogenicidade , Fatores de Transcrição/genética , Terminação da Transcrição Genética , Regulação para Cima , Fatores de Virulência/genética
3.
Nat Commun ; 10(1): 3728, 2019 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-31427601

RESUMO

Discovery of CRISPR-Cas systems is one of paramount importance in the field of microbiology. Currently, how CRISPR-Cas systems are finely regulated remains to be defined. Here we use small regulatory RNA (sRNA) library to screen sRNAs targeting type I-F CRISPR-Cas system through proximity ligation by T4 RNA ligase and find 34 sRNAs linking to CRISPR loci. Among 34 sRNAs for potential regulators of CRISPR, sRNA pant463 and PhrS enhance CRISPR loci transcription, while pant391 represses their transcription. We identify PhrS as a regulator of CRISPR-Cas by binding CRISPR leaders to suppress Rho-dependent transcription termination. PhrS-mediated anti-termination facilitates CRISPR locus transcription to generate CRISPR RNA (crRNA) and subsequently promotes CRISPR-Cas adaptive immunity against bacteriophage invasion. Furthermore, this also exists in type I-C/-E CRISPR-Cas, suggesting general regulatory mechanisms in bacteria kingdom. Our findings identify sRNAs as important regulators of CRISPR-Cas, extending roles of sRNAs in controlling bacterial physiology by promoting CRISPR-Cas adaptation priming.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Escherichia coli/genética , Pseudomonas aeruginosa/genética , RNA Bacteriano/biossíntese , Pequeno RNA não Traduzido/genética , Fator Rho/antagonistas & inibidores , Terminação da Transcrição Genética/fisiologia , Bacteriófagos/genética , Sistemas CRISPR-Cas/genética , Ensaios de Triagem em Larga Escala , RNA Bacteriano/genética
4.
FEBS J ; 286(12): 2277-2294, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30828992

RESUMO

The endothelium functions as a semipermeable barrier regulating fluid homeostasis, nutrient, and gas supply to the tissue. Endothelial permeability is increased in several pathological conditions including inflammation and tumors; despite its clinical relevance, however, there are no specific therapies preventing vascular leakage. Here, we show that endothelial cell-restricted ablation of BRAF, a kinase frequently activated in cancer, prevents vascular leaking as well metastatic spread. BRAF regulates endothelial permeability by promoting the cytoskeletal rearrangements necessary for the remodeling of VE-Cadherin-containing endothelial cell-cell junctions and the formation of intercellular gaps. BRAF kinase activity and the ability to form complexes with RAS/RAP1 and dimers with its paralog RAF1 are required for proper permeability control, achieved mechanistically by modulating the interaction between RAF1 and the RHO effector ROKα. Thus, RAF dimerization impinges on RHO pathways to regulate cytoskeletal rearrangements, junctional plasticity, and endothelial permeability. The data advocate the development of RAF dimerization inhibitors, which would combine tumor cell autonomous effect with stabilization of the vasculature and antimetastatic spread.


Assuntos
Antígenos CD/genética , Caderinas/genética , Citoesqueleto/genética , Proteínas Proto-Oncogênicas B-raf/genética , Quinases Associadas a rho/genética , Animais , Permeabilidade Capilar/genética , Citoesqueleto/metabolismo , Dimerização , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Junções Intercelulares/genética , Camundongos , Fosforilação/genética , Fator Rho/genética , Transdução de Sinais , Proteínas rap1 de Ligação ao GTP/genética
5.
BMC Bioinformatics ; 20(1): 117, 2019 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-30845912

