RESUMEN
Transcription is an essential and multistep process carried out by RNA polymerase (RNAP). In bacterial RNAP, in addition to the catalytic core domain, multiple other conserved domains are also identified to play regulatory roles in transcription. One such domain is the zinc-binding domain (ZBD) located at the N terminus of the largest subunit ß' in bacterial RNAP, whose homolog is also reported in eukaryotic RNAPs. Recent structural and biochemical studies have revealed various key roles of the conserved ß' ZBD during different steps of transcription. In this review, we summarize recent progress on the regulatory roles of this ß' ZBD in bacterial transcription.
Asunto(s)
ARN Polimerasas Dirigidas por ADN , ARN Bacteriano , Bacterias/genética , Bacterias/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , Transcripción Genética , ZincRESUMEN
Transcription activation is a crucial step of regulation during transcription initiation and a classic check point in response to different stimuli and stress factors. The Escherichia coli NarL is a nitrate-responsive global transcription factor that controls the expression of nearly 100 genes. However, the molecular mechanism of NarL-mediated transcription activation is not well defined. Here we present a cryo-EM structure of NarL-dependent transcription activation complex (TAC) assembled on the yeaR promoter at 3.2 Å resolution. Our structure shows that the NarL dimer binds at the -43.5 site of the promoter DNA with its C-terminal domain (CTD) not only binding to the DNA but also making interactions with RNA polymerase subunit alpha CTD (αCTD). The key role of these NarL-mediated interactions in transcription activation was further confirmed by in vivo and in vitro transcription assays. Additionally, the NarL dimer binds DNA in a different plane from that observed in the structure of class II TACs. Unlike the canonical class II activation mechanism, NarL does not interact with σ4, while RNAP αCTD is bound to DNA on the opposite side of NarL. Our findings provide a structural basis for detailed mechanistic understanding of NarL-dependent transcription activation on yeaR promoter and reveal a potentially novel mechanism of transcription activation.
Asunto(s)
Proteínas de Escherichia coli , Nitratos , Activación Transcripcional , Proteínas Bacterianas/metabolismo , ADN/genética , ADN/metabolismo , Proteínas de Unión al ADN/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Nitratos/metabolismoRESUMEN
The copper efflux regulator (CueR) is a classical member of the MerR family of metalloregulators and is common in gram-negative bacteria. Through its C-terminal effector-binding domain, CueR senses cytoplasmic copper ions to regulate the transcription of genes contributing to copper homeostasis, an essential process for survival of all cells. In this chapter, we review the regulatory roles of CueR in the model organism Escherichia coli and the mechanisms for CueR in copper binding, DNA recognition, and interplay with RNA polymerase in regulating transcription. In light of biochemical and structural analyses, we provide molecular details for how CueR represses transcription in the absence of copper ions, how copper ions mediate CueR conformational change to form holo CueR, and how CueR bends and twists promoter DNA to activate transcription. We also characterize the functional domains and key residues involved in these processes. Since CueR is a representative member of the MerR family, elucidating its regulatory mechanisms could help to understand the CueR-like regulators in other organisms and facilitate the understanding of other metalloregulators in the same family.
Asunto(s)
Cobre , Proteínas de Escherichia coli , Escherichia coli , Regulación Bacteriana de la Expresión Génica , Cobre/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Transcripción Genética , Regiones Promotoras Genéticas , TransactivadoresRESUMEN
In bacteria, σ28 is the flagella-specific sigma factor that targets RNA polymerase (RNAP) to control the expression of flagella-related genes involving bacterial motility and chemotaxis. However, the structural mechanism of σ28 -dependent promoter recognition remains uncharacterized. Here, we report cryo-EM structures of E. coli σ28 -dependent transcribing complexes on a complete flagella-specific promoter. These structures reveal how σ28 -RNAP recognizes promoter DNA through strong interactions with the -10 element, but weak contacts with the -35 element, to initiate transcription. In addition, we observed a distinct architecture in which the ß' zinc-binding domain (ZBD) of RNAP stretches out from its canonical position to interact with the upstream non-template strand. Further in vitro and in vivo assays demonstrate that this interaction has the overall effect of facilitating closed-to-open isomerization of the RNAP-promoter complex by compensating for the weak interaction between σ4 and -35 element. This suggests that ZBD relocation may be a general mechanism employed by σ70 family factors to enhance transcription from promoters with weak σ4/-35 element interactions.
