RESUMEN
Present in all organisms, DNA ligases catalyse the formation of a phosphodiester bond between a 3' hydroxyl and a 5' phosphate, a reaction that is essential for maintaining genome integrity during replication and repair. Eubacterial DNA ligases use NAD+ as a cofactor and possess low sequence and structural homology relative to eukaryotic DNA ligases which use ATP as a cofactor. These key differences enable specific targeting of bacterial DNA ligases as an antibacterial strategy. In this study, four small molecule accessible sites within functionally important regions of Escherichia coli ligase (EC-LigA) were identified using in silico methods. Molecular docking was then used to screen for small molecules predicted to bind to these sites. Eight candidate inhibitors were then screened for inhibitory activity in an in vitro ligase assay. Five of these (geneticin, chlorhexidine, glutathione (reduced), imidazolidinyl urea and 2-(aminomethyl)imidazole) showed dose-dependent inhibition of EC-LigA with half maximal inhibitory concentrations (IC50) in the micromolar to millimolar range (11-2600 µM). Two (geneticin and chlorhexidine) were predicted to bind to a region of EC-LigA that has not been directly investigated previously, raising the possibility that there may be amino acids within this region that are important for EC-LigA activity or that the function of essential residues proximal to this region are impacted by inhibitor interactions with this region. We anticipate that the identified small molecule binding sites and inhibitors could be pursued as part of an antibacterial strategy targeting bacterial DNA ligases.
Asunto(s)
ADN Ligasas/antagonistas & inhibidores , Inhibidores Enzimáticos/aislamiento & purificación , Escherichia coli/enzimología , Sitios de Unión/efectos de los fármacos , ADN Ligasas/química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Escherichia coli/efectos de los fármacos , Simulación del Acoplamiento MolecularRESUMEN
The increase in antibacterial resistance is a serious challenge for both the health and defence sectors and there is a need for both novel antibacterial targets and antibacterial strategies. RNA degradation and ribonucleases, such as the essential endoribonuclease RNase E, encoded by the rne gene, are emerging as potential antibacterial targets while antisense oligonucleotides may provide alternative antibacterial strategies. As rne mRNA has not been previously targeted using an antisense approach, we decided to explore using antisense oligonucleotides to target the translation initiation region of the Escherichia coli rne mRNA. Antisense oligonucleotides were rationally designed and were synthesised as locked nucleic acid (LNA) gapmers to enable inhibition of rne mRNA translation through two mechanisms. Either LNA gapmer binding could sterically block translation and/or LNA gapmer binding could facilitate RNase H-mediated cleavage of the rne mRNA. This may prove to be an advantage over the majority of previous antibacterial antisense oligonucleotide approaches which used oligonucleotide chemistries that restrict the mode-of-action of the antisense oligonucleotide to steric blocking of translation. Using an electrophoretic mobility shift assay, we demonstrate that the LNA gapmers bind to the translation initiation region of E. coli rne mRNA. We then use a cell-free transcription translation reporter assay to show that this binding is capable of inhibiting translation. Finally, in an in vitro RNase H cleavage assay, the LNA gapmers facilitate RNase H-mediated mRNA cleavage. Although the challenges of antisense oligonucleotide delivery remain to be addressed, overall, this work lays the foundations for the development of a novel antibacterial strategy targeting rne mRNA with antisense oligonucleotides.
Asunto(s)
Antibacterianos/farmacología , Endorribonucleasas/genética , Escherichia coli/enzimología , Oligonucleótidos/farmacología , Sistema Libre de Células , Endorribonucleasas/antagonistas & inhibidores , Escherichia coli/efectos de los fármacos , Proteínas de Escherichia coli/genética , Oligonucleótidos/síntesis química , Iniciación de la Cadena Peptídica Traduccional/efectos de los fármacos , ARN Mensajero/antagonistas & inhibidoresRESUMEN
The discovery and engineering of more and more functions of RNA has highlighted the utility of RNA-targeting small molecules. Recently, several fluorogen-binding RNA aptamers have been developed that have been applied to live cell imaging of RNA and metabolites as RNA tags or biosensors, respectively. Although the design and application of these fluorogen-binding RNA aptamer-based devices is straightforward in theory, in practice, careful optimisation is required. For this reason, high throughput in vitro screening techniques, capable of quantifying fluorogen-RNA aptamer interactions, would be beneficial. We recently developed a method for generating functional-RNA arrays and demonstrated that they could be used to detect fluorogen-RNA aptamer interactions. Specifically, we were able to visualise the interaction between malachite green and the malachite green-binding aptamer. Here we expand this study to demonstrate that functional-RNA arrays can be used to quantify fluorogen-aptamer interactions. As proof-of-concept, we provide detailed protocols for the production of malachite green-binding RNA aptamer and DFHBI-binding Spinach RNA aptamer arrays. Furthermore, we discuss the potential utility of the technology to fluorogen-binding RNA aptamers, including application as a molecular biosensor platform. We anticipate that functional-RNA array technology will be beneficial for a wide variety of biological disciplines.