RESUMO

BACKGROUND: In bacterial genomes, there are two mechanisms to terminate the DNA transcription: the "intrinsic" or Rho-independent termination and the Rho-dependent termination. Intrinsic terminators are characterized by a RNA hairpin followed by a run of 6-8 U residues relatively easy to identify using one of the numerous available prediction programs. In contrast, Rho-dependent termination is mediated by the Rho protein factor that, firstly, binds to ribosome-free mRNA in a site characterized by a C > G content and then reaches the RNA polymerase to induce its release. Conversely on intrinsic terminators, the computational prediction of Rho-dependent terminators in prokaryotes is a very difficult problem because the sequence features required for the function of Rho are complex and poorly defined. This is the reason why it still does not exist an exhaustive Rho-dependent terminators prediction program. RESULTS: In this study we introduce RhoTermPredict, the first published algorithm for an exhaustive Rho-dependent terminators prediction in bacterial genomes. RhoTermPredict identifies these elements based on a previously proposed consensus motif common to all Rho-dependent transcription terminators. It essentially searches for a 78 nt long RUT site characterized by a C > G content and with regularly spaced C residues, followed by a putative pause site for the RNA polymerase. We tested RhoTermPredict performances by using available genomic and transcriptomic data of the microorganism Escherichia coli K-12, both in limited-length sequences and in the whole-genome, and available genomic sequences from Bacillus subtilis 168 and Salmonella enterica LT2 genomes. We also estimated the overlap between the predictions of RhoTermPredict and those obtained by the predictor of intrinsic terminators ARNold webtool. Our results demonstrated that RhoTermPredict is a very performing algorithm both for limited-length sequences (F1-score obtained about 0.7) and for a genome-wide analysis. Furthermore the degree of overlap with ARNold predictions was very low. CONCLUSIONS: Our analysis shows that RhoTermPredict is a powerful tool for Rho-dependent terminators search in the three analyzed genomes and could fill this gap in computational genomics. We conclude that RhoTermPredict could be used in combination with an intrinsic terminators predictor in order to predict all the transcription terminators in bacterial genomes.


Assuntos
Algoritmos , Bacillus subtilis/genética , Bases de Dados Genéticas , Escherichia coli K12/genética , Escherichia coli/genética , Fator Rho/metabolismo , Salmonella enterica/genética , Regiões Terminadoras Genéticas , Sequência de Bases , Genoma Bacteriano , RNA Bacteriano , Análise de Sequência de RNA
6.
Proc Natl Acad Sci U S A ; 116(8): 3042-3051, 2019 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-30718400

RESUMO

Transcription termination is a critical step in the control of gene expression. One of the major termination mechanisms is mediated by Rho factor that dissociates the complex mRNA-DNA-RNA polymerase upon binding with RNA polymerase. Rho promotes termination at the end of operons, but it can also terminate transcription within leader regions, performing regulatory functions and avoiding pervasive transcription. Transcription of rho is autoregulated through a Rho-dependent attenuation in the leader region of the transcript. In this study, we have included an additional player in this pathway. By performing MS2-affinity purification coupled with RNA sequencing (MAPS), rho transcript was shown to directly interact with the small noncoding RNA SraL. Using bioinformatic in vivo and in vitro experimental analyses, SraL was shown to base pair with the 5'-UTR of rho mRNA upregulating its expression in several growth conditions. This base pairing was shown to prevent the action of Rho over its own message. Moreover, the results obtained indicate that both ProQ and Hfq are associated with this regulation. We propose a model that contemplates the action of Salmonella SraL sRNA in the protection of rho mRNA from premature transcription termination by Rho. Note that since the interaction region between both RNAs corresponds to a very-well-conserved sequence, it is plausible to admit that this regulation also occurs in other enterobacteria.


Assuntos
DNA/genética , Pequeno RNA não Traduzido/genética , Fator Rho/genética , Terminação da Transcrição Genética , DNA/biossíntese , RNA Polimerases Dirigidas por DNA/genética , Regulação Bacteriana da Expressão Gênica/genética , Conformação de Ácido Nucleico , RNA Mensageiro/genética , Salmonella enterica/genética , Análise de Sequência de RNA , Transcrição Genética
7.
Curr Genet ; 65(3): 729-733, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30739200

RESUMO

The bacterial hexameric helicase known as Rho is an archetypal sequence-specific transcription terminator that typically halts the synthesis of a defined set of transcripts, particularly those bearing cytosine-rich 3'-untranslated regions. However, under conditions of translational stress, Rho can also terminate transcription at cytosine-poor sites when assisted by the transcription factor NusG. Recent structural, biochemical, and computational studies of the Rho·NusG interaction in Escherichia coli have helped establish how NusG reprograms Rho activity. NusG is found to be an allosteric activator of Rho that directly binds to the ATPase motor domain of the helicase and facilitates closure of the Rho ring around non-ideal (purine-rich) target RNAs. The manner in which NusG acts on Rho helps to explain how the transcription terminator is excluded from acting on RNA polymerase by exogenous factors, such as the antitermination protein NusE, the NusG paralog RfaH, and RNA polymerase-coupled ribosomes. Collectively, an understanding of the link between NusG and Rho provides new insights into how transcriptional and translational fidelity are maintained during gene expression in bacteria.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Fatores de Alongamento de Peptídeos/metabolismo , Fator Rho/metabolismo , Fatores de Transcrição/metabolismo , Terminação da Transcrição Genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Fatores de Alongamento de Peptídeos/genética , Fator Rho/genética , Fatores de Transcrição/genética
8.
Biochemistry ; 58(7): 865-874, 2019 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-30624903