Asunto(s)
Proteínas Bacterianas , ADN Bacteriano , Escherichia coli , Regiones Promotoras Genéticas , Factor sigma , Transcripción Genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/ultraestructura , Microscopía por Crioelectrón , ADN Bacteriano/metabolismo , ADN Bacteriano/ultraestructura , Escherichia coli/metabolismo , Escherichia coli/ultraestructura , Dominios Proteicos , Factor sigma/metabolismo , Factor sigma/ultraestructuraRESUMEN
RapA is a bacterial RNA polymerase (RNAP)-associated Swi2/Snf2 ATPase that stimulates RNAP recycling. The ATPase activity of RapA is autoinhibited by its N-terminal domain (NTD) but activated with RNAP bound. Here, we report a 3.4-Å cryo-EM structure of Escherichia coli RapA-RNAP elongation complex, in which the ATPase active site of RapA is structurally remodeled. In this process, the NTD of RapA is wedged open by RNAP ß' zinc-binding domain (ZBD). In addition, RNAP ß flap tip helix (FTH) forms extensive hydrophobic interactions with RapA ATPase core domains. Functional assay demonstrates that removing the ZBD or FTH of RNAP significantly impairs its ability to activate the ATPase activity of RapA. Our results provide the structural basis of RapA ATPase activation by RNAP, through the active site remodeling driven by the ZBD-buttressed large-scale opening of NTD and the direct interactions between FTH and ATPase core domains.
Asunto(s)
Adenosina Trifosfatasas/química , ARN Polimerasas Dirigidas por ADN/química , Proteínas de Escherichia coli/química , Adenosina Trifosfatasas/metabolismo , Dominio Catalítico , Microscopía por Crioelectrón , ARN Polimerasas Dirigidas por ADN/metabolismo , Escherichia coli/enzimología , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Dominios ProteicosRESUMEN
BACKGROUND: Hypoxic stress plays a critical role in the persistence of Mycobacterium tuberculosis (Mtb) infection, but the mechanisms underlying this adaptive response remain ill defined. MATERIAL AND METHODS: In this study, using M. marinum as a surrogate, we analyzed hypoxic responses at the transcriptional level by Cappable-seq and regular RNA-seq analyses. RESULTS: A total of 6808 transcriptional start sites (TSSs) were identified under normoxic and hypoxic conditions. Among these TSSs, 1112 were upregulated and 1265 were downregulated in response to hypoxic stress. Using SigE-recognized consensus sequence, we identified 59 SigE-dependent promoters and all were upregulated under hypoxic stress, suggesting an important role for SigE in this process. We also compared the performance of Cappable-seq and regular RNA-seq using the same RNA samples collected from normoxic and hypoxic conditions, and confirmed that Cappable-seq is a valuable approach for global transcriptional regulation analyses. CONCLUSIONS: Our results provide insights and information for further characterization of responses to hypoxia in mycobacteria, and prove that Cappable-seq is a valuable approach for global transcriptional studies in mycobacteria.
Asunto(s)
Mycobacterium marinum , Humanos , Hipoxia/genética , Mycobacterium marinum/genética , Regiones Promotoras Genéticas , Análisis de Secuencia de ARN , Sitio de Iniciación de la TranscripciónRESUMEN
The CpxRA two-component regulatory system and the Rcs phosphorelay system are both employed by the Enterobacteriaceae family to preserve bacterial envelope integrity and function when growing under stress. Although both systems regulate several overlapping physiological processes, evidence demonstrating a molecular connection between Cpx and Rcs signalling outputs is scarce. Here, we show that CpxR negatively regulates the transcription of the rcsB gene in the Rcs phosphorelay system in Yersinia pseudotuberculosis. Interestingly, transcription of rcsB is under the control of three promoters, which were all repressed by CpxR. Critically, synthetic activation of Cpx signalling through mislocalization of the NlpE lipoprotein to the inner membrane resulted in an active form of CpxR that repressed activity of rcsB promoters. On the other hand, a site-directed mutation of the phosphorylation site at residue 51 in CpxR generated an inactive non-phosphorylated variant that was unable to regulate output from these rcsB promoters. Importantly, CpxR-mediated inhibition of rcsB transcription in turn restricted activation of the Ysc-Yop type III secretion system (T3SS). Moreover, active CpxR blocks zinc-mediated activation of Rcs signalling and the subsequent activation of lcrF transcription. Our results demonstrate a novel regulatory cascade linking CpxR-RcsB-LcrF to control production of the Ysc-Yop T3SS.