Asunto(s)
Aptámeros de Nucleótidos/química , Técnicas Biosensibles/métodos , ARN/química , Bibliotecas de Moléculas Pequeñas/farmacología , Colorantes Fluorescentes/química , Humanos , ARN/efectos de los fármacos , Colorantes de Rosanilina/química , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/aislamiento & purificaciónRESUMEN
We are just beginning to unravel the myriad of interactions in which non-coding RNAs participate. The intricate RNA interactome is the foundation of many biological processes, including bacterial virulence and human disease, and represents unexploited resources for the development of potential therapeutic interventions. However, identifying specific associations of a given RNA from the multitude of possible binding partners within the cell requires robust high-throughput systems for their rapid screening. Here, we present the first demonstration of functional-RNA arrays as a novel platform technology designed for the study of such interactions using immobilized, active RNAs. We have generated high-density RNA arrays by an innovative method involving surface-capture of in vitro transcribed RNAs. This approach has significant advantages over existing technologies, particularly in its versatility in regards to binding partner character. Indeed, proof-of-principle application of RNA arrays to both RNA-small molecule and RNA-RNA pairings is demonstrated, highlighting their potential as a platform technology for mapping RNA-based networks and for pharmaceutical screening. Furthermore, the simplicity of the method supports greater user-accessibility over currently available technologies. We anticipate that functional-RNA arrays will find broad utility in the expanding field of RNA characterization.
Asunto(s)
Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , ARN Pequeño no Traducido/análisis , Regiones no Traducidas 5' , Aptámeros de Nucleótidos/análisis , ARN Bacteriano/análisisRESUMEN
Ribonucleases play essential roles in all aspects of RNA metabolism, including the coordination of post-transcriptional gene regulation that allows organisms to respond to internal changes and environmental stimuli. However, as inherently destructive enzymes, their activity must be carefully controlled. Recent research exemplifies the repertoire of regulatory strategies employed by ribonucleases. The activity of the phosphorolytic exoribonuclease, polynucleotide phosphorylase (PNPase), has previously been shown to be modulated by the Krebs cycle metabolite citrate in Escherichia coli. Here, we provide evidence for the existence of citrate-mediated inhibition of ribonucleases in all three domains of life. In silico molecular docking studies predict that citrate will bind not only to bacterial PNPases from E. coli and Streptomyces antibioticus, but also PNPase from human mitochondria and the structurally and functionally related archaeal exosome complex from Sulfolobus solfataricus. Critically, we show experimentally that citrate also inhibits the exoribonuclease activity of bacterial, eukaryotic and archaeal PNPase homologues in vitro. Furthermore, bioinformatics data, showing key citrate-binding motifs conserved across a broad range of PNPase homologues, suggests that this regulatory mechanism may be widespread. Overall, our data highlight a communicative link between ribonuclease activity and central metabolism that may have been conserved through the course of evolution.
