RESUMO
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.
Assuntos
Escherichia coli , Conformação de Ácido Nucleico , Dobramento de RNA , Riboswitch , Tiamina Pirofosfato , Riboswitch/genética , Tiamina Pirofosfato/metabolismo , Tiamina Pirofosfato/química , Escherichia coli/genética , Escherichia coli/metabolismo , Transcrição Gênica , RNA Bacteriano/química , RNA Bacteriano/metabolismo , RNA Bacteriano/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/química , Regulação Bacteriana da Expressão Gênica , Proteínas de BactériasRESUMO
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.
Assuntos
Cobamidas , Proteínas de Escherichia coli , Escherichia coli , Regulação Bacteriana da Expressão Gênica , RNA Bacteriano , RNA Mensageiro , Riboswitch , Riboswitch/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Cobamidas/metabolismo , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , Pequeno RNA não Traduzido/genética , Pequeno RNA não Traduzido/metabolismo , Iniciação Traducional da Cadeia Peptídica , RNA Helicases/genética , RNA Helicases/metabolismo , Endorribonucleases/metabolismo , Endorribonucleases/genética , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Proteínas da Membrana Bacteriana Externa , Polirribonucleotídeo Nucleotidiltransferase , Proteínas de Membrana TransportadorasRESUMO
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.
Assuntos
Riboswitch , Riboswitch/genética , Sequência de Bases , Escherichia coli/genética , Escherichia coli/metabolismo , Transcrição Gênica , Bactérias/genética , Regulação Bacteriana da Expressão Gênica , RNA Bacteriano/metabolismoRESUMO
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.
Assuntos
RNA Polimerases Dirigidas por DNA , Proteínas de Escherichia coli , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Conformação de Ácido Nucleico , RNA/genética , RNA Bacteriano/química , RNA Bacteriano/genética , Transcrição Gênica , Fatores de Elongação da Transcrição/genéticaRESUMO
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.
Assuntos
Dobramento de RNA , Riboswitch , Bibliotecas de Moléculas Pequenas , Pareamento de Bases , Ligantes , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade , Tiamina Pirofosfato/química , Transcrição GênicaRESUMO
Transcriptional pausing is crucial for the timely expression of genetic information. Biochemical methods quantify the half-life of paused RNA polymerase (RNAP) by monitoring restarting complexes across time. However, this approach may produce apparent half-lives that are longer than true pause escape rates in biological contexts where multiple consecutive pause sites are present. We show here that the 6-nitropiperonyloxymethyl (NPOM) photolabile group provides an approach to monitor transcriptional pausing in biological systems containing multiple pause sites. We validate our approach using the well-studied his pause and show that an upstream RNA sequence modulates the pause half-life. NPOM was also used to study a transcriptional region within the Escherichia coli thiC riboswitch containing multiple consecutive pause sites. We find that an RNA hairpin structure located upstream to the region affects the half-life of the 5' most proximal pause site-but not of the 3' pause site-in contrast to results obtained using conventional approaches not preventing asynchronous transcription. Our results show that NPOM is a powerful tool to study transcription elongation dynamics within biologically complex systems.
Assuntos
Proteínas de Escherichia coli , Transcrição Gênica , RNA Polimerases Dirigidas por DNA/química , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Conformação de Ácido NucleicoRESUMO
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.
Assuntos
Proteínas de Escherichia coli/metabolismo , Riboswitch/fisiologia , Imagem Individual de Molécula/métodos , Elongação da Transcrição Genética , Carbocianinas , Escherichia coli , Proteínas de Escherichia coli/análise , Transferência Ressonante de Energia de Fluorescência , Corantes FluorescentesRESUMO
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.
Assuntos
Carbono-Nitrogênio Ligases/genética , Clostridioides difficile/fisiologia , Infecções por Clostridium/microbiologia , Regulação Bacteriana da Expressão Gênica , Riboswitch , Animais , Carbono-Nitrogênio Ligases/metabolismo , Genoma Bacteriano , Genômica/métodos , Guanina , Camundongos , Viabilidade Microbiana/genética , Mutação , Transcrição Gênica , Virulência/genéticaRESUMO
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.
Assuntos
Adenina/química , Aptâmeros de Nucleotídeos/química , Vibrio vulnificus/química , Sítios de Ligação , Ligantes , Modelos Moleculares , Mutação , Conformação de Ácido Nucleico , Dobramento de RNA , Riboswitch , Imagem Individual de Molécula , Software , Espectroscopia de Infravermelho com Transformada de Fourier , Vibrio vulnificus/genéticaRESUMO
Riboswitches are RNA sensors that have been shown to modulate the expression of downstream genes by altering their structure upon metabolite binding. Riboswitches are unique among cellular regulators in that metabolite detection is strictly performed using RNA interactions with the sensed metabolite and in which no regulatory protein is needed to mediate the interaction. However, recent studies have shed light on riboswitch control mechanisms relying on protein regulators to harness metabolite binding for the mediation of gene expression, thereby increasing the range of cellular factors involved in riboswitch regulation. The interaction between riboswitches and proteins adds another level of evolutionary pressure as riboswitches must maintain key residues for metabolite detection, structural switching and protein binding sites. Here, we review regulatory mechanisms involving Escherichia coli riboswitches that have recently been shown to rely on regulatory proteins. We also discuss the implication of such protein-based riboswitch regulatory mechanisms for genetic regulation.
