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Riboswitches are metabolite-binding RNA regulators that modulate gene expression at the levels of transcription and translation. One of the hallmarks of riboswitch regulation is that they undergo structural changes upon metabolite binding. While a lot of effort has been put to characterize how the metabolite is recognized by the riboswitch, there is still relatively little information regarding how ligand sensing is performed within a transcriptional context. Here, we study the ligand-dependent cotranscriptional folding of the FMN-sensing ribB riboswitch of Escherichia coli Using RNase H assays to study nascent ribB riboswitch transcripts, DNA probes targeting the P1 and sequestering stems indicate that FMN binding leads to the protection of these regions from RNase H cleavage, consistent with the riboswitch inhibiting translation initiation when bound to FMN. Our results show that ligand sensing is strongly affected by the position of elongating RNA polymerase, which is defining an FMN-binding transcriptional window that is bordered in its 3' extremity by a transcriptional pause site. Also, using successively overlapping DNA probes targeting a subdomain of the riboswitch, our data suggest the presence of a previously unsuspected helical region involving the 3' strand of the P1 stem. Our results show that this helical region is conserved across bacterial species, thus suggesting that this predicted structure, the anti*-P1 stem, is involved in the FMN-free conformation of the ribB riboswitch. Overall, our study further demonstrates that intricate folding strategies may be used by riboswitches to perform metabolite sensing during the transcriptional process., (© 2024 Eschbach et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Transcription elongation is one of the most important processes in the cell. During RNA polymerase elongation, the folding of nascent transcripts plays crucial roles in the genetic decision. Bacterial riboswitches are prime examples of RNA regulators that control gene expression by altering their structure upon metabolite sensing. It was previously revealed that the thiamin pyrophosphate-sensing tbpA riboswitch in Escherichia coli cotranscriptionally adopts three main structures leading to metabolite sensing. Here, using single-molecule FRET, we characterize the transition in which the first nascent structure, a 5' stem-loop, is unfolded during transcription elongation to form the ligand-binding competent structure. Our results suggest that the structural transition occurs in a relatively abrupt manner, i.e., within a 1-2 nucleotide window. Furthermore, a highly dynamic structural exchange is observed, indicating that riboswitch transcripts perform rapid sampling of nascent co-occurring structures. We also observe that the presence of the RNAP stabilizes the 5' stem-loop along the elongation process, consistent with RNAP interacting with the 5' stem-loop. Our study emphasizes the role of early folding stem-loop structures in the cotranscriptional formation of complex RNA molecules involved in genetic regulation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Riboswitches regulate gene expression by modulating their structure upon metabolite binding. These RNA orchestrate several layers of regulation to achieve genetic control. Although Escherichia coli riboswitches modulate translation initiation, several cases have been reported where riboswitches also modulate mRNA levels. Here, we characterize the regulation mechanisms of the thiamin pyrophosphate (TPP) tbpA riboswitch in E. coli. Our results indicate that the tbpA riboswitch modulates both levels of translation and transcription and that TPP sensing is achieved more efficiently cotranscriptionally than post-transcriptionally. The preference for cotranscriptional binding is also observed when monitoring the TPP-dependent inhibition of translation initiation. Using single-molecule approaches, we observe that the aptamer domain freely fluctuates between two main structures involved in TPP recognition. Our results suggest that translation initiation is controlled through the ligand-dependent stabilization of the riboswitch structure. This study demonstrates that riboswitch cotranscriptional sensing is the primary determinant in controlling translation and mRNA levels., (© 2024. The Author(s).)

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.