Asunto(s)
Ácido Cítrico/química , Escherichia coli/enzimología , Polirribonucleótido Nucleotidiltransferasa/química , ARN/química , Streptomyces antibioticus/enzimología , Sulfolobus solfataricus/enzimología , Secuencia de Aminoácidos , Sitios de Unión , Evolución Biológica , Ácido Cítrico/metabolismo , Clonación Molecular , Biología Computacional , Secuencia Conservada , Escherichia coli/genética , Exosomas/química , Exosomas/enzimología , Expresión Génica , Humanos , Cinética , Mitocondrias/química , Mitocondrias/enzimología , Simulación del Acoplamiento Molecular , Polirribonucleótido Nucleotidiltransferasa/genética , Polirribonucleótido Nucleotidiltransferasa/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de Proteína , ARN/metabolismo , Estabilidad del ARN/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Streptomyces antibioticus/genética , Homología Estructural de Proteína , Especificidad por Sustrato , Sulfolobus solfataricus/genética , TermodinámicaRESUMEN
OxyS and RprA are two small noncoding RNAs (sRNAs) that modulate the expression of rpoS, encoding an alternative sigma factor that activates transcription of multiple Escherichia coli stress-response genes. While RprA activates rpoS for translation, OxyS down-regulates the transcript. Crucially, the RNA binding protein Hfq is required for both sRNAs to function, although the specific role played by Hfq remains unclear. We have investigated RprA and OxyS interactions with Hfq using biochemical and biophysical approaches. In particular, we have obtained the molecular envelopes of the Hfq-sRNA complexes using small-angle scattering methods, which reveal key molecular details. These data indicate that Hfq does not substantially change shape upon complex formation, whereas the sRNAs do. We link the impact of Hfq binding, and the sRNA structural changes induced, to transcript stability with respect to RNase E degradation. In light of these findings, we discuss the role of Hfq in the opposing regulatory functions played by RprA and OxyS in rpoS regulation.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteína de Factor 1 del Huésped/metabolismo , ARN Bacteriano/metabolismo , ARN Pequeño no Traducido/metabolismo , Proteínas Represoras/metabolismo , Factor sigma/metabolismo , Proteínas Bacterianas/genética , Secuencia de Bases , Sitios de Unión , Fenómenos Biofísicos , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Proteína de Factor 1 del Huésped/química , Proteína de Factor 1 del Huésped/genética , Modelos Moleculares , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Estructura Cuaternaria de Proteína , Estabilidad del ARN , ARN Bacteriano/química , ARN Bacteriano/genética , ARN Pequeño no Traducido/química , ARN Pequeño no Traducido/genética , Proteínas Represoras/genética , Dispersión del Ángulo Pequeño , Factor sigma/genéticaRESUMEN
MicA is a small non-coding RNA that regulates ompA mRNA translation in Escherichia coli. MicA has an inhibitory function, base pairing to the translation initiation region of target mRNAs through short sequences of complementarity, blocking their ribosome-binding sites. The MicA structure contains two stem loops, which impede its interaction with target mRNAs, and it is thought that the RNA chaperone protein Hfq, known to be involved in MicA regulation of ompA, may structurally remodel MicA to reveal the ompA-binding site for cognate pairing. To further characterize these interactions, we undertook biochemical and biophysical studies using native MicA and a 'stabilized' version, modified to mimic the conformational state of MicA where the ompA-binding site is exposed. Our data corroborate two proposed roles for Hfq: first, to bring both MicA and ompA into close proximity, and second, to restructure MicA to allow exposure of the ompA-binding site for pairing, thereby demonstrating the RNA chaperone function of Hfq. Additionally, at accumulated MicA levels, we identified a Mg(2+)-dependent self-association that occludes the ompA-recognition region. We discuss the potential contribution of an Mg(2+)-mediated conformational switch of MicA for the regulation of MicA function.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/genética , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , ARN Pequeño no Traducido/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Secuencia de Bases , Sitios de Unión , Ensayo de Cambio de Movilidad Electroforética , Proteínas de Escherichia coli/química , Proteína de Factor 1 del Huésped/química , Secuencias Invertidas Repetidas , Magnesio/química , Modelos Moleculares , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Unión Proteica , ARN Pequeño no Traducido/químicaRESUMEN
In Vibrio cholerae, the RNA binding protein and chaperone Hfq (VcHfq) facilitates the pairing of the quorum regulatory RNA (Qrr) small regulatory RNAs (sRNAs) to the 5' untranslated regions of the mRNAs for a number of global regulators that modulate the expression of virulence genes. This Qrr-mediated sRNA circuit is an attractive antimicrobial target, but characterization at the molecular level is required for this to be realized. Here, we investigate the interactions between VcHfq and the Qrr sRNAs using a variety of biochemical and biophysical techniques. We show that the ring-shaped VcHfq hexamer binds the Qrrs with 1:1 stoichiometry through its proximal face, and the molecular envelope of the VcHfq-Qrr complex is experimentally determined from small angle scattering data to present the first structural glimpse of a Hfq-sRNA complex. This structure reveals that the VcHfq protein does not change shape on complex formation but the RNA does, suggesting that a chaperone role for VcHfq is a critical part of the VcHfq-Qrr interaction. Overall, these studies enhance our understanding of VcHfq-Qrr interactions.
