RESUMEN
Bacterial riboswitches are molecular structures that play a crucial role in controlling gene expression to maintain cellular balance. The Escherichia coli lysC riboswitch has been previously shown to regulate gene expression through translation initiation and mRNA decay. Recent research suggests that lysC gene expression is also influenced by Rho-dependent transcription termination. Through a series of in silico, in vitro, and in vivo experiments, we provide experimental evidence that the lysC riboswitch directly and indirectly modulates Rho transcription termination. Our study demonstrates that Rho-dependent transcription termination plays a significant role in the cotranscriptional regulation of lysC expression. Together with previous studies, our work suggests that lysC expression is governed by a lysine-sensing riboswitch that regulates translation initiation, transcription termination, and mRNA degradation. Notably, both Rho and RNase E target the same region of the RNA molecule, implying that RNase E may degrade Rho-terminated transcripts, providing a means to selectively eliminate these incomplete messenger RNAs. Overall, this study sheds light on the complex regulatory mechanisms used by bacterial riboswitches, emphasizing the role of transcription termination in the control of gene expression and mRNA stability.
Asunto(s)
Riboswitch , Riboswitch/genética , Secuencia de Bases , Escherichia coli/genética , Escherichia coli/metabolismo , Transcripción Genética , Bacterias/genética , Regulación Bacteriana de la Expresión Génica , ARN Bacteriano/metabolismoRESUMEN
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
The transcriptome represents an attractive but underused set of targets for small-molecule ligands. Here, we devise a technology that leverages fragment-based screening and SHAPE-MaP RNA structure probing to discover small-molecule fragments that bind an RNA structure of interest. We identified fragments and cooperatively binding fragment pairs that bind to the thiamine pyrophosphate (TPP) riboswitch with millimolar to micromolar affinities. We then used structure-activity relationship information to efficiently design a linked-fragment ligand, with no resemblance to the native ligand, with high ligand efficiency and druglikeness, that binds to the TPP thiM riboswitch with high nanomolar affinity and that modulates RNA conformation during cotranscriptional folding. Principles from this work are broadly applicable, leveraging cooperativity and multisite binding, for developing high-quality ligands for diverse RNA targets.
Asunto(s)
Pliegue del ARN , Riboswitch , Bibliotecas de Moléculas Pequeñas , Emparejamiento Base , Ligandos , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-Actividad , Tiamina Pirofosfato/química , Transcripción GenéticaRESUMEN
Riboswitches are RNA-regulating elements that mostly rely on structural changes to modulate gene expression at various levels. Recent studies have revealed that riboswitches may control several regulatory mechanisms cotranscriptionally, i.e., during the transcription elongation of the riboswitch or early in the coding region of the regulated gene. Here, we study the structure of the nascent thiamin pyrophosphate (TPP)-sensing thiC riboswitch in Escherichia coli by using biochemical and enzymatic conventional probing approaches. Our chemical (in-line and lead probing) and enzymatic (nucleases S1, A, T1, and RNase H) probing data provide a comprehensive model of how TPP binding modulates the structure of the thiC riboswitch. Furthermore, by using transcriptional roadblocks along the riboswitch sequence, we find that a certain portion of nascent RNA is needed to sense TPP that coincides with the formation of the P5 stem loop. Together, our data suggest that conventional techniques may readily be used to study cotranscriptional folding of nascent RNAs.