Asunto(s)
Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Sistemas de Secreción Tipo III/metabolismo , Yersinia pseudotuberculosis/metabolismo , Proteínas Bacterianas/genética , Fosforilación , Regiones Promotoras Genéticas , Sistemas de Secreción Tipo III/genética , Yersinia pseudotuberculosis/genéticaRESUMEN
The (p)ppGpp-mediated stringent response (SR) is a highly conserved regulatory mechanism in bacterial pathogens, enabling adaptation to adverse environments, and is linked to pathogenesis. Actinobacillus pleuropneumoniae can cause damage to the lungs of pigs, its only known natural host. Pig lungs are known to have a low concentration of free branched-chain amino acids (BCAAs) compared to the level in plasma. We had investigated the role for (p)ppGpp in viability and biofilm formation of A. pleuropneumoniae Now, we sought to determine whether (p)ppGpp was a trigger signal for the SR in A. pleuropneumoniae in the absence of BCAAs. Combining transcriptome and phenotypic analyses of the wild type (WT) and an relA spoT double mutant [which does not produce (p)ppGpp], we found that (p)ppGpp could repress de novo purine biosynthesis and activate antioxidant pathways. There was a positive correlation between GTP and endogenous hydrogen peroxide content. Furthermore, the growth, viability, morphology, and virulence were altered by the inability to produce (p)ppGpp. Genes involved in the biosynthesis of BCAAs were constitutively upregulated, regardless of the existence of BCAAs, without accumulation of (p)ppGpp beyond a basal level. Collectively, our study shows that the absence of BCAAs was not a sufficient signal to trigger the SR in A. pleuropneumoniae (p)ppGpp-mediated regulation in A. pleuropneumoniae is different from that described for the model organism Escherichia coli Further work will establish whether the (p)ppGpp-dependent SR mechanism in A. pleuropneumoniae is conserved among other veterinary pathogens, especially those in the Pasteurellaceae family.IMPORTANCE (p)ppGpp is a key player in reprogramming transcriptomes to respond to nutritional challenges. Here, we present transcriptional and phenotypic differences of A. pleuropneumoniae grown in different chemically defined media in the absence of (p)ppGpp. We show that the deprivation of branched-chain amino acids (BCAAs) does not elicit a change in the basal-level (p)ppGpp, but this level is sufficient to regulate the expression of BCAA biosynthesis. The mechanism found in A. pleuropneumoniae is different from that of the model organism Escherichia coli but similar to that found in some Gram-positive bacteria. This study not only broadens the research scope of (p)ppGpp but also further validates the complexity and multiplicity of (p)ppGpp regulation in microorganisms that occupy different biological niches.
Asunto(s)
Actinobacillus pleuropneumoniae/metabolismo , Aminoácidos de Cadena Ramificada/metabolismo , Guanosina Pentafosfato/metabolismo , Guanosina Trifosfato/metabolismo , Actinobacillus pleuropneumoniae/crecimiento & desarrollo , Peróxido de Hidrógeno/metabolismoRESUMEN
The transcriptional activator RbpA associates with Mycobacterium tuberculosis RNA polymerase (MtbRNAP) during transcription initiation, and stimulates formation of the MtbRNAP-promoter open complex (RPo). Here, we explored the influence of promoter motifs on RbpA-mediated activation of MtbRNAP containing the stress-response σB subunit. We show that both the 'extended -10' promoter motif (T-17G-16T-15G-14) and RbpA stabilized RPo and allowed promoter opening at suboptimal temperatures. Furthermore, in the presence of the T-17G-16T-15G-14 motif, RbpA was dispensable for RNA synthesis initiation, while exerting a stabilization effect on RPo. On the other hand, RbpA compensated for the lack of sequence-specific interactions of domains 3 and 4 of σB with the extended -10 and the -35 motifs, respectively. Mutations of the positively charged residues K73, K74 and R79 in RbpA basic linker (BL) had little effect on RPo formation, but affected MtbRNAP capacity for de novo transcription initiation. We propose that RbpA stimulates transcription by strengthening the non-specific interaction of the σ subunit with promoter DNA upstream of the -10 element, and by indirectly optimizing MtbRNAP interaction with initiation substrates. Consequently, RbpA renders MtbRNAP promiscuous in promoter selection, thus compensating for the weak conservation of the -35 motif in mycobacteria.