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., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Fluorescent detection in cells has been tremendously developed over the years and now benefits from a large array of reporters that can provide sensitive and specific detection in real time. However, the intracellular monitoring of metabolite levels still poses great challenges due to the often complex nature of detected metabolites. Here, we provide a systematic analysis of thiamin pyrophosphate (TPP) metabolism in Escherichia coli by using a TPP-sensing riboswitch that controls the expression of the fluorescent gfp reporter. By comparing different combinations of reporter fusions and TPP-sensing riboswitches, we determine key elements that are associated with strong TPP-dependent sensing. Furthermore, by using the Keio collection as a proxy for growth conditions differing in TPP levels, we perform a high-throughput screen analysis using high-density solid agar plates. Our study reveals several genes whose deletion leads to increased or decreased TPP levels. The approach developed here could be applicable to other riboswitches and reporter genes, thus representing a framework onto which further development could lead to highly sophisticated detection platforms allowing metabolic screens and identification of orphan riboswitches., (© 2024. The Author(s).)

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., (© 2024 Ghosh et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

In Escherichia coli , transport of magnesium ions across the cellular membrane relies on MgtA and CorA transporters. While the expression of mgtA is controlled by the two-component system PhoQ/PhoP and 5' upstream region elements, corA expression is considered to be constitutive and not to depend on cellular factors. Importantly, the 5' upstream region of corA is predicted to fold into structures highly similar to the magnesium-sensing mgtA 5' upstream region. Here using biochemical and genetic assays, we show that the intracellular concentration of magnesium ions affects corA expression. Similarly to mgtA , we find that the effect of magnesium ions on corA expression is mediated by the 5' upstream region. We demonstrate that the RNA structure is important for regulation and that the Rho transcription factor is involved in the modulation of transcription termination. Consistent with previous studies, we find that translation of corL , a short ORF located within the 5' upstream region, is important for corA regulation. Our data indicate that the efficiency of corL translation is inversely proportional to corA expression, similar to what has been described for mgtA and corA in Salmonella enterica . Using a novel assay to control the import of magnesium ions, we show that while the expression of mgtA is regulated by both extra- and intracellular magnesium ions, corA is regulated by variations in intracellular magnesium ions. Our results support a model in which the expression of corA is regulated by the 5' upstream region that senses variations of intracellular magnesium ions.

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.

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., (© 2022. Crown.)

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.

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 K d 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.

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., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

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., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier B.V.)

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.

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., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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.

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., (© 2017 Dussault et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

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., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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.

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., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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.

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.

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., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

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., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

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.

The study of biologically significant and native structures is vital to characterize RNA-based regulatory mechanisms. Riboswitches are cis-acting RNA molecules that are involved in the biosynthesis and transport of cellular metabolites. Because riboswitches regulate gene expression by modulating their structure, it is vital to employ native probing assays to determine how native riboswitch structures perform highly efficient and specific ligand recognition. By employing RNase H probing, it is possible to determine the accessibility of specific RNA domains in various structural contexts. Herein, we describe how to employ RNase H probing to characterize nascent mRNA riboswitch molecules as a way to obtain information regarding the riboswitch regulation control mechanism.

Riboswitches are highly structured RNA molecules that control genetic expression by altering their structure as a function of metabolite binding. Accumulating evidence suggests that riboswitch structures are highly dynamic and perform conformational exchange between structural states that are important for the outcome of genetic regulation. To understand how ligand binding influences the folding of riboswitches, it is important to monitor in real time the riboswitch folding pathway as a function of experimental conditions. Single-molecule FRET (sm-FRET) is unique among biophysical techniques to study riboswitch conformational changes as it allows to both monitor steady-state populations of riboswitch conformers and associated interconversion dynamics. Since FRET fluorophores can be attached to virtually any nucleotide position, FRET assays can be adapted to monitor specific conformational changes, thus enabling to deduce complex riboswitch folding pathways. Herein, we show how to employ sm-FRET to study the folding pathway of the S-adenosylmethionine (SAM) and how this can be used to understand very specific conformational changes that are at the heart of riboswitch regulation mechanism.