Asunto(s)
Escherichia coli , Conformación de Ácido Nucleico , Pliegue del ARN , Riboswitch , Tiamina Pirofosfato , Riboswitch/genética , Tiamina Pirofosfato/metabolismo , Tiamina Pirofosfato/química , Escherichia coli/genética , Escherichia coli/metabolismo , Transcripción Genética , ARN Bacteriano/química , ARN Bacteriano/metabolismo , ARN Bacteriano/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/química , Regulación Bacteriana de la Expresión Génica , Proteínas BacterianasRESUMEN
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
Riboswitches are RNA sequences that regulate gene expression by undergoing structural changes upon the specific binding of cellular metabolites. Crystal structures of purine-sensing riboswitches have revealed an intricate network of interactions surrounding the ligand in the bound complex. The mechanistic details about how the aptamer folding pathway is involved in the formation of the metabolite binding site have been previously shown to be highly important for the riboswitch regulatory activity. Here, a combination of single-molecule FRET and SHAPE assays have been used to characterize the folding pathway of the adenine riboswitch from Vibrio vulnificus. Experimental evidences suggest a folding process characterized by the presence of a structural intermediate involved in ligand recognition. This intermediate state acts as an open conformation to ensure ligand accessibility to the aptamer and folds into a structure nearly identical to the ligand-bound complex through a series of structural changes. This study demonstrates that the add riboswitch relies on the folding of a structural intermediate that pre-organizes the aptamer global structure and the ligand binding site to allow efficient metabolite sensing and riboswitch genetic regulation.
Asunto(s)
Adenina/química , Aptámeros de Nucleótidos/química , Vibrio vulnificus/química , Sitios de Unión , Ligandos , Modelos Moleculares , Mutación , Conformación de Ácido Nucleico , Pliegue del ARN , Riboswitch , Imagen Individual de Molécula , Programas Informáticos , Espectroscopía Infrarroja por Transformada de Fourier , Vibrio vulnificus/genéticaRESUMEN
Transcriptional pausing occurs across the bacterial genome but the importance of this mechanism is still poorly understood. Only few pauses were observed during the previous decades, leaving an important gap in understanding transcription mechanisms. Using the well-known Escherichia coli hisL and trpL pause sites as models, we describe here the relation of pause sites with upstream RNA structures suspected to stabilize pausing. We find that the transcription factor NusA influences the pause half-life at leuL, pheL and thrL pause sites. Using a mutagenesis approach, we observe that transcriptional pausing is affected in all tested pause sites, suggesting that the upstream RNA sequence is important for transcriptional pausing. Compensatory mutations assessing the presence of RNA hairpins did not yield clear conclusions, indicating that complex RNA structures or transcriptional features may be playing a role in pausing. Moreover, using a bioinformatic approach, we explored the relation between a DNA consensus sequence important for pausing and putative hairpins among thousands of pause sites in E. coli. We identified 2125 sites presenting hairpin-dependent transcriptional pausing without consensus sequence, suggesting that this mechanism is widespread across E. coli. This study paves the way to understand the role of RNA structures in transcriptional pausing.
Asunto(s)
ARN Polimerasas Dirigidas por ADN , Proteínas de Escherichia coli , ARN Polimerasas Dirigidas por ADN/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Conformación de Ácido Nucleico , ARN/genética , ARN Bacteriano/química , ARN Bacteriano/genética , Transcripción Genética , Factores de Elongación Transcripcional/genéticaRESUMEN
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
Riboswitches are cis-acting regulatory RNA biosensors that rival the efficiency of those found in proteins. At the heart of their regulatory function is the formation of a highly specific aptamer-ligand complex. Understanding how these RNAs recognize the ligand to regulate gene expression at physiological concentrations of Mg2+ ions and ligand is critical given their broad impact on bacterial gene expression and their potential as antibiotic targets. In this work, we used single-molecule FRET and biochemical techniques to demonstrate that Mg2+ ions act as fine-tuning elements of the amino acid-sensing lysC aptamer's ligand-free structure in the mesophile Bacillus subtilis. Mg2+ interactions with the aptamer produce encounter complexes with strikingly different sensitivities to the ligand in different, yet equally accessible, physiological ionic conditions. Our results demonstrate that the aptamer adapts its structure and folding landscape on a Mg2+-tunable scale to efficiently respond to changes in intracellular lysine of more than two orders of magnitude. The remarkable tunability of the lysC aptamer by sub-millimolar variations in the physiological concentration of Mg2+ ions suggests that some single-aptamer riboswitches have exploited the coupling of cellular levels of ligand and divalent metal ions to tightly control gene expression.