Asunto(s)
Proteínas Bacterianas/genética , ARN Polimerasas Dirigidas por ADN/genética , Regulación Bacteriana de la Expresión Génica , Mycobacterium tuberculosis/genética , Proteínas de Unión al ARN/genética , Factor sigma/genética , Sustitución de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Sitios de Unión , ADN Bacteriano/química , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , ARN Polimerasas Dirigidas por ADN/química , ARN Polimerasas Dirigidas por ADN/metabolismo , Cinética , Lisina/química , Lisina/metabolismo , Modelos Moleculares , Mutación , Mycobacterium tuberculosis/metabolismo , Motivos de Nucleótidos , Regiones Promotoras Genéticas , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de Proteína , ARN Bacteriano/biosíntesis , ARN Bacteriano/química , ARN Bacteriano/genética , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Factor sigma/química , Factor sigma/metabolismo , Especificidad por Sustrato , Temperatura , Activación TranscripcionalRESUMEN
To facilitate survival under drug stresses, a small population of Mycobacterium tuberculosis can tolerate bactericidal concentrations of drugs without genetic mutations. These drug-tolerant mycobacteria can be induced by environmental stresses and contribute to recalcitrant infections. However, mechanisms underlying the development of drug-tolerant mycobacteria remain obscure. Herein, we characterized a regulatory pathway which is important for the tolerance to isoniazid (INH) in Mycobacterium smegmatis. We found that the RNA polymerase binding protein RbpA associates with the stress response sigma factor σB , to activate the transcription of ppk1, the gene encoding polyphosphate kinase. Subsequently, intracellular levels of inorganic polyphosphate increase to promote INH-tolerant mycobacteria. Interestingly, σB and ppk1 expression varied proportionately in mycobacterial populations and positively correlated with tolerance to INH in individual mycobacteria. Moreover, sigB and ppk1 transcription are both induced upon nutrient depletion, a condition that stimulates the formation of INH-tolerant mycobacteria. Over-expression of ppk1 in rbpA knockdown or sigB deleted strains successfully restored the number of INH-tolerant mycobacteria under both normal growth and nutrient starved conditions. These data suggest that RbpA and σB regulate ppk1 expression to control drug tolerance both during the logarithmic growth phase and under the nutrition starved conditions.
Asunto(s)
Proteínas Bacterianas/metabolismo , Isoniazida/farmacología , Mycobacterium smegmatis/metabolismo , Mycobacterium tuberculosis/metabolismo , Polifosfatos/metabolismo , Factor sigma/metabolismo , Antituberculosos/farmacología , Proteínas Bacterianas/genética , Farmacorresistencia Bacteriana , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Mycobacterium smegmatis/efectos de los fármacos , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/crecimiento & desarrollo , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/crecimiento & desarrollo , Fosfotransferasas (Aceptor del Grupo Fosfato)/genética , Fosfotransferasas (Aceptor del Grupo Fosfato)/metabolismo , Regiones Promotoras Genéticas , Factor sigma/genéticaRESUMEN
During infection, Mycobacterium tuberculosis colonizes macrophages or necrotic granulomas, in which low pH is one of the major challenges. The PhoPR two-component regulatory system and the cytosolic redox sensor WhiB3 both play important roles in the response to low pH by M. tuberculosis However, whether close association exists between PhoPR and WhiB3 remains unclear. In this study, the positive regulation of whiB3 by PhoPR in mycobacteria was characterized. We observed that the expression patterns of the whiB3 gene under acidic conditions are different among mycobacterial species, suggesting that the regulation of whiB3 differs among mycobacteria. A sequence analysis of the whiB3 promoters (whiB3p) from M. tuberculosis and two closely related species, namely, M. marinum and M. smegmatis, showed that the whiB3p regions from M. tuberculosis and M. marinum contain a new type of PhoP box that is absent in the M. smegmatiswhiB3p Direct binding of PhoP to whiB3p from M. tuberculosis and M. marinum but not that from M. smegmatis was validated by in vitro protein-DNA binding assays. The direct activation of whiB3 by PhoPR under acidic conditions was further verified by reverse transcription-quantitative PCR (qRT-PCR) analysis in M. marinum Moreover, mutating the residues important for the phosphorylation pathway of PhoPR in M. marinum abolished the activation of whiB3 expression by PhoPR under acidic conditions, suggesting that low pH triggers the phosphorylation of PhoPR, which in turn activates the transcription of whiB3 Since the PhoP box was only identified in whiB3p of pathogenic mycobacteria, we suggest that the PhoPR-whiB3 regulatory pathway may have evolved to facilitate mycobacterial infection.IMPORTANCE The low pH in macrophages is an important barrier for infection by microbes. The PhoPR two-component regulatory system is required for the response to low pH and plays a role in redox homeostasis in Mycobacterium tuberculosis WhiB3, a cytosolic redox-sensing transcriptional regulator, is also involved in these processes. However, there is no direct evidence to demonstrate the regulation of WhiB3 by PhoPR. In this study, we found that PhoPR directly activates whiB3 expression in response to low pH. An atypical PhoP box in the whiB3 promoters has been identified and is only found in pathogenic mycobacteria, which suggests that the PhoPR-whiB3 regulatory pathway may facilitate mycobacterial infection. This study provides novel information for further characterization of the PhoPR regulon.