Asunto(s)
Proteína de Factor 1 del Huésped/química , ARN Pequeño no Traducido/química , Vibrio cholerae , Sitios de Unión , Proteína de Factor 1 del Huésped/metabolismo , Modelos Moleculares , Conformación de Ácido Nucleico , Conformación Proteica , ARN Pequeño no Traducido/metabolismo , Dispersión del Ángulo PequeñoRESUMEN
The RNA degradosome is a multi-enzyme assembly that contributes to key processes of RNA metabolism, and it engages numerous partners in serving its varied functional roles. Small domains within the assembly recognize collectively a diverse range of macromolecules, including the core protein components, the cytoplasmic lipid membrane, mRNAs, non-coding regulatory RNAs and precursors of structured RNAs. We present evidence that the degradosome can form a stable complex with the 70S ribosome and polysomes, and we demonstrate the proximity in vivo of ribosomal proteins and the scaffold of the degradosome, RNase E. The principal interactions are mapped to two, independent, RNA-binding domains from RNase E. RhlB, the RNA helicase component of the degradosome, also contributes to ribosome binding, and this is favoured through an activating interaction with RNase E. The catalytic activity of RNase E for processing 9S RNA (the ribosomal 5S RNA precursor) is repressed in the presence of the ribosome, whereas there is little affect on the cleavage of single-stranded substrates mediated by non-coding RNA, suggestings that the enzyme retains capacity to cleave unstructured substrates when associated with the ribosome. We propose that polysomes may act as antennae that enhance the rates of capture of the limited number of degradosomes, so that they become recruited to sites of active translation to act on mRNAs as they become exposed or tagged for degradation.
Asunto(s)
Endorribonucleasas/metabolismo , Escherichia coli/enzimología , Complejos Multienzimáticos/metabolismo , Polirribonucleótido Nucleotidiltransferasa/metabolismo , Polirribosomas/metabolismo , ARN Helicasas/metabolismo , Ribosomas/metabolismo , Sitios de Unión , Reactivos de Enlaces Cruzados , ARN Helicasas DEAD-box/metabolismo , Endorribonucleasas/química , Endorribonucleasas/aislamiento & purificación , Proteínas de Escherichia coli/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Dominios y Motivos de Interacción de Proteínas , ARN/metabolismo , Procesamiento Postranscripcional del ARN , ARN Pequeño no Traducido/metabolismo , Proteínas Ribosómicas/metabolismo , Subunidades Ribosómicas Grandes Bacterianas/metabolismo , Subunidades Ribosómicas Pequeñas Bacterianas/metabolismoRESUMEN
RNA performs a wide variety of vital cellular functions. These functions typically require interactions with other biological macromolecules, often as part of an intricate communication network. High-throughput techniques capable of analyzing RNA-based interactions are therefore essential. Functional-RNA arrays address this need, providing the capability of performing hundreds of miniature assays in parallel. Here we describe a method to generate functional-RNA arrays using in vitro transcription of a DNA template array and in situ RNA capture. We also suggest how functional-RNA arrays could be applied to investigating RNA-RNA interactions.
Asunto(s)
ARN no Traducido , ARN , ARN/genética , Bioensayo , Replicación del ADNRESUMEN
RNA turnover is an essential element of cellular homeostasis and response to environmental change. Whether the ribonucleases that mediate RNA turnover can respond to cellular metabolic status is an unresolved question. Here we present evidence that the Krebs cycle metabolite citrate affects the activity of Escherichia coli polynucleotide phosphorylase (PNPase) and, conversely, that cellular metabolism is affected widely by PNPase activity. An E. coli strain that requires PNPase for viability has suppressed growth in the presence of increased citrate concentration. Transcriptome analysis reveals a PNPase-mediated response to citrate, and PNPase deletion broadly impacts on the metabolome. In vitro, citrate directly binds and modulates PNPase activity, as predicted by crystallographic data. Binding of metal-chelated citrate in the active site at physiological concentrations appears to inhibit enzyme activity. However, metal-free citrate is bound at a vestigial active site, where it stimulates PNPase activity. Mutagenesis data confirmed a potential role of this vestigial site as an allosteric binding pocket that recognizes metal-free citrate. Collectively, these findings suggest that RNA degradative pathways communicate with central metabolism. This communication appears to be part of a feedback network that may contribute to global regulation of metabolism and cellular energy efficiency.