Asunto(s)
Regulación Bacteriana de la Expresión Génica , Magnesio/fisiología , Riboswitch , Bacillus subtilis/química , Bacillus subtilis/genética , Transferencia Resonante de Energía de Fluorescencia , Ligandos , Magnesio/análisis , Pliegue del ARN , Transcripción GenéticaRESUMEN
Riboswitches are noncoding mRNA elements that control gene expression by altering their structure upon metabolite binding. Although riboswitch crystal structures provide detailed information about RNA-ligand interactions, little knowledge has been gathered to understand how riboswitches modulate gene expression. Here, we study the molecular recognition mechanism of the S-adenosylmethionine SAM-I riboswitch by characterizing the formation of a helical stacking interaction involving the ligand-binding process. We show that ligand binding is intimately linked to the formation of the helical stacking, which is dependent on the presence of three conserved purine residues that are flanked by stacked helices. We also find that these residues are important for the formation of a crucial long-range base pair formed upon SAM binding. Together, our results lend strong support to a critical role for helical stacking in the folding pathway and suggest a particularly important function in the formation of the long-range base pair.
Asunto(s)
Pliegue del ARN , Riboswitch/fisiología , S-Adenosilmetionina/metabolismo , Aptámeros de Nucleótidos/química , Emparejamiento Base , Transferencia Resonante de Energía de Fluorescencia , Ligandos , Conformación de Ácido Nucleico , Purinas/química , S-Adenosilmetionina/química , Uracilo/químicaRESUMEN
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 are RNA regulators that control gene expression by modulating their structure in response to metabolite binding. The study of mechanisms by which riboswitches modulate gene expression is crucial to understand how riboswitches are involved in maintaining cellular homeostasis. Previous reports indicate that riboswitches can control gene expression at the level of translation, transcription or mRNA decay. However, there are very few described examples where riboswitches regulate multiple steps in gene expression. Recent studies of a translation-regulating, TPP-dependent riboswitch have revealed that ligand binding is also involved in the control of mRNA levels. In this model, TPP binding to the riboswitch leads to the inhibition of translation, which in turn allows for Rho-dependent transcription termination. Thus, mRNA levels are indirectly controlled through ribosome occupancy. This is in contrast to other riboswitches that directly control mRNA levels by modulating the access of regulatory sequences involved in either Rho-dependent transcription termination or RNase E cleavage activity. Together, these findings indicate that riboswitches modulate both translation initiation and mRNA levels using multiple strategies that direct the outcome of gene expression.
Asunto(s)
Bacterias/metabolismo , Regulación Bacteriana de la Expresión Génica/fisiología , Biosíntesis de Proteínas/fisiología , Estabilidad del ARN/fisiología , Riboswitch/fisiología , Transcripción Genética/fisiología , Bacterias/genéticaRESUMEN
Members of the ribonuclease III (RNase III) family regulate gene expression by triggering the degradation of double stranded RNA (dsRNA). Hundreds of RNase III cleavage targets have been identified and their impact on RNA maturation and stability is now established. However, the mechanism defining substrates' reactivity remains unclear. In this study, we developed a real-time FRET assay for the detection of dsRNA degradation by yeast RNase III (Rnt1p) and characterized the kinetic bottlenecks controlling the reactivity of different substrates. Surprisingly, the results indicate that Rnt1p cleavage reaction is not only limited by the rate of catalysis but can also depend on base-pairing of product termini. Cleavage products terminating with paired nucleotides, like the degradation signals found in coding mRNA sequence, were less reactive and more prone to inhibition than products having unpaired nucleotides found in non-coding RNA substrates. Mutational analysis of U5 snRNA and Mig2 mRNA confirms the pairing of the cleavage site as a major determinant for the difference between cleavage rates of coding and non-coding RNA. Together the data indicate that the base-pairing of Rnt1p substrates encodes reactivity determinants that permit both constitutive processing of non-coding RNA while limiting the rate of mRNA degradation.