RESUMO

Transcription termination mediated by the ring-shaped, ATP-dependent Rho motor is a multipurpose regulatory mechanism specific to bacteria and constitutes an interesting target for the development of new antibiotics. Although Rho-dependent termination can punctuate gene expression or contribute to the protection of the genome at hundreds of sites within a given bacterium, its exact perimeter and site- or species-specific features remain insufficiently characterized. New advanced approaches are required to explore thoroughly the diversity of Rho-dependent terminators and the complexity of associated mechanisms. Current in vitro analyses of Rho-dependent termination rely on radiolabeling, gel electrophoresis, and phosphorimaging of transcription reaction products and are thus hazardous, inconvenient, and low-throughput. To address these limitations, we have developed the first in vitro assay using a fluorescence detection modality to study Rho-dependent transcription termination. This powerful experimental tool accurately estimates terminator strengths in a matter of minutes and is optimized for a microplate reader format allowing multiplexed characterization of putative terminator sequences and mechanisms or high-throughput screening of new drugs targeting Rho-dependent termination.


Assuntos
Bioquímica/métodos , Corantes Fluorescentes , Fator Rho/genética , Terminação da Transcrição Genética , Sondas Moleculares/genética , Fator Rho/metabolismo , Espectrometria de Fluorescência , p-Dimetilaminoazobenzeno/análogos & derivados
9.
RNA Biol ; 16(4): 404-412, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30252595

RESUMO

The Class 2 Type V-A CRISPR effector protein Cas12a/Cpf1 has gained widespread attention in part because of the ease in achieving multiplexed genome editing, gene regulation, and DNA detection. Multiplexing derives from the ability of Cas12a alone to generate multiple guide RNAs from a transcribed CRISPR array encoding alternating conserved repeats and targeting spacers. While array design has focused on how to optimize guide-RNA sequences, little attention has been paid to sequences outside of the CRISPR array. Here, we show that a structured hairpin located immediately downstream of the 3' repeat interferes with utilization of the adjacent encoded guide RNA by Francisella novicida (Fn)Cas12a. We first observed that a synthetic Rho-independent terminator immediately downstream of an array impaired DNA cleavage based on plasmid clearance in E. coli and DNA cleavage in a cell-free transcription-translation (TXTL) system. TXTL-based cleavage assays further revealed that inhibition was associated with incomplete processing of the transcribed CRISPR array and could be attributed to the stable hairpin formed by the terminator. We also found that the inhibitory effect partially extended to upstream spacers in a multi-spacer array. Finally, we found that removing the terminal repeat from the array increased the inhibitory effect, while replacing this repeat with an unprocessable terminal repeat from a native FnCas12a array restored cleavage activity directed by the adjacent encoded guide RNA. Our study thus revealed that sequences surrounding a CRISPR array can interfere with the function of a CRISPR nuclease, with implications for the design and evolution of CRISPR arrays.


Assuntos
Proteínas Associadas a CRISPR/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Francisella/genética , Sequências Repetidas Terminais/genética , Clivagem do DNA , DNA Intergênico/genética , Conformação de Ácido Nucleico , Processamento Pós-Transcricional do RNA/genética , RNA Guia/metabolismo , Fator Rho/metabolismo , Terminação da Transcrição Genética
10.
mBio ; 9(5)2018 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-30228237