Asunto(s)
Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Mycobacterium tuberculosis/genética , Proteínas Bacterianas/genética , Proteínas de Unión al ADN/metabolismo , Concentración de Iones de Hidrógeno , Mutación , Mycobacterium marinum/genética , Mycobacterium marinum/patogenicidad , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/patogenicidad , Mycobacterium tuberculosis/patogenicidad , Fosforilación/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The ω subunit is the smallest subunit of bacterial RNA polymerase (RNAP). Although homologs of ω are essential in both eukaryotes and archaea, this subunit has been known to be dispensable for RNAP in Escherichia coli and in other bacteria. In this study, we characterized an indispensable role of the ω subunit in Mycobacterium tuberculosis Unlike the well-studied E. coli RNAP, the M. tuberculosis RNAP core enzyme cannot be functionally assembled in the absence of the ω subunit. Importantly, substitution of M. tuberculosis ω with ω subunits from E. coli or Thermus thermophilus cannot restore the assembly of M. tuberculosis RNAP. Furthermore, by replacing different regions in M. tuberculosis ω with the corresponding regions from E. coli ω, we found a nonconserved loop region in M. tuberculosis ω essential for its function in RNAP assembly. From RNAP structures, we noticed that the location of the C-terminal region of the ß' subunit (ß'CTD) in M. tuberculosis RNAP but not in E. coli or T. thermophilus RNAP is close to the ω loop region. Deletion of this ß'CTD in M. tuberculosis RNAP destabilized the binding of M. tuberculosis ω on RNAP and compromised M. tuberculosis core assembly, suggesting that these two regions may function together to play a role in ω-dependent RNAP assembly in M. tuberculosis Sequence alignment of the ω loop and the ß'CTD regions suggests that the essential role of ω is probably restricted to mycobacteria. Together, our study characterized an essential role of M. tuberculosis ω and highlighted the importance of the ω loop region in M. tuberculosis RNAP assembly.IMPORTANCE DNA-dependent RNA polymerase (RNAP), which consists of a multisubunit core enzyme (α2ßß'ω) and a dissociable σ subunit, is the only enzyme in charge of transcription in bacteria. As the smallest subunit, the roles of ω remain the least well studied. In Escherichia coli and some other bacteria, the ω subunit is known to be nonessential for RNAP. In this study, we revealed an essential role of the ω subunit for RNAP assembly in the human pathogen Mycobacterium tuberculosis, and a mycobacterium-specific ω loop that plays a role in this function was also characterized. Our study provides fresh insights for further characterizing the roles of bacterial ω subunit.
Asunto(s)
Proteínas Bacterianas/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , Mycobacterium tuberculosis/metabolismo , Factor sigma/química , Factor sigma/metabolismo , Secuencias de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , ARN Polimerasas Dirigidas por ADN/química , ARN Polimerasas Dirigidas por ADN/genética , Escherichia coli/enzimología , Escherichia coli/genética , Escherichia coli/metabolismo , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/enzimología , Mycobacterium tuberculosis/genética , Unión Proteica , Factor sigma/genética , Transcripción GenéticaRESUMEN
We aimed to compare the diagnostic efficiency of proGRP and NSE on SCLC and to investigate whether the change of proGRP level would predict therapeutic response. Patients who were firstly diagnosed pathologically in Nanjing Chest Hospital and measured proGRP level consecutively were enrolled in the study. ProGRP level was detected using Elecsys ProGRP Assay. Totally 75 SCLC, 234 NSCLC and 264 benign lung diseases (BLD) were enrolled. Both proGRP and NSE levels in SCLC were significantly higher than those in NSCLC and BLD, and proGRP in extensive stage SCLC was higher than which in limited stage (P ≤ .001). The diagnostic efficiency of proGRP on SCLC was higher than that of NSE, but when the two biomarkers were bind together, the diagnostic efficiency was the best. When SCLC was differentiated from NSCLC and BLD, the cut-off values were 114.35 pg/mL and 162.55 pg/mL respectively. For treatment responsive patients, proGRP level decreased markedly after the first cycle of therapy and kept a continued momentum of decline during treatment. But for unresponsive patients, no obvious decline was observed. ProGRP had higher diagnostic efficiency on SCLC when compared to NSE, and it could better predict therapeutic response of pulmonary target lesions on chemotherapy.