Asunto(s)
Biocatálisis , Ribonucleasa III/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Emparejamiento Base/genética , Secuencia de Bases , Fluorescencia , Genes Reporteros , Cinética , Estabilidad del ARN/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Especificidad por SustratoRESUMEN
RNA-based genetic regulation is exemplified by metabolite-binding riboswitches that modulate gene expression through conformational changes. Crystal structures show that the Escherichia coli btuB riboswitch contains a kissing loop interaction that is in close proximity to the bound ligand. To analyze the role of the kissing loop interaction in the riboswitch regulatory mechanism, we used RNase H cleavage assays to probe the structure of nascent riboswitch transcripts produced by the E. coli RNA polymerase. By monitoring the folding of the aptamer, kissing loop, and riboswitch expression platform, we established the conformation of each structural component in the absence or presence of bound adenosylcobalamin. We found that the kissing loop interaction is not essential for ligand binding. However, we showed that kissing loop formation improves ligand binding efficiency and is required to couple ligand binding to the riboswitch conformational changes involved in regulating gene expression. These results support a mechanism by which the btuB riboswitch modulates the formation of a tertiary structure to perform metabolite sensing and regulate gene expression.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/química , Cobamidas/química , Proteínas de Escherichia coli/química , Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Proteínas de Transporte de Membrana/química , ARN Bacteriano/química , Riboswitch , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Secuencia de Bases , Transporte Biológico , Cobamidas/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Ligandos , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Datos de Secuencia Molecular , Mutación , Conformación de Ácido Nucleico , Plásmidos/química , Plásmidos/metabolismo , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , Ribonucleasa H/química , Ribonucleasa H/metabolismo , Transcripción GenéticaRESUMEN
The last decade has witnessed the discovery of a variety of non-coding RNA sequences that perform a broad range of crucial biological functions. Among these, the ability of certain RNA sequences, so-called riboswitches, has attracted considerable interest. Riboswitches control gene expression in response to the concentration of particular metabolites to which they bind without the need for any protein. These RNA switches not only need to adopt a very specific tridimensional structure to perform their function, but also their sequence has been evolutionary optimized to recognize a particular metabolite with high affinity and selectivity. Thus, riboswitches offer a unique opportunity to get fundamental insights into RNA plasticity and how folding dynamics and ligand recognition mechanisms have been efficiently merged to control gene regulation. Because riboswitch sequences have been mostly found in bacterial organisms controlling the expression of genes associated to the synthesis, degradation or transport of crucial metabolites for bacterial survival, they offer exciting new routes for antibiotic development in an era where bacterial resistance is more than ever challenging conventional drug discovery strategies. Here, we give an overview of the architecture, diversity and regulatory mechanisms employed by riboswitches with particular emphasis on the biophysical methods currently available to characterise their structure and functional dynamics.
Asunto(s)
Bacterias/genética , Proteínas Bacterianas/genética , ADN Bacteriano/genética , Regulación Bacteriana de la Expresión Génica , Imagen Molecular/métodos , ARN Bacteriano/genética , Riboswitch/genética , Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Bacterias/metabolismo , Proteínas Bacterianas/biosíntesis , ADN Bacteriano/química , ADN Bacteriano/metabolismo , Resistencia a Antineoplásicos/genética , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Ligandos , Conformación de Ácido Nucleico , ARN Bacteriano/química , ARN Bacteriano/metabolismo , Relación Estructura-ActividadRESUMEN
Clostridium difficile is an anaerobic Gram-positive bacterium that causes intestinal infections with symptoms ranging from mild diarrhea to fulminant colitis. Cyclic diguanosine monophosphate (c-di-GMP) is a bacterial second messenger that typically regulates the switch from motile, free-living to sessile and multicellular behaviors in Gram-negative bacteria. Increased intracellular c-di-GMP concentration in C. difficile was recently shown to reduce flagellar motility and to increase cell aggregation. In this work, we investigated the role of the primary type IV pilus (T4P) locus in c-di-GMP-dependent cell aggregation. Inactivation of two T4P genes, pilA1 (CD3513) and pilB1 (CD3512), abolished pilus formation and significantly reduced cell aggregation under high c-di-GMP conditions. pilA1 is preceded by a putative c-di-GMP riboswitch, predicted to be transcriptionally active upon c-di-GMP binding. Consistent with our prediction, high intracellular c-di-GMP concentration increased transcript levels of T4P genes. In addition, single-round in vitro transcription assays confirmed that transcription downstream of the predicted transcription terminator was dose dependent and specific to c-di-GMP binding to the riboswitch aptamer. These results support a model in which T4P gene transcription is upregulated by c-di-GMP as a result of its binding to an upstream transcriptionally activating riboswitch, promoting cell aggregation in C. difficile.