RESUMO

Staphylococcus aureus causes various diseases ranging from skin and soft tissue infections to life-threatening infections. Adaptation to the different host niches is controlled by a complex network of transcriptional regulators. Global profiling of condition-dependent transcription revealed adaptation of S. aureus HG001 at the levels of transcription initiation and termination. In particular, deletion of the gene encoding the Rho transcription termination factor triggered a remarkable overall increase in antisense transcription and gene expression changes attributable to indirect regulatory effects. The goal of the present study was a detailed comparative analysis of S. aureus HG001 and its isogenic rho deletion mutant. Proteome analysis revealed significant differences in cellular and extracellular protein profiles, most notably increased amounts of the proteins belonging to the SaeR regulon in the Rho-deficient strain. The SaeRS two-component system acts as a major regulator of virulence gene expression in staphylococci. Higher levels of SaeRS-dependent virulence factors such as adhesins, toxins, and immune evasion proteins in the rho mutant resulted in higher virulence in a murine bacteremia model, which was alleviated in a rho complemented strain. Inhibition of Rho activity by bicyclomycin, a specific inhibitor of Rho activity, also induced the expression of SaeRS-dependent genes, at both the mRNA and protein levels, to the same extent as observed in the rho mutant. Taken together, these findings indicate that activation of the Sae system in the absence of Rho is directly linked to Rho's transcription termination activity and establish a new link between antibiotic action and virulence gene expression in S. aureusIMPORTANCE The major human pathogen Staphylococcus aureus is a widespread commensal bacterium but also the most common cause of nosocomial infections. It adapts to the different host niches through a complex gene regulatory network. We show here that the Rho transcription termination factor, which represses pervasive antisense transcription in various bacteria, including S. aureus, plays a role in controlling SaeRS-dependent virulence gene expression. A Rho-deficient strain produces larger amounts of secreted virulence factors in vitro and shows increased virulence in mice. We also show that treatment of S. aureus with the antibiotic bicyclomycin, which inhibits Rho activity and is effective against Gram-negative bacteria, induces the same changes in the proteome as observed in the Rho-deficient strain. Our results reveal for the first time a link between transcription termination and virulence regulation in S. aureus, which implies a novel mechanism by which an antibiotic can modulate the expression of virulence factors.


Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Proteínas Quinases/metabolismo , Fator Rho/metabolismo , Staphylococcus aureus/genética , Fatores de Transcrição/metabolismo , Terminação da Transcrição Genética , Fatores de Virulência/biossíntese , Animais , Antibacterianos/metabolismo , Bacteriemia/microbiologia , Bacteriemia/patologia , Proteínas de Bactérias/genética , Modelos Animais de Doenças , Deleção de Genes , Perfilação da Expressão Gênica , Teste de Complementação Genética , Proteínas Quinases/genética , Proteoma/análise , Regulon , Fator Rho/genética , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/patologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/metabolismo , Staphylococcus aureus/patogenicidade , Fatores de Transcrição/genética , Virulência
11.
Mol Cell ; 71(6): 911-922.e4, 2018 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-30122535

RESUMO

NusG/Spt5 proteins are the only transcription factors utilized by all cellular organisms. In enterobacteria, NusG antagonizes the transcription termination activity of Rho, a hexameric helicase, during the synthesis of ribosomal and actively translated mRNAs. Paradoxically, NusG helps Rho act on untranslated transcripts, including non-canonical antisense RNAs and those arising from translational stress; how NusG fulfills these disparate functions is unknown. Here, we demonstrate that NusG activates Rho by assisting helicase isomerization from an open-ring, RNA-loading state to a closed-ring, catalytically active translocase. A crystal structure of closed-ring Rho in complex with NusG reveals the physical basis for this activation and further explains how Rho is excluded from translationally competent RNAs. This study demonstrates how a universally conserved transcription factor acts to modulate the activity of a ring-shaped ATPase motor and establishes how the innate sequence bias of a termination factor can be modulated to silence pervasive, aberrant transcription.


Assuntos
Proteínas Cromossômicas não Histona/fisiologia , Proteínas de Escherichia coli/fisiologia , Fatores de Alongamento de Peptídeos/fisiologia , Fatores de Transcrição/fisiologia , Terminação da Transcrição Genética/fisiologia , Fatores de Elongação da Transcrição/fisiologia , Proteínas de Bactérias , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Fatores de Alongamento de Peptídeos/metabolismo , Conformação Proteica , RNA Bacteriano , Fator Rho/metabolismo , Fator Rho/fisiologia , Fatores de Transcrição/metabolismo , Transcrição Genética/genética , Transcrição Genética/fisiologia
12.
Zhen Ci Yan Jiu ; 43(7): 445-9, 2018 Jul 25.
Artigo em Chinês | MEDLINE | ID: mdl-30094982