Protein-DNA interactions underpin life and play key roles in all cellular processes and functions including DNA transcription, packaging, replication, and repair. Identifying and examining the nature of these interactions is therefore a crucial prerequisite to understand the molecular basis of how these fundamental processes take place. The application of fluorescence techniques and in particular fluorescence resonance energy transfer (FRET) to provide structural and kinetic information has experienced a stunning growth during the past decade. This has been mostly promoted by new advances in the preparation of dye-labeled nucleic acids and proteins and in optical sensitivity, where its implementation at the level of individual molecules has opened a new biophysical frontier. Nowadays, the application of FRET-based techniques to the analysis of protein-DNA interactions spans from the classical steady-state and time-resolved methods averaging over large ensembles to the analysis of distances, conformational changes, and enzymatic reactions in individual protein-DNA complexes. This chapter introduces the practical aspects of applying these methods for the study of protein-DNA interactions.

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.

Single-molecule fluorescence studies of nucleic acids are revolutionizing our understanding of fundamental cellular processes related to DNA and RNA processing mechanisms. Detailed molecular insights into DNA repair, replication, transcription, and RNA folding and function are continuously being uncovered by using the full repertoire of single-molecule fluorescence techniques. The fundamental reason behind the stunning growth in the application of single-molecule techniques to study nucleic acid structure and dynamics is the unmatched ability of single-molecule fluorescence, and mostly single-molecule FRET, to resolve heterogeneous static and dynamic populations and identify transient and low-populated states without the need for sample synchronization. New advances in DNA and RNA synthesis, post-synthetic dye-labeling methods, immobilization and passivation strategies, improved dye photophysics, and standardized analysis methods have enabled the implementation of single-molecule techniques beyond specialized laboratories. In this chapter, we introduce the practical aspects of applying single-molecule techniques to investigate nucleic acid structure, dynamics, and function.

RNA folding studies aim to clarify the relationship among sequence, tridimensional structure, and biological function. In the last decade, the application of single-molecule fluorescence resonance energy transfer (sm-FRET) techniques to investigate RNA structure and folding has revealed the details of conformational changes and timescale of the process leading to the formation of biologically active RNA structures with subnanometer resolution on millisecond timescales. In this review, we initially summarize the first wave of single-molecule FRET-based RNA techniques that focused on analyzing the influence of mono- and divalent metal ions on RNA function, and how these studies have provided very valuable information about folding pathways and the presence of intermediate and low-populated states. Next, we describe a second generation of single-molecule techniques that combine sm-FRET with the use of chemical denaturants as an emerging powerful approach to reveal information about the dynamics and energetics of RNA folding that remains hidden using conventional sm-FRET approaches. The main advantages of using the competing interplay between folding agents such as metal ions and denaturants to observe and manipulate the dynamics of RNA folding and RNA-ligand interactions is discussed in the context of the adenine riboswitch aptamer.

The study of biologically relevant native RNA structures is important to understand their cellular function(s). Native gel electrophoresis provides information about such native structures in solution as a function of experimental conditions. The application of native gel electrophoresis in a comparative manner allows to obtain precise information on relative angles subtended between given pair of stems in an RNA molecule. By adapting this approach, it is possible to obtain very specific structural information such as the amplitude of dihedral angles and helical rotation. As an example, we will describe how native gel electrophoresis can be used to study the folding of the S-adenosylmethionine (SAM) sensing riboswitch.

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.

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.

To date, single-molecule RNA science has been developed almost exclusively around the effect of metal ions as folding promoters and stabilizers of the RNA structure. Here, we introduce a novel strategy that combines single-molecule Förster resonance energy transfer (FRET) and chemical denaturation to observe and manipulate RNA dynamics. We demonstrate that the competing interplay between metal ions and denaturant agents provides a platform to extract information that otherwise will remain hidden with current methods. Using the adenine-sensing riboswitch aptamer as a model, we provide strong evidence for a rate-limiting folding step of the aptamer domain being modulated through ligand binding, a feature that is important for regulation of the controlled gene. In the absence of ligand, the rate-determining step is dominated by the formation of long-range key tertiary contacts between peripheral stem-loop elements. In contrast, when the adenine ligand interacts with partially folded messenger RNAs, the aptamer requires specifically bound Mg(2+) ions, as those observed in the crystal structure, to progress further towards the native form. Moreover, despite that the ligand-free and ligand-bound states are indistinguishable by FRET, their different stability against urea-induced denaturation allowed us to discriminate them, even when they coexist within a single FRET trajectory; a feature not accessible by existing methods.