Asunto(s)
Escherichia coli/enzimología , Regulación Enzimológica de la Expresión Génica , Polirribonucleótido Nucleotidiltransferasa/metabolismo , Sitio Alostérico , Ácido Cítrico/química , Clonación Molecular , Cristalografía por Rayos X/métodos , Eliminación de Gen , Metabolómica/métodos , Metales/química , Modelos Químicos , Mutagénesis , Análisis de Secuencia por Matrices de Oligonucleótidos , Polímeros/química , Unión ProteicaRESUMEN
The Escherichia coli endoribonuclease RNase E is an essential enzyme having key roles in mRNA turnover and the processing of several structured RNA precursors, and it provides the scaffold to assemble the multienzyme RNA degradosome. The activity of RNase E is inhibited by the protein RraA, which can interact with the ribonuclease's degradosome-scaffolding domain. Here, we report that RraA can bind to the RNA helicase component of the degradosome (RhlB) and the two RNA-binding sites in the degradosome-scaffolding domain of RNase E. In the presence of ATP, the helicase can facilitate the exchange of RraA for RNA stably bound to the degradosome. Our data suggest that RraA can affect multiple components of the RNA degradosome in a dynamic, energy-dependent equilibrium. The multidentate interactions of RraA impede the RNA-binding and ribonuclease activities of the degradosome and may result in complex modulation and rerouting of degradosome activity.
Asunto(s)
Endorribonucleasas/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Complejos Multienzimáticos/metabolismo , Polirribonucleótido Nucleotidiltransferasa/metabolismo , ARN Helicasas/metabolismo , Adenosina Trifosfato/metabolismo , ARN Helicasas DEAD-box/metabolismoRESUMEN
The RNA chaperone Hfq promotes the association of small RNAs (sRNAs) with cognate mRNAs, controlling the expression of bacterial phenotype. Actinobacillus pleuropneumoniae hfq mutants strains are attenuated for virulence in pigs, impaired in the ability to form biofilms, and more susceptible to stress, but knowledge of the extent of sRNA involvement is limited. Here, using A. pleuropneumoniae strain MIDG2331 (serovar 8), 14 sRNAs were identified by co-immunoprecipitation with Hfq and the expression of eight, identified as trans-acting sRNAs, were confirmed by Northern blotting. We focused on one of these sRNAs, named Rna01, containing a putative promoter for RpoE (stress regulon) recognition. Knockout mutants of rna01 and a double knockout mutant of rna01 and hfq, both had decreased biofilm formation and hemolytic activity, attenuation for virulence in Galleria mellonella, altered stress susceptibility, and an altered outer membrane protein profile. Rna01 affected extracellular vesicle production, size and toxicity in G. mellonella. qRT-PCR analysis of rna01 and putative cognate mRNA targets indicated that Rna01 is associated with the extracytoplasmic stress response. This work increases our understanding of the multilayered and complex nature of the influence of Hfq-dependent sRNAs on the physiology and virulence of A. pleuropneumoniae.
RESUMEN
The coordinated regulation of gene expression is required for homeostasis, growth and development in all organisms. Such coordination may be partly achieved at the level of messenger RNA stability, in which the targeted destruction of subsets of transcripts generates the potential for cross-regulating metabolic pathways. In Escherichia coli, the balance and composition of the transcript population is affected by RNase E, an essential endoribonuclease that not only turns over RNA but also processes certain key RNA precursors. RNase E cleaves RNA internally, but its catalytic power is determined by the 5' terminus of the substrate, even if this lies at a distance from the cutting site. Here we report crystal structures of the catalytic domain of RNase E as trapped allosteric intermediates with RNA substrates. Four subunits of RNase E catalytic domain associate into an interwoven quaternary structure, explaining why the subunit organization is required for catalytic activity. The subdomain encompassing the active site is structurally congruent to a deoxyribonuclease, making an unexpected link in the evolutionary history of RNA and DNA nucleases. The structure explains how the recognition of the 5' terminus of the substrate may trigger catalysis and also sheds light on the question of how RNase E might selectively process, rather than destroy, specific RNA precursors.