RESUMO

OBJECTIVE: To observe the effect of electroacupuncture (EA) of "Jiaji" (EX-B 2) on limb locomotor function and expression of Ras homolog gene family member A (RhoA), Rho-associated kinase Ⅱ (ROCK Ⅱ) and myosin light chain (MLC) proteins in the anterior horn of spinal cord in acute spinal cord injury (ASCI) rats, so as to explore its mechanisms under-lying improvement of SCI-induced limb locomotor dysfunction. METHODS: Forty-eight female Wistar rats were randomly divided into sham operation (sham), ASCI model (model), EA EX-B 2 (EA) and ROCK inhibitor (Fasudil) groups which were further divided into 14 d and 28 d subgroups (n=6 in each). The ASCI model was made by using weight drop striking method. Three hours after modeling, EA (100 Hz, 0.4, 0.6 mA) was applied to EX-B 2 (T 9, T 11) for 30 min, once daily for 14 d and 28 d, respectively. The ROCK inhibitor (hydrochloride Fasudil, 10 mg/kg) was administrated by intraperitoneal injection immediately after modeling, once a day, continuously for 14 d or 28 d. The expression of RhoA, ROCK Ⅱ and MLC proteins in the spinal cord anterior horn tissue (T 10) was detected by immunohistochemistry. The rats' hindlimb locomotor function was assessed according to Basso, Beattie and Bresnahan (BBB) locomotor rating scale (21-points). RESULTS: After ASCI, the BBB scores were significantly lower in the model group than in the sham group on day 14 and 28 (P<0.05), and obviously higher in the EA and inhibitor groups than in the model group (P<0.05), suggesting an improvement of the hindlimb locomotor function after EA intervention or suppression of ROCK. Immunohistochemical results indicated that the numbers of RhoA, ROCK Ⅱ and MLC immune-reaction positive cells in the anterior horn of spinal cord were significantly more in the model group than in the sham group (P<0.05), and remarkably decreased in both EA and inhibitor groups on day 14 and 28 relevant to the model group (P<0.05). The therapeutic effects of EA were markedly weaker than those of inhibitor Fasudil in up-regulating BBB score and down-regulating the number of RhoA, ROCK Ⅱ and MLC positive cells (P<0.05). CONCLUSION: EA of EX-B 2 can improve the hindlimb locomotor function in ASCI rats, which may be associated with its effect in down-regulating the expression of RhoA, ROCK Ⅱ and MLC proteins (i.e., inhibiting the RhoA/ROCK signaling pathway) in the anterior horn of spinal cord.


Assuntos
Eletroacupuntura , Traumatismos da Medula Espinal , Animais , Células do Corno Anterior , Feminino , Membro Posterior , Locomoção , Ratos , Ratos Wistar , Fator Rho , Transdução de Sinais , Medula Espinal , Traumatismos da Medula Espinal/terapia , Quinases Associadas a rho
13.
Dev Cell ; 46(2): 162-172.e5, 2018 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-29983336

RESUMO

The dynamics of extracellular signal-regulated kinase (ERK) signaling underlies its versatile functions in cell differentiation, cell proliferation, and cell motility. Classical studies in Drosophila established that a gradient of epidermal growth factor receptor (EGFR)-ERK signaling is essential for these cellular responses. However, we challenge this view by the real-time monitoring of ERK activation; we show that a switch-like ERK activation is essential for the invagination movement of the Drosophila tracheal placode. This switch-like ERK activation stems from the positive feedback regulation of the EGFR-ERK signaling and a resultant relay of EGFR-ERK signaling among tracheal cells. A key transcription factor Trachealess (Trh) permissively regulates the iteration of the relay, and the ERK activation becomes graded in trh mutant. A mathematical model based on these observations and a molecular link between ERK activation dynamics and myosin shows that the relay mechanism efficiently promotes epithelial invagination while the gradient mechanism does not.