Staphylococcus aureus is a leading cause of intramammary infections (IMI). We recently demonstrated that Staph. aureus strains express the gene guaA during bovine IMI. This gene codes for a guanosine monophosphate synthetase and its expression is regulated by a guanine riboswitch. The guanine analog 2,5,6-triaminopyrimidine-4-one (PC1) is a ligand of the guanine riboswitch. Interactions between PC1 and its target result in inhibition of guanosine monophosphate synthesis and subsequent death of the bacterium. The present study describes the investigational use of PC1 for therapy of Staph. aureus IMI in lactating cows. The in vitro minimal inhibitory concentration of PC1 ranged from 0.5 to 4 μg/mL for a variety of Staph. aureus and Staphylococcus epidermidis strains and required a reducing agent for stability and full potency. A safety assessment study was performed, whereby the healthy quarters of 4 cows were infused with increasing doses of PC1 (0, 150, 250, and 500 mg). Over the 44 h following infusions, no obvious adverse effect was observed. Ten Holstein multiparous cows in mid lactation were then experimentally infused into 3 of the quarters with approximately 50 cfu of Staph. aureus strain SHY97-3906 and infection was allowed to progress for 2 wk before starting PC1 treatment. Bacterial counts reached then about 10(3) to 10(4) cfu/mL of milk. Infected quarters were treated with 1 of 3 doses of PC1 (0, 250, or 500 mg) after each morning and evening milking for 7d (i.e., 14 intramammary infusions of PC1). During the treatment period, milk from PC1-treated quarters showed a significant reduction in bacterial concentrations. However, this reduction of Staph. aureus count in milk was not maintained during the 4 wk following the end of the treatment and only 15% of the PC1-treated quarters underwent bacteriological cure. The somatic cell count and the quarter milk production were not affected by treatments. Although bacterial clearance was not achieved following treatment with PC1, these results demonstrate that the Staph. aureus guanine riboswitch represents a relevant and promising drug target for a novel class of antibiotics for the animal food industry., (Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Riboswitches are mRNA regulatory elements that control gene expression by altering their structure in response to specific metabolite binding. In bacteria, riboswitches consist of an aptamer that performs ligand recognition and an expression platform that regulates either transcription termination or translation initiation. Here, we describe a dual-acting riboswitch from Escherichia coli that, in addition to modulating translation initiation, also is directly involved in the control of initial mRNA decay. Upon lysine binding, the lysC riboswitch adopts a conformation that not only inhibits translation initiation but also exposes RNase E cleavage sites located in the riboswitch expression platform. However, in the absence of lysine, the riboswitch folds into an alternative conformation that simultaneously allows translation initiation and sequesters RNase E cleavage sites. Both regulatory activities can be individually inhibited, indicating that translation initiation and mRNA decay can be modulated independently using the same conformational switch. Because RNase E cleavage sites are located in the riboswitch sequence, this riboswitch provides a unique means for the riboswitch to modulate RNase E cleavage activity directly as a function of lysine. This dual inhibition is in contrast to other riboswitches, such as the thiamin pyrophosphate-sensing thiM riboswitch, which triggers mRNA decay only as a consequence of translation inhibition. The riboswitch control of RNase E cleavage activity is an example of a mechanism by which metabolite sensing is used to regulate gene expression of single genes or even large polycistronic mRNAs as a function of environmental changes.