Asunto(s)
Dominio Catalítico , Endorribonucleasas/química , Endorribonucleasas/metabolismo , Escherichia coli/enzimología , Estabilidad del ARN , ARN/metabolismo , Regulación Alostérica , Sitios de Unión , Catálisis , Dominio Catalítico/genética , Endorribonucleasas/genética , Escherichia coli/genética , Modelos Moleculares , Conformación de Ácido Nucleico , Conformación Proteica , ARN/químicaRESUMEN
Regulatory RNA-based interactions are critical for coordinating gene expression and are increasingly being targeted in synthetic biology, antimicrobial, and therapeutic fields. Bacterial trans-encoded small RNAs (sRNAs) regulate the translation and/or stability of mRNA targets through base-pairing interactions. These interactions are often integral to complex gene circuits which coordinate critical bacterial processes. The ability to predictably modulate these gene circuits has potential for reprogramming gene expression for synthetic biology and antibacterial purposes. Here, we present a novel pipeline for targeting such RNA-based interactions with antisense oligonucleotides (ASOs) in order to reprogram gene expression. As proof-of-concept, we selected sRNA-mRNA interactions that are central to the Vibrio cholerae quorum sensing pathway, required for V. cholerae pathogenesis, as a regulatory RNA-based interaction input. We rationally designed anti-sRNA ASOs to target the sRNAs and synthesized them as peptide nucleic acids (PNAs). Next, we devised an RNA array-based interaction assay to allow screening of the anti-sRNA ASOs in vitro. Finally, an Escherichia coli-based gene expression reporter assay was developed and used to validate anti-sRNA ASO regulatory activity in a cellular environment. The output from the pipeline was an anti-sRNA ASO that targets sRNAs to inhibit sRNA-mRNA interactions and modulate gene expression. This anti-sRNA ASO has potential for reprogramming gene expression for synthetic biology and/or antibacterial purposes. We anticipate that this pipeline will find widespread use in fields targeting RNA-based interactions as modulators of gene expression.
Asunto(s)
Regulación Bacteriana de la Expresión Génica , Oligodesoxirribonucleótidos Antisentido/química , Ácidos Nucleicos de Péptidos/química , ARN Bacteriano/biosíntesis , Vibrio cholerae , ARN Bacteriano/genética , Vibrio cholerae/genética , Vibrio cholerae/metabolismoRESUMEN
RNase E is an essential bacterial endoribonuclease involved in the turnover of messenger RNA and the maturation of structured RNA precursors in Escherichia coli. Here, we present the crystal structure of the E. coli RNase E catalytic domain in the apo-state at 3.3 A. This structure indicates that, upon catalytic activation, RNase E undergoes a marked conformational change characterized by the coupled movement of two RNA-binding domains to organize the active site. The structural data suggest a mechanism of RNA recognition and cleavage that explains the enzyme's preference for substrates possessing a 5'-monophosphate and accounts for the protective effect of a triphosphate cap for most transcripts. Internal flexibility within the quaternary structure is also observed, a finding that has implications for recognition of structured RNA substrates and for the mechanism of internal entry for a subset of substrates that are cleaved without 5'-end requirements.
Asunto(s)
Apoproteínas/química , Endorribonucleasas/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimología , Estructura Cuaternaria de Proteína , Estabilidad del ARN , ARN/metabolismo , Secuencia de Aminoácidos , Apoproteínas/genética , Apoproteínas/metabolismo , Cristalografía por Rayos X , Endorribonucleasas/genética , Endorribonucleasas/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Datos de Secuencia Molecular , ARN/química , Especificidad por SustratoRESUMEN
Increasing resistance of bacteria to antibiotics is a serious global challenge and there is a need to unlock the potential of novel antibacterial targets. One such target is the essential prokaryotic endoribonuclease RNase E. Using a combination of in silico high-throughput screening and in vitro validation we have identified three novel small molecule inhibitors of RNase E that are active against RNase E from Escherichia coli, Francisella tularensis and Acinetobacter baumannii. Two of the inhibitors are non-natural small molecules that could be suitable as lead compounds for the development of broad-spectrum antibiotics targeting RNase E. The third small molecule inhibitor is glucosamine-6-phosphate, a precursor of bacterial cell envelope peptidoglycans and lipopolysaccharides, hinting at a novel metabolite-mediated mechanism of regulation of RNase E.