Assuntos
Proteínas de Drosophila/metabolismo , Receptores ErbB/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Receptores de Peptídeos de Invertebrados/metabolismo , Animais , Movimento Celular , Proliferação de Células , Drosophila/metabolismo , Fator de Crescimento Epidérmico/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Miosinas/metabolismo , Fosforilação , Fator Rho/metabolismo , Fatores de Transcrição/metabolismo
14.
Nucleic Acids Res ; 46(16): 8245-8260, 2018 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-29931073

RESUMO

Bacterial transcription termination proceeds via two main mechanisms triggered either by simple, well-conserved (intrinsic) nucleic acid motifs or by the motor protein Rho. Although bacterial genomes can harbor hundreds of termination signals of either type, only intrinsic terminators are reliably predicted. Computational tools to detect the more complex and diversiform Rho-dependent terminators are lacking. To tackle this issue, we devised a prediction method based on Orthogonal Projections to Latent Structures Discriminant Analysis [OPLS-DA] of a large set of in vitro termination data. Using previously uncharacterized genomic sequences for biochemical evaluation and OPLS-DA, we identified new Rho-dependent signals and quantitative sequence descriptors with significant predictive value. Most relevant descriptors specify features of transcript C>G skewness, secondary structure, and richness in regularly-spaced 5'CC/UC dinucleotides that are consistent with known principles for Rho-RNA interaction. Descriptors collectively warrant OPLS-DA predictions of Rho-dependent termination with a ∼85% success rate. Scanning of the Escherichia coli genome with the OPLS-DA model identifies significantly more termination-competent regions than anticipated from transcriptomics and predicts that regions intrinsically refractory to Rho are primarily located in open reading frames. Altogether, this work delineates features important for Rho activity and describes the first method able to predict Rho-dependent terminators in bacterial genomes.


Assuntos
Biologia Computacional/métodos , Proteínas de Escherichia coli/genética , Genoma Bacteriano/genética , Genômica/métodos , Fator Rho/genética , Terminação da Transcrição Genética , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Modelos Genéticos , Análise Multivariada , Fator Rho/metabolismo
15.
Infect Immun ; 86(7)2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29712731

RESUMO

As an obligate intracellular, developmentally regulated bacterium, Chlamydia is sensitive to amino acid fluctuations within its host cell. When human epithelial cells are treated with the cytokine interferon gamma (IFN-γ), the tryptophan (Trp)-degrading enzyme, indoleamine-2,3-dioxygenase, is induced. Chlamydiae within such cells are starved for Trp and enter a state of so-called persistence. Chlamydia lacks the stringent response used by many eubacteria to respond to this stress. Unusually, chlamydial transcription is globally elevated during Trp starvation with transcripts for Trp codon-containing genes disproportionately increased. Yet, the presence of Trp codons destabilized 3' ends of transcripts in operons or large genes. We initially hypothesized that ribosome stalling on Trp codons rendered the 3' ends sensitive to RNase activity. The half-life of chlamydial transcripts containing different numbers of Trp codons was thus measured in untreated and IFN-γ-treated infected cells to determine whether Trp codons influenced the stability of transcripts. However, no effect of Trp codon content was detected. Therefore, we investigated whether Rho-dependent transcription termination could play a role in mediating transcript instability. Rho is expressed as a midcycle gene product, interacts with itself as predicted, and is present in all chlamydial species. Inhibition of Rho via the Rho-specific antibiotic, bicyclomycin, and overexpression of Rho are detrimental to chlamydiae. Finally, when we measured transcript abundance 3' to Trp codons in the presence of bicyclomycin, we observed that transcript abundance increased. These data are the first to demonstrate the importance of Rho in Chlamydia and the role of Rho-dependent transcription polarity during persistence.


Assuntos
Chlamydia trachomatis/genética , Interferon gama/farmacologia , Fator Rho/fisiologia , Transcrição Genética , Triptofano/genética , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Células Cultivadas , Chlamydia trachomatis/crescimento & desenvolvimento , Códon , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/fisiologia , Fases de Leitura Aberta
16.
Nucleic Acids Res ; 46(7): 3400-3411, 2018 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-29474582

RESUMO

Transcription termination by Rho is essential for viability in various bacteria, including some major pathogens. Since Rho acts by targeting nascent RNAs that are not simultaneously translated, it also regulates antisense transcription. Here we show that RNase H-deficient mutants of Escherichia coli exhibit heightened sensitivity to the Rho inhibitor bicyclomycin, and that Rho deficiency provokes increased formation of RNA-DNA hybrids (R-loops) which is ameliorated by expression of the phage T4-derived R-loop helicase UvsW. We also provide evidence that in Rho-deficient cells, R-loop formation blocks subsequent rounds of antisense transcription at more than 500 chromosomal loci. Hence these antisense transcripts, which can extend beyond 10 kb in their length, are only detected when Rho function is absent or compromised and the UvsW helicase is concurrently expressed. Thus the potential for antisense transcription in bacteria is much greater than hitherto recognized; and the cells are able to retain viability even when nearly one-quarter of their total non-rRNA abundance is accounted for by antisense transcripts, provided that R-loop formation from them is curtailed.