Asunto(s)
Biomarcadores de Tumor/sangre , Carcinoma de Pulmón de Células no Pequeñas/diagnóstico , Neoplasias Pulmonares/diagnóstico , Neoplasias/diagnóstico , Fragmentos de Péptidos/sangre , Fosfopiruvato Hidratasa/sangre , Carcinoma Pulmonar de Células Pequeñas/diagnóstico , Adulto , Anciano , Antineoplásicos/uso terapéutico , Área Bajo la Curva , Biomarcadores de Tumor/genética , Carcinoma de Pulmón de Células no Pequeñas/sangre , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/patología , China , Diagnóstico Diferencial , Femenino , Estudios de Seguimiento , Humanos , Neoplasias Pulmonares/sangre , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Masculino , Persona de Mediana Edad , Neoplasias/sangre , Neoplasias/patología , Fragmentos de Péptidos/genética , Fosfopiruvato Hidratasa/genética , Proteínas Recombinantes/sangre , Proteínas Recombinantes/genética , Estudios Retrospectivos , Sensibilidad y Especificidad , Carcinoma Pulmonar de Células Pequeñas/sangre , Carcinoma Pulmonar de Células Pequeñas/tratamiento farmacológico , Carcinoma Pulmonar de Células Pequeñas/patologíaRESUMEN
Several studies have shown the tumorigenesis role of transcriptional enhancer associate domain (TEAD) proteins; here, we initially explored expression, function and signalling mechanisms of TEAD4 in lung adenocarcinoma (LAD). Both the mRNA and protein levels of TEAD4 were increased in LAD tissues than those in adjacent nontumourous tissues. Besides, database search indicated a poorer clinical outcome in LAD patients with higher TEAD4 expression, revealing its potential tumour-promoting role. Therefore, we conducted cellular experiments to further investigate its effect on tumour phenotypes. Accordingly, TEAD4 showed little impact on LAD cell cycle, proliferation, or apoptosis. However, silencing TEAD4 remarkably attenuated cell migration and invasion capacities. Consistently, several important epithelial-mesenchymal transition (EMT) markers such as E-cadherin and Slug were consequently altered by silencing TEAD4. Furthermore, we identified a novel TEAD4-targeted microRNA, namely miR6839-3p, and confirmed its function in suppressing TEAD4 expression. Finally, the impact of overexpressing miR6839-3p mimics on LAD progression was validated, which showed a similar pattern with TEAD4 knockdown cells. Taken together, our data not only revealed the significant role of TEAD4 in promoting LAD progression and predicting clinical outcome but also distinguished miR6839-3p mimics as a promising therapeutic direction.
Asunto(s)
Adenocarcinoma del Pulmón/patología , Proteínas de Unión al ADN/genética , Neoplasias Pulmonares/patología , MicroARNs/genética , Proteínas Musculares/genética , Factores de Transcripción/genética , Adenocarcinoma del Pulmón/genética , Adenocarcinoma del Pulmón/mortalidad , Apoptosis/genética , Línea Celular Tumoral , Movimiento Celular/genética , Proteínas de Unión al ADN/metabolismo , Transición Epitelial-Mesenquimal/genética , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Estimación de Kaplan-Meier , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/mortalidad , Proteínas Musculares/metabolismo , Pronóstico , Factores de Transcripción de Dominio TEA , Factores de Transcripción/metabolismoRESUMEN
As a master regulator, the alternative sigma factor RpoS coordinates the transcription of genes associated with protection against environmental stresses in bacteria. In Pseudomonas aeruginosa, RpoS is also involved in quorum sensing and virulence. The cellular RpoS level is regulated at multiple levels, whereas the post-transcriptional regulation of rpoS in P. aeruginosa remains unclear. To identify and characterize small regulatory RNAs (sRNAs) regulating RpoS in P. aeruginosa, an sRNA library expressing a total of 263 sRNAs was constructed to examine their regulatory roles on rpoS expression. Our results demonstrate that rpoS expression is repressed by the RpoS-dependent sRNA RgsA at the post-transcriptional level. Unlike OxyS, an sRNA previously known to repress rpoS expression under oxidative stress in Escherichia coli, RgsA represses rpoS expression during the exponential phase. This repression requires the RNA chaperone Hfq. Furthermore, the 71-77 conserved region of RgsA is necessary for full repression of rpoS expression, and the -25 to +27 region of rpoS mRNA is sufficient for RgsA-mediated rpoS repression. Together, our results not only add RgsA to the RpoS regulatory circuits but also highlight the complexity of interplay between sRNAs and transcriptional regulators in bacteria.