RESUMEN
The development of programmable regulators that precisely and predictably control gene expression is a major goal of synthetic biology. Consequently, rapid high-throughput biochemical methods capable of quantitatively analyzing all components of gene expression would be of value in the characterization and optimization of regulator performance. In this study we demonstrate a novel application of RNA arrays, involving the production of reporter-protein arrays, to gene expression analysis. This method enables simultaneous quantification of both the transcription and post-transcription/translation components of gene expression, and it also allows the assessment of the orthogonality of multiple regulators. We use our method to directly compare the performance of a series of previously characterized synthetic post-transcriptional riboregulators, thus demonstrating its utility in the development of synthetic regulatory modules and evaluation of gene expression regulation in general.
Asunto(s)
Hibridación de Ácido Nucleico/métodos , Análisis por Matrices de Proteínas/métodos , Conformación de Ácido Nucleico , ARN Mensajero/metabolismo , Biología SintéticaRESUMEN
Regulation of gene expression through processing and turnover of RNA is a key mechanism that allows bacteria to rapidly adapt to changing environmental conditions. Consequently, RNA degrading enzymes (ribonucleases; RNases) such as the endoribonuclease RNase E, frequently play critical roles in pathogenic bacterial virulence and are potential antibacterial targets. RNase E consists of a highly conserved catalytic domain and a variable non-catalytic domain that functions as the structural scaffold for the multienzyme degradosome complex. Despite conservation of the catalytic domain, a recent study identified differences in the response of RNase E homologues from different species to the same inhibitory compound(s). While RNase E from Escherichia coli has been well-characterised, far less is known about RNase E homologues from other bacterial species. In this study, we structurally and biochemically characterise the RNase E catalytic domains from four pathogenic bacteria: Yersinia pestis, Francisella tularensis, Burkholderia pseudomallei and Acinetobacter baumannii, with a view to exploiting RNase E as an antibacterial target. Bioinformatics, small-angle x-ray scattering and biochemical RNA cleavage assays reveal globally similar structural and catalytic properties. Surprisingly, subtle species-specific differences in both structure and substrate specificity were also identified that may be important for the development of effective antibacterial drugs targeting RNase E.
Asunto(s)
Acinetobacter baumannii/enzimología , Proteínas Bacterianas/química , Burkholderia pseudomallei/enzimología , Endorribonucleasas/química , Francisella tularensis/enzimología , Yersinia pestis/enzimología , Acinetobacter baumannii/genética , Acinetobacter baumannii/patogenicidad , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Burkholderia pseudomallei/genética , Burkholderia pseudomallei/patogenicidad , Dominio Catalítico , Clonación Molecular , Endorribonucleasas/genética , Endorribonucleasas/metabolismo , Escherichia coli/enzimología , Escherichia coli/genética , Escherichia coli/patogenicidad , Francisella tularensis/genética , Francisella tularensis/patogenicidad , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Cinética , Modelos Moleculares , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , ARN/química , ARN/genética , ARN/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología Estructural de Proteína , Especificidad por Sustrato , Virulencia , Yersinia pestis/genética , Yersinia pestis/patogenicidadRESUMEN
The hydrolytic endoribonuclease RNase E, which is widely distributed in bacteria and plants, plays key roles in mRNA degradation and RNA processing in Escherichia coli. The enzymatic activity of RNase E is contained within the conserved amino-terminal half of the 118 kDa protein, and the carboxy-terminal half organizes the RNA degradosome, a multi-enzyme complex that degrades mRNA co-operatively and processes ribosomal and other RNA. The study described herein demonstrates that the carboxy-terminal domain of RNase E has little structure under native conditions and is unlikely to be extensively folded within the degradosome. However, three isolated segments of 10-40 residues, and a larger fourth segment of 80 residues, are predicted to be regions of increased structural propensity. The larger of these segments appears to be a protein-RNA interaction site while the other segments possibly correspond to sites of self-recognition and interaction with the other degradosome proteins. The carboxy-terminal domain of RNase E may thus act as a flexible tether of the degradosome components. The implications of these and other observations for the organization of the RNA degradosome are discussed.