Assuntos
Genoma Bacteriano/genética , Fator Rho/genética , Terminação da Transcrição Genética , Transcrição Genética , Bacteriófago T4/genética , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Cromossomos/genética , DNA Helicases/genética , Replicação do DNA/genética , DNA Antissenso/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Genes de RNAr/genética , Genoma Bacteriano/efeitos dos fármacos , Fator Rho/antagonistas & inibidores , Ribonuclease H/genética , Proteínas Virais/genética
17.
J Antibiot (Tokyo) ; 71(4): 447-455, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29371644

RESUMO

The alarming issue of antibiotic resistance expansion requires a continuous search for new and efficient antibacterial agents. Here we describe the design of new tools to screen for target-specific inhibitors of the bacterial Rho factor directly inside eukaryotic cells. Rho factor is a global regulator of gene expression which is essential to most bacteria, especially Gram-negative. Since Rho has no functional or structural homolog in eukaryotes, it constitutes a valuable and well known bacterial target as evidenced by its inhibition by the natural antibiotic, Bicyclomycin. Our screening tools are based on perturbation of mRNA processing and packaging reactions in the nucleus of eukaryotic cells by the RNA-dependent helicase/translocase activity of bacterial Rho factor leading to a growth defect phenotype. In this approach, any compound that impedes Rho activity should restore growth to yeast or human cells expressing Rho protein, providing valuable means to screen for target-specific antibacterial agents within the environment of a eukaryotic cell. The yeast tool expressing E. coli Rho factor was validated using Bicyclomycin as the control antibacterial agent. The validation of the screening tool was further extended with a stable human cell line expressing Rho factor conditionally. Finally, we show that Rho factors from different bacterial pathogens can also be designed as yeast-based screening tools which can reveal subtle variations in the functional features of the proteins.


Assuntos
Antibacterianos/farmacologia , Fator Rho/efeitos dos fármacos , Leveduras/efeitos dos fármacos , Infecções Bacterianas/microbiologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Bactérias Gram-Negativas/efeitos dos fármacos , Células HEK293 , Humanos , Saccharomyces cerevisiae/efeitos dos fármacos , Transcrição Genética
18.
Transcription ; 9(3): 152-158, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-28886303

RESUMO

Helicases are enzymes that remodel nucleic acids or protein-nucleic acid complexes in an ATP-dependent manner. They are ubiquitous and can play many diverse functions related to the metabolism of nucleic acids. A few helicases from both the prokaryotic and the eukaryotic worlds have the ability to induce transcription termination. Here we discuss how the same biological function is achieved by different helicases with quite divergent structures and mechanisms of action.


Assuntos
Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , DNA Helicases/metabolismo , RNA Helicases/metabolismo , Fator Rho/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Terminação da Transcrição Genética , Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Saccharomyces cerevisiae/genética
19.
J Bacteriol ; 200(1)2018 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-29038252