Asunto(s)
Proteínas Bacterianas/metabolismo , Clostridioides difficile/fisiología , GMP Cíclico/metabolismo , Fimbrias Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Riboswitch , Proteínas Bacterianas/genética , Clostridioides difficile/genética , Fimbrias Bacterianas/genética , Conformación de Ácido Nucleico , ARN Bacteriano/química , ARN Bacteriano/genética , ARN Bacteriano/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
Riboswitches are mRNA elements that specifically bind cellular metabolites and control gene expression by modifying their structure. As riboswitches often control essential genes in pathogenic bacteria, riboswitches have been proposed as new targets for antibiotics. High-throughput screening provides a powerful approach to identify riboswitch ligand analogs that could act as powerful antibacterial drugs. Biochemical assays have already been used to find riboswitch-binding analogs, but those methods do take into account the transcriptional context for riboswitch regulation. As the importance of co-transcriptional ligand binding has been shown for several riboswitches, it is vital to develop an assay that screens riboswitch-binding analogs during the transcriptional process. Here, we describe the development of a dual molecular beacon system monitoring the transcriptional regulation activity of the Bacillus subtilis pbuE adenine riboswitch. This system relies on two molecular beacons that enable the monitoring of transcription efficiency, as well as the regulatory activity of the riboswitch. Different analogs were tested using our system, and a good correlation was observed between riboswitch activity and reported metabolite affinities. This method is specific, reliable and could be applied at the high-throughput level for the identification of new potential antibiotics targeting any riboswitch-regulating gene expression at the mRNA level.
Asunto(s)
Colorantes Fluorescentes/química , Regulación de la Expresión Génica/efectos de los fármacos , Sondas de Ácido Nucleico/química , Riboswitch/efectos de los fármacos , Transcripción Genética/efectos de los fármacos , Adenina/análogos & derivados , Adenina/farmacología , Bacillus subtilis/genética , Evaluación Preclínica de Medicamentos , Guanina/análogos & derivados , Guanina/farmacología , Ligandos , Proteínas de Transporte de Nucleobases/genética , Espectrometría de FluorescenciaRESUMEN
The stability of chromosome ends, the telomeres, is dependent on the ribonucleoprotein telomerase. In vitro, telomerase requires at least one RNA molecule and a reverse transcriptase-like protein. However, for telomere homeostasis in vivo, additional proteins are required. Telomerase RNAs of different species vary in size and sequence and only few features common to all telomerases are known. Here we show that stem-loop IVc of the Saccharomyces cerevisiae telomerase RNA contains a structural element that is required for telomerase function in vivo. Indeed, the distal portion of stem-loop IVc stimulates telomerase activity in vitro in a way that is independent of Est1 binding on more proximal portions of this stem-loop. Functional analyses of the RNA in vivo reveal that this distal element we call telomerase-stimulating structure (TeSS) must contain a bulged area in single stranded form and also show that Est1-dependent functions such as telomerase import or recruitment are not affected by TeSS. This study thus uncovers a new structural telomerase RNA element implicated in catalytic activity. Given previous evidence for TeSS elements in ciliate and mammalian RNAs, we speculate that this substructure is a conserved feature that is required for optimal telomerase holoenzyme function.