Asunto(s)
Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Pseudomonas aeruginosa/genética , ARN Bacteriano/metabolismo , ARN Pequeño no Traducido/metabolismo , Factor sigma/genética , Regiones no Traducidas 5' , Secuencia Conservada , Regulación hacia Abajo , Biblioteca de Genes , Proteína de Factor 1 del Huésped/metabolismo , Pseudomonas aeruginosa/crecimiento & desarrollo , Procesamiento Postranscripcional del ARN , ARN Bacteriano/química , ARN Bacteriano/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Pequeño no Traducido/química , ARN Pequeño no Traducido/genéticaRESUMEN
OBJECTIVE: We investigated the effect of topotecan on injury and inflammation in a model of ventilator-inducedlunginjury (VILI). METHODS: Acute lung injury (ALI) was induced in mice by high-tidal volume ventilation, and the mice were then treated with topotecan or PBS. Lung tissue and bronchoalveolar lavage fluid were collected to assess pulmonary vascular leaks, inflammation, and cell apoptosis. RESULTS: Compared to PBS treatment, topotecan significantly decreased the ALI score, myeloperoxidase (MPO) content, total protein concentration, and presence of inflammatory cells and inflammatory cytokines in bronchoalveolar lavage fluid. Topotecan also reduced caspase-3 activation and type â ¡ alveolar epithelial cell apoptosis. Moreover, topotecan inhibited NF-κB expression and activation in the VILI model. CONCLUSION: Topotecan alleviates acute lung injury in the model of VILI through the inhibition of the NF-κB pathway.
Asunto(s)
Lesión Pulmonar Aguda/tratamiento farmacológico , Lesión Pulmonar Aguda/metabolismo , FN-kappa B/metabolismo , Topotecan/farmacología , Lesión Pulmonar Inducida por Ventilación Mecánica/tratamiento farmacológico , Lesión Pulmonar Inducida por Ventilación Mecánica/metabolismo , Animales , Apoptosis/efectos de los fármacos , Líquido del Lavado Bronquioalveolar/química , Caspasa 3/metabolismo , Citocinas/metabolismo , Modelos Animales de Enfermedad , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Peroxidasa/metabolismoRESUMEN
Three key promoter elements, i.e., -10, -35, and T-15G-14N, are recognized by the σ subunit of RNA polymerase. Among them, promoters with the -10 element and either -35 or T-15G-14N are known to initiate transcription efficiently, but recent systematic analyses have identified a large group of promoters in Mycobacterium tuberculosis that contain only a -10 consensus. How these promoters initiate transcription remains poorly understood. Here, we show that promoters containing the -10 element and an upstream G located at the -14 or -13 position can successfully initiate transcription in mycobacteria. Importantly, this new type of promoter is active in the absence of other promoter consensuses, suggesting that it is a minimal promoter type. Mutation of the upstream G in promoters decreased the efficiencies of their binding with RNA polymerase and their abilities to initiate transcription in both in vitro and in vivo analyses. A glutamic acid in σ region 3.0 is essential for recognizing G-14 and G-13 and is conserved in both principal and principal-like σ factors in mycobacteria, indicating that recognition of this minimal type of promoter might be a common mechanism for transcription initiation. Consistently, more than 70% of the identified promoters in M. tuberculosis contained G-14 or G-13 upstream of the conserved -10 element, and thousands of promoters in representative mycobacterial species have been predicted using the -10 consensus and G-14 or G-13 Altogether, our study presents a universal mechanism for transcription initiation from a minimal promoter in mycobacteria, which might also be applicable to other bacteria.IMPORTANCE In contrast to the detailed information for recognizing classic promoters in the model organism Escherichia coli, very little is known about how transcription is initiated in the human pathogen Mycobacterium tuberculosis In this study, we characterized a new type of promoter in mycobacteria that requires only a -10 consensus and an upstream G-14 or G-13 Residues important for recognizing the -10 element and the upstream G are conserved in σA and σB from mycobacterial species. According to such features, thousands of promoters in mycobacteria can be predicted using the -10 consensus and G-14 or G-13, which suggests that transcription from this new type of promoter might be widespread. Our findings provide insightful information for characterizing promoters in mycobacteria.