RESUMO

Rho is a hexameric molecular motor that functions as a conserved transcription terminator in the majority of bacterial species and is a potential drug target. Psu is a bacteriophage P4 capsid protein that inhibits Escherichia coli Rho by obstructing its ATPase and translocase activities. In this study, we explored the anti-Rho activity of Psu for Rho proteins from different pathogens. Sequence alignment and homology modeling of Rho proteins from pathogenic bacteria revealed the conserved nature of the Psu-interacting regions in all these proteins. We chose Rho proteins from various pathogens, including Mycobacterium smegmatis, Mycobacterium bovis, Mycobacterium tuberculosis, Xanthomonas campestris, Xanthomonas oryzae, Corynebacterium glutamicum, Vibrio cholerae, Salmonella enterica, and Pseudomonas syringae The purified recombinant Rho proteins of these organisms showed variable rates of ATP hydrolysis on poly(rC) as the substrate and were capable of releasing RNA from the E. coli transcription elongation complexes. Psu was capable of inhibiting these two functions of all these Rho proteins. In vivo pulldown assays revealed direct binding of Psu with many of these Rho proteins. In vivo expression of psu induced killing of M. smegmatis, M. bovis, X. campestris, and E. coli expressing S. enterica Rho indicating Psu-induced inhibition of Rho proteins of these strains under physiological conditions. We propose that the "universal" inhibitory function of the Psu protein against the Rho proteins from both Gram-negative and Gram-positive bacteria could be useful for designing peptides with antimicrobial functions and that these peptides could contribute to synergistic antibiotic treatment of the pathogens by compromising the Rho functions.IMPORTANCE Bacteriophage-derived protein factors modulating different bacterial processes could be converted into unique antimicrobial agents. Bacteriophage P4 capsid protein Psu is an inhibitor of the E. coli transcription terminator Rho. Here we show that apart from antagonizing E. coli Rho, Psu is able to inhibit Rho proteins from various phylogenetically unrelated Gram-negative and Gram-positive pathogens. Upon binding to these Rho proteins, Psu inhibited them by affecting their ATPase and RNA release functions. The expression of Psu in vivo kills various pathogens, such as Mycobacterium and Xanthomonas species. Hence, Psu could be useful for identifying peptide sequences with anti-Rho activities and might constitute part of synergistic antibiotic treatment against pathogens.


Assuntos
Proteínas do Capsídeo/farmacologia , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Fator Rho/antagonistas & inibidores , Terminação da Transcrição Genética/efeitos dos fármacos , Adenosina Trifosfatases/antagonistas & inibidores , Bacteriófagos/química , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/genética , Escherichia coli/patogenicidade , Bactérias Gram-Negativas/genética , Bactérias Gram-Negativas/patogenicidade , Bactérias Gram-Positivas/genética , Bactérias Gram-Positivas/patogenicidade , Mycobacterium/efeitos dos fármacos , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/patogenicidade , Ligação Proteica , Proteínas Recombinantes/metabolismo , Fator Rho/genética , Fator Rho/metabolismo , Homologia de Sequência do Ácido Nucleico , Vibrio cholerae/efeitos dos fármacos , Vibrio cholerae/genética , Vibrio cholerae/patogenicidade , Xanthomonas/efeitos dos fármacos
20.
Biochemistry ; 57(1): 61-65, 2018 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-29053243

RESUMO

Diketopiperazines (DKPs) make up a large group of natural products with diverse structures and biological activities. Bicyclomycin is a broad-spectrum DKP antibiotic with unique structure and function: it contains a highly oxidized bicyclic [4.2.2] ring and is the only known selective inhibitor of the bacterial transcription termination factor, Rho. Here, we identify the biosynthetic gene cluster for bicyclomycin containing six iron-dependent oxidases. We demonstrate that the DKP core is made by a tRNA-dependent cyclodipeptide synthase, and hydroxylations on two unactivated sp3 carbons are performed by two mononuclear iron, α-ketoglutarate-dependent hydroxylases. Using bioinformatics, we also identify a homologous gene cluster prevalent in a human pathogen Pseudomonas aeruginosa. We detect bicyclomycin by overexpressing this gene cluster and establish P. aeruginosa as a new producer of bicyclomycin. Our work uncovers the biosynthetic pathway for bicyclomycin and sheds light on the intriguing oxidation chemistry that converts a simple DKP into a powerful antibiotic.


Assuntos
Antibacterianos/biossíntese , Proteínas de Bactérias/metabolismo , Inibidores Enzimáticos/metabolismo , Pseudomonas aeruginosa/enzimologia , Fator Rho/antagonistas & inibidores , Antibacterianos/química , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , Compostos Bicíclicos Heterocíclicos com Pontes/química , Compostos Bicíclicos Heterocíclicos com Pontes/metabolismo , Biologia Computacional , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Inibidores Enzimáticos/química , Hidroxilação , Ácidos Cetoglutáricos/metabolismo , Estrutura Molecular , Família Multigênica , Oxirredução , Oxigenases/genética , Oxigenases/metabolismo , Peptídeo Sintases/metabolismo , Pseudomonas aeruginosa/metabolismo , Proteínas Recombinantes/metabolismo , Fator Rho/química , Fator Rho/metabolismo , Especificidade da Espécie , Especificidade por Substrato
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