Assuntos
Regulação da Expressão Gênica , Riboswitch , Endorribonucleases/metabolismo , Escherichia coli/genética , Pequeno RNA não Traduzido/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fator Rho/metabolismo , Terminação da Transcrição GenéticaRESUMO
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.
Assuntos
Regulação Bacteriana da Expressão Gênica , Magnésio/fisiologia , Riboswitch , Bacillus subtilis/química , Bacillus subtilis/genética , Transferência Ressonante de Energia de Fluorescência , Ligantes , Magnésio/análise , Dobramento de RNA , Transcrição GênicaRESUMO
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.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Escherichia coli/genética , Riboswitch/genética , Transcrição Gênica , Fatores de Elongação da Transcrição/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/genética , Conformação de Ácido Nucleico , Tiamina Pirofosfato/genética , Fatores de Transcrição/genéticaRESUMO
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.
Assuntos
Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Biossíntese de Proteínas/fisiologia , Estabilidade de RNA/fisiologia , Riboswitch/fisiologia , Transcrição Gênica/fisiologia , Bactérias/genéticaRESUMO
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.
Assuntos
Dobramento de RNA , Riboswitch/fisiologia , S-Adenosilmetionina/metabolismo , Aptâmeros de Nucleotídeos/química , Pareamento de Bases , Transferência Ressonante de Energia de Fluorescência , Ligantes , Conformação de Ácido Nucleico , Purinas/química , S-Adenosilmetionina/química , Uracila/químicaRESUMO
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.
Assuntos
Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Biossíntese de Proteínas , Fator Rho/genética , Riboswitch , Terminação da Transcrição Genética , Sequência de Bases , Escherichia coli/metabolismo , Genes Reporter , Conformação de Ácido Nucleico , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fator Rho/metabolismo , Tiamina Pirofosfato/metabolismoRESUMO
On the basis of nascent transcript sequencing, it has been postulated but never demonstrated that transcriptional pausing at translation start sites is important for gene regulation. Here we show that the Escherichia coli thiamin pyrophosphate (TPP) thiC riboswitch contains a regulatory pause site in the translation initiation region that acts as a checkpoint for thiC expression. By biochemically probing nascent transcription complexes halted at defined positions, we find a narrow transcriptional window for metabolite binding, in which the downstream boundary is delimited by the checkpoint. We show that transcription complexes at the regulatory pause site favour the formation of a riboswitch intramolecular lock that strongly prevents TPP binding. In contrast, cotranscriptional metabolite binding increases RNA polymerase pausing and induces Rho-dependent transcription termination at the checkpoint. Early transcriptional pausing may provide a general mechanism, whereby transient transcriptional windows directly coordinate the sensing of environmental cues and bacterial mRNA regulation.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Riboswitch/genética , Proteínas de Bactérias/metabolismo , Códon de Iniciação , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Mutação , Biossíntese de Proteínas , Conformação Proteica , Ribonuclease H/genética , Ribonuclease H/metabolismo , Tiamina Pirofosfato/metabolismo , Transcrição GênicaRESUMO
In addition to the helical nature of double-stranded DNA and RNA, single-stranded oligonucleotides can arrange themselves into tridimensional structures containing loops, bulges, internal hairpins and many other motifs. This ability has been used for more than two decades to generate oligonucleotide sequences, so-called aptamers, that can recognize certain metabolites with high affinity and specificity. More recently, this library of artificially-generated nucleic acid aptamers has been expanded by the discovery that naturally occurring RNA sequences control bacterial gene expression in response to cellular concentration of a given metabolite. The application of fluorescence methods has been pivotal to characterize in detail the structure and dynamics of these aptamer-ligand complexes in solution. This is mostly due to the intrinsic high sensitivity of fluorescence methods and also to significant improvements in solid-phase synthesis, post-synthetic labeling strategies and optical instrumentation that took place during the last decade. In this work, we provide an overview of the most widely employed fluorescence methods to investigate aptamer structure and function by describing the use of aptamers labeled with a single dye in fluorescence quenching and anisotropy assays. The use of 2-aminopurine as a fluorescent analog of adenine to monitor local changes in structure and fluorescence resonance energy transfer (FRET) to follow long-range conformational changes is also covered in detail. The last part of the review is dedicated to the application of fluorescence techniques based on single-molecule microscopy, a technique that has revolutionized our understanding of nucleic acid structure and dynamics. We finally describe the advantages of monitoring ligand-binding and conformational changes, one molecule at a time, to decipher the complexity of regulatory aptamers and summarize the emerging folding and ligand-binding models arising from the application of these single-molecule FRET microscopy techniques.
RESUMO
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.
Assuntos
Biocatálise , Ribonuclease III/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Pareamento de Bases/genética , Sequência de Bases , Fluorescência , Genes Reporter , Cinética , Estabilidade de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Especificidade por SubstratoRESUMO
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.
Assuntos
Bactérias/genética , Proteínas de Bactérias/genética , DNA Bacteriano/genética , Regulação Bacteriana da Expressão Gênica , Imagem Molecular/métodos , RNA Bacteriano/genética , Riboswitch/genética , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Bactérias/metabolismo , Proteínas de Bactérias/biossíntese , DNA Bacteriano/química , DNA Bacteriano/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Ligantes , Conformação de Ácido Nucleico , RNA Bacteriano/química , RNA Bacteriano/metabolismo , Relação Estrutura-AtividadeRESUMO
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.