Asunto(s)
ADN Bacteriano/genética , Guanina , Mycobacterium tuberculosis/genética , Regiones Promotoras Genéticas , Transcripción Genética , Análisis Mutacional de ADN , ADN Bacteriano/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , Unión ProteicaRESUMEN
RgsA is a phylogenetically conserved small regulatory RNA (sRNA) in Pseudomonas species. This sRNA has been shown to be directly controlled by the major stationary phase and stress sigma factor σS (RpoS), and also indirectly regulated by the GacS/GacA two-component system. However, the role and the regulatory targets of this sRNA remain unclear. Here, two direct regulatory targets of RgsA, the mRNAs coding for the global transcriptional regulator Fis and the acyl carrier protein AcpP, were identified in P. aeruginosa. RgsA downregulates the synthesis of Fis and AcpP by base-pairing, and this regulation requires the RNA chaperone protein Hfq. Alignment of RgsA homologs in Pseudomonas revealed a conserved core region, which is strictly required for RgsA target recognition. Specifically, RgsA inhibits fis expression via an 11 + 11 bp RNA duplex, whereas this interaction region is not sufficient for repression and the 35 nt downstream region is also required. Interestingly, two functional start codons initiate fis mRNA translation and both are repressed by RgsA. Furthermore, deletion of rgsA significantly increased swarming motility in P. aeruginosa. Together, this study suggests a novel regulatory role of sRNA in which the versatile transcriptional regulator Fis and the stress regulator RpoS are connected by RgsA.
Asunto(s)
Proteínas Bacterianas/metabolismo , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , ARN Pequeño no Traducido/genética , Factor sigma/metabolismo , Proteína Transportadora de Acilo/metabolismo , Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Biosíntesis de Proteínas , ARN Bacteriano/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Pequeño no Traducido/metabolismo , Secuencias Reguladoras de Ácido Ribonucleico , Factor sigma/genéticaRESUMEN
Mycobacterium tuberculosis adopts various strategies to cope with oxidative stress during infection. Transcriptional regulators, including σ factors, make important contributions to this stress response, but how these proteins cooperate with each other is largely unknown. In this study, the role of RbpA and its cooperation with σ factors in response to oxidative stress are investigated. Knock down expression of rbpA in Mycobacterium smegmatis attenuated bacterial survival in the presence of H2 O2 . Additionally, transcription of the rbpA gene was induced by H2 O2 in a σ(E) -dependent manner. After induction, RbpA interacts with the principal sigma factor, σ(A) , to control the transcription of furA-katG operon, which encodes an H2 O2 scavenging enzyme. Moreover, this regulation is responsible for the role of σ(E) in oxidative response because bacterial survival was attenuated and transcription of the furA-katG operon was down-regulated with H2 O2 treatment in sigE deletion mutant (ΔsigE), and over-expression of RbpA in ΔsigE strain restored all of these phenotypes. Taken together, our study first illustrated a mechanism for σ(E) in response to oxidative stress through regulation of rbpA transcription. This study was also the first to demonstrate that RbpA is required for the full response to oxidative stress by cooperating with the principal σ(A) .
Asunto(s)
Proteínas Bacterianas/metabolismo , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/metabolismo , Factor sigma/metabolismo , Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Operón , Estrés Oxidativo/genética , Estrés Oxidativo/fisiología , Regiones Promotoras Genéticas , Factor sigma/genética , Transcripción GenéticaRESUMEN
Although the de novo folate biosynthesis pathway has been well studied in bacteria, little is known about its regulation. In the present study, the sigB gene in Mycobacterium tuberculosis was deleted. Subsequent drug susceptibility tests revealed that the M. tuberculosis ΔsigB strain was more sensitive to para-aminosalicylic acid (PAS) and sulfamethoxazole. Comparative transcriptional analysis was performed, and downregulation of pabB was observed in the ΔsigB strain, which was further verified by a quantitative reverse transcription-PCR and Western blot assay. Then, the production levels of para-aminobenzoic acid (pABA) were compared between the sigB deletion mutant and wild-type strain, and the results showed that sigB deletion resulted in decreased production of pABA. In addition, SigB was able to recognize the promoter of pabB in vitro Furthermore, we found that deleting pabC also caused increased susceptibility to PAS. Taken together, our data revealed that, in M. tuberculosis, sigB affects susceptibility to antifolates through multiple ways, primarily by regulating the expression of pabB To our knowledge, this is the first report showing that SigB modulates pABA biosynthesis and thus affecting susceptibility to antifolates, which broadens our understanding of the regulation of bacterial folate metabolism and mechanisms of susceptibility to antifolates.