RESUMEN
Small RNAs (sRNAs) and riboswitches represent distinct classes of RNA regulators that control gene expression upon sensing metabolic or environmental variations. While sRNAs and riboswitches regulate gene expression by affecting mRNA and protein levels, existing studies have been limited to the characterization of each regulatory system in isolation, suggesting that sRNAs and riboswitches target distinct mRNA populations. We report that the expression of btuB in Escherichia coli, which is regulated by an adenosylcobalamin (AdoCbl) riboswitch, is also controlled by the small RNAs OmrA and, to a lesser extent, OmrB. Strikingly, we find that the riboswitch and sRNAs reduce mRNA levels through distinct pathways. Our data show that while the riboswitch triggers Rho-dependent transcription termination, sRNAs rely on the degradosome to modulate mRNA levels. Importantly, OmrA pairs with the btuB mRNA through its central region, which is not conserved in OmrB, indicating that these two sRNAs may have specific targets in addition to their common regulon. In contrast to canonical sRNA regulation, we find that OmrA repression of btuB is lost using an mRNA binding-deficient Hfq variant. Together, our study demonstrates that riboswitch and sRNAs modulate btuB expression, providing an example of cis- and trans-acting RNA-based regulatory systems maintaining cellular homeostasis.
Asunto(s)
Cobamidas , Proteínas de Escherichia coli , Escherichia coli , Regulación Bacteriana de la Expresión Génica , ARN Bacteriano , ARN Mensajero , Riboswitch , Riboswitch/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , ARN Mensajero/metabolismo , ARN Mensajero/genética , Cobamidas/metabolismo , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , ARN Pequeño no Traducido/genética , ARN Pequeño no Traducido/metabolismo , Iniciación de la Cadena Peptídica Traduccional , ARN Helicasas/genética , ARN Helicasas/metabolismo , Endorribonucleasas/metabolismo , Endorribonucleasas/genética , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/metabolismo , Proteínas de la Membrana Bacteriana Externa , Polirribonucleótido Nucleotidiltransferasa , Proteínas de Transporte de MembranaRESUMEN
Cotranscriptional RNA folding is crucial for the timely control of biological processes, but because of its transient nature, its study has remained challenging. While single-molecule Förster resonance energy transfer (smFRET) is unique to investigate transient RNA structures, its application to cotranscriptional studies has been limited to nonnative systems lacking RNA polymerase (RNAP)-dependent features, which are crucial for gene regulation. Here, we present an approach that enables site-specific labeling and smFRET studies of kilobase-length transcripts within native bacterial complexes. By monitoring Escherichia coli nascent riboswitches, we reveal an inverse relationship between elongation speed and metabolite-sensing efficiency and show that pause sites upstream of the translation start codon delimit a sequence hotspot for metabolite sensing during transcription. Furthermore, we demonstrate a crucial role of the bacterial RNAP actively delaying the formation, within the hotspot sequence, of competing structures precluding metabolite binding. Our approach allows the investigation of cotranscriptional regulatory mechanisms in bacterial and eukaryotic elongation complexes.
Asunto(s)
Proteínas de Escherichia coli/metabolismo , Riboswitch/fisiología , Imagen Individual de Molécula/métodos , Elongación de la Transcripción Genética , Carbocianinas , Escherichia coli , Proteínas de Escherichia coli/análisis , Transferencia Resonante de Energía de Fluorescencia , Colorantes FluorescentesRESUMEN
Clostridioides difficile is the main cause of nosocomial antibiotic-associated diarrhoea. There is a need for new antimicrobials to tackle this pathogen. Guanine riboswitches have been proposed as promising new antimicrobial targets, but experimental evidence of their importance in C. difficile is missing. The genome of C. difficile encodes four distinct guanine riboswitches, each controlling a single gene involved in purine metabolism and transport. One of them controls the expression of guaA, encoding a guanosine monophosphate (GMP) synthase. Here, using in-line probing and GusA reporter assays, we show that these riboswitches are functional in C. difficile and cause premature transcription termination upon binding of guanine. All riboswitches exhibit a high affinity for guanine characterized by Kd values in the low nanomolar range. Xanthine and guanosine also bind the guanine riboswitches, although with less affinity. Inactivating the GMP synthase (guaA) in C. difficile strain 630 led to cell death in minimal growth conditions, but not in rich medium. Importantly, the capacity of a guaA mutant to colonize the mouse gut was significantly reduced. Together, these results demonstrate the importance of de novo GMP biosynthesis in C. difficile during infection, suggesting that targeting guanine riboswitches with analogues could be a viable therapeutic strategy.
Asunto(s)
Ligasas de Carbono-Nitrógeno/genética , Clostridioides difficile/fisiología , Infecciones por Clostridium/microbiología , Regulación Bacteriana de la Expresión Génica , Riboswitch , Animales , Ligasas de Carbono-Nitrógeno/metabolismo , Genoma Bacteriano , Genómica/métodos , Guanina , Ratones , Viabilidad Microbiana/genética , Mutación , Transcripción Genética , Virulencia/genéticaRESUMEN
Transcriptional pauses have been reported in bacterial riboswitches and, in some cases, their specific positioning has been shown to be important for gene regulation. Here, we show that a hairpin structure in the Escherichia coli thiamin pyrophosphate (TPP) thiC riboswitch is involved in transcriptional pausing and ligand sensitivity. Using in vitro transcription kinetic experiments, we show that all three major transcriptional pauses in the thiC riboswitch are affected by NusA, a transcriptional factor known to stimulate hairpin-stabilized pauses. Using a truncated region of the riboswitch, we isolated the hairpin structure responsible for stabilization of the most upstream pause. Destabilization of this structure led to a weaker pause and a decreased NusA effect. In the context of the full-length riboswitch, this same mutation also led to a weaker pause, as well as a decreased TPP binding affinity. Our work suggests that RNA structures involved in transcriptional pausing in riboswitches are important for ligand sensitivity, most likely by increasing the time allowed to the ligand for binding to the riboswitch.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas de Escherichia coli/genética , Riboswitch/genética , Transcripción Genética , Factores de Elongación Transcripcional/genética , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica/genética , Conformación de Ácido Nucleico , Tiamina Pirofosfato/genética , Factores de Transcripción/genéticaRESUMEN
Riboswitches are regulatory elements that control gene expression by altering RNA structure upon the binding of specific metabolites. Although Bacillus subtilis riboswitches have been shown to control premature transcription termination, less is known about regulatory mechanisms employed by Escherichia coli riboswitches, which are predicted to regulate mostly at the level of translation initiation. Here, we present experimental evidence suggesting that the majority of known E. coli riboswitches control transcription termination by using the Rho transcription factor. In the case of the thiamin pyrophosphate-dependent thiM riboswitch, we find that Rho-dependent transcription termination is triggered as a consequence of translation repression. Using in vitro and in vivo assays, we show that the Rho-mediated regulation relies on RNA target elements located at the beginning of thiM coding region. Gene reporter assays indicate that relocating Rho target elements to a different gene induces transcription termination, demonstrating that such elements are modular domains controlling Rho. Our work provides strong evidence that translationally regulating riboswitches also regulate mRNA levels through an indirect control mechanism ensuring tight control of gene expression.
Asunto(s)
Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Biosíntesis de Proteínas , Factor Rho/genética , Riboswitch , Terminación de la Transcripción Genética , Secuencia de Bases , Escherichia coli/metabolismo , Genes Reporteros , Conformación de Ácido Nucleico , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Factor Rho/metabolismo , Tiamina Pirofosfato/metabolismoRESUMEN
Riboswitches regulate gene expression by rearranging their structure upon metabolite binding. The lysine-sensing lysC riboswitch is a rare example of an RNA aptamer organized around a 5-way helical junction in which ligand binding is performed exclusively through nucleotides located at the junction core. We have probed whether the nucleotides involved in ligand binding play any role in the global folding of the riboswitch. As predicted, our findings indicate that ligand-binding residues are critical for the lysine-dependent gene regulation mechanism. We also find that these residues are not important for the establishment of key magnesium-dependent tertiary interactions, suggesting that folding and ligand recognition are uncoupled in this riboswitch for the formation of specific interactions. However, FRET assays show that lysine binding results in an additional conformational change, indicating that lysine binding may also participate in a specific folding transition. Thus, in contrast to helical junctions being primary determinants in ribozymes and rRNA folding, we speculate that the helical junction of the lysine-sensing lysC riboswitch is not employed as structural a scaffold to direct global folding, but rather has a different role in establishing RNA-ligand interactions required for riboswitch regulation. Our work suggests that helical junctions may adopt different functions such as the coordination of global architecture or the formation of specific ligand binding site.
Asunto(s)
Lisina/metabolismo , Conformación de Ácido Nucleico , Pliegue del ARN , Riboswitch/genética , Aptámeros de Nucleótidos/metabolismo , Secuencia de Bases , Transferencia Resonante de Energía de Fluorescencia , Iones , Lisina/farmacología , Magnesio/farmacología , Datos de Secuencia Molecular , Mutación/genética , Pliegue del ARN/efectos de los fármacos , Terminación de la Transcripción Genética/efectos de los fármacosRESUMEN
Noroviruses and sapoviruses are members of the family Caliciviridae and emerging enteric pathogens of humans and animals. Since their discovery and characterization in swine, relatively few strains have been described in detail. In order to investigate their genetic diversity, a total of 266 fecal samples collected in the province of Quebec, Canada, between 2005 and 2007 were screened for the presence of caliciviruses by RT-PCR using broadly reactive primers. Genetically heterogeneous caliciviruses were detected on the majority of farms. Typical noroviruses related to known swine genotypes were present on 20% of the farms. Sapoviruses were detected on 75% of the farms and were the most heterogeneous group. Further characterization of selected strains in their 3' end parts was carried out for their classification and unveiled possibly new clusters of sapoviruses. No human-like noroviruses or sapoviruses were detected in the present study.
Asunto(s)
Infecciones por Caliciviridae/veterinaria , Variación Genética , Norovirus/clasificación , Norovirus/genética , Sapovirus/clasificación , Sapovirus/genética , Enfermedades de los Porcinos/virología , Animales , Infecciones por Caliciviridae/virología , Análisis por Conglomerados , Heces/virología , Genotipo , Datos de Secuencia Molecular , Norovirus/aislamiento & purificación , Filogenia , Quebec , ARN Viral/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Sapovirus/aislamiento & purificación , Análisis de Secuencia de ADN , Homología de Secuencia , PorcinosRESUMEN
This study reports the molecular characterization of novel caliciviruses, the St-Valérien-like viruses, which were isolated from pig feces in the province of Quebec, Canada between 2005 and 2007. The genomes of St-Valérien-like viruses contain 6409 nucleotides and include two main open reading frames (ORFs). ORF1 encodes the non structural (NS) polyprotein and the major capsid protein (VP1) while ORF2 encodes the putative basic minor capsid protein. Typical conserved amino acid motifs predict a gene order reminiscent of calicivirus genomes. Phylogenetic, pairwise homology, and distance analyses performed on complete genomic sequences and partial amino acid sequences from the NTPase, polymerase, and major capsid protein segregated the St-Valérien-like viruses in a unique cluster sharing a common root with the Tulane virus and the noroviruses. Based on the genomic analyses presented, the St-Valérien-like viruses are members of a new genus of Caliciviridae for which we propose the name Valovirus.
Asunto(s)
Infecciones por Caliciviridae/veterinaria , Caliciviridae/clasificación , Caliciviridae/genética , Enfermedades de los Porcinos/virología , Animales , Caliciviridae/aislamiento & purificación , Infecciones por Caliciviridae/virología , Análisis por Conglomerados , Genoma Viral , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Filogenia , Quebec , ARN Viral/genética , Análisis de Secuencia de ADN , Homología de Secuencia , Porcinos , Proteínas Virales/genéticaRESUMEN
Riboswitches recently emerged as possible targets for the development of alternative antimicrobial approaches. Guanine-sensing riboswitches in the bacterial pathogen Clostridioides difficile (formerly known as Clostridium difficile) constitute potential targets based on their involvement in the regulation of basal metabolic control of purine compounds. In this study, we deciphered the structure-activity relationship of several guanine derivatives on the guanine riboswitch and determined their antimicrobial activity. We describe the synthesis of purine analogs modified in ring B as well as positions 2 and 6. Their biological activity was determined by measuring their affinity for the C. difficile guanine riboswitch and their inhibitory effect on bacterial growth, including a counter-screen to discriminate against riboswitch-independent antibacterial effects. Altogether, our results suggest that improvements in riboswitch binding affinity in vitro do not necessarily translate into improved antibacterial activity in bacteria, despite the fact that some structure-activity relationship was observed at least with respect to binding affinity.
Asunto(s)
Antibacterianos/farmacología , Clostridioides difficile/efectos de los fármacos , Guanina/antagonistas & inhibidores , Purinas/farmacología , Riboswitch/efectos de los fármacos , Antibacterianos/síntesis química , Antibacterianos/química , Clostridioides difficile/crecimiento & desarrollo , Clostridioides difficile/metabolismo , Relación Dosis-Respuesta a Droga , Guanina/metabolismo , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Purinas/síntesis química , Purinas/química , Relación Estructura-ActividadRESUMEN
Molecular docking calculations combined with chemically focused libraries can bring insight in the exploration of the structure-activity relationships for a series of related compounds against an RNA target. Yet, the in silico engine must be fueled by experimental observations to drive the research into a more effective ligand-discovery path. Here we show how molecular docking predictions can be coupled with in-line probing assays to explore the available chemical and configurational space in a riboswitch binding pocket.