Riboswitches are ligand-dependent RNA genetic regulators that control gene expression by altering their structures. The elucidation of riboswitch conformational changes before and after ligand recognition is crucial to understand how riboswitches can achieve high ligand binding affinity and discrimination against cellular analogs. The detailed characterization of riboswitch folding pathways suggest that they may use their intrinsic conformational dynamics to sample a large array of structures, some of which being nearly identical to ligand-bound molecules. Some of these structural conformers can be "captured" upon ligand binding, which is crucial for the outcome of gene regulation. Recent studies about the SAM-I riboswitch have revealed unexpected and previously unknown RNA folding mechanisms. For instance, the observed helical twist of the P1 stem upon ligand binding to the SAM-I aptamer adds a new element in the repertoire of RNA strategies for recognition of small metabolites. From an RNA folding perspective, these findings also strongly indicate that the SAM-I riboswitch could achieve ligand recognition by using an optimized combination of conformational capture and induced-fit approaches, a feature that may be shared by other RNA regulatory sequences.

The exquisite specificity of the adenine-responsive riboswitch toward its cognate metabolite has been shown to arise from the formation of a Watson-Crick interaction between the adenine ligand and residue U65. A recent crystal structure of a U65C adenine aptamer variant has provided a rationale for the phylogenetic conservation observed at position 39 for purine aptamers. The G39-C65 variant adopts a compact ligand-free structure in which G39 is accommodated by the ligand binding site and is base-paired to the cytosine at position 65. Here, we demonstrate using a combination of biochemical and biophysical techniques that the G39-C65 base pair not only severely impairs ligand binding but also disrupts the functioning of the riboswitch in vivo by constitutively activating gene expression. Folding studies using single-molecule FRET revealed that the G39-C65 variant displays a low level of dynamic heterogeneity, a feature reminiscent of ligand-bound wild-type complexes. A restricted conformational freedom together with an ability to significantly fold in monovalent ions are exclusive to the G39-C65 variant. This work provides a mechanistic framework to rationalize the evolutionary exclusion of certain nucleotide combinations in favor of sequences that preserve ligand binding and gene regulation functionalities.

S-adenosylmethionine (SAM) riboswitches are widespread in bacteria, and up to five different SAM riboswitch families have been reported, highlighting the relevance of SAM regulation. On the basis of crystallographic and biochemical data, it has been postulated, but never demonstrated, that ligand recognition by SAM riboswitches involves key conformational changes in the RNA architecture. We show here that the aptamer follows a two-step hierarchical folding selectively induced by metal ions and ligand binding, each of them leading to the formation of one of the two helical stacks observed in the crystal structure. Moreover, we find that the anti-antiterminator P1 stem is rotated along its helical axis upon ligand binding, a mechanistic feature that could be common to other riboswitches. We also show that the nonconserved P4 helical domain is used as an auxiliary element to enhance the ligand-binding affinity. This work provides the first comprehensive characterization, to our knowledge, of a ligand-controlled riboswitch folding pathway.

Riboswitches are genetic elements located in non-coding regions of some messenger RNAs (mRNAs) that are present in all three domains of life. The binding of ligands to riboswitches induces conformational changes in the mRNA molecule, resulting in modulation of gene transcription, or RNA splicing, translation or stability. This mechanism of regulation is particularly widespread in bacteria and allows a direct response to various metabolic changes. A large number of riboswitches have been discovered in the last few years, suggesting the existence of a huge diversity of regulatory ligands and genetic mechanisms of regulation. This review focuses on recent discoveries in riboswitch regulatory mechanisms as well as current outstanding challenges., (© 2011 Blackwell Publishing Ltd.)

The Bacillus subtilis lysC lysine riboswitch modulates its own gene expression upon lysine binding through a transcription attenuation mechanism. The riboswitch aptamer is organized around a single five-way junction that provides the scaffold for two long-range tertiary interactions (loop L2-loop L3 and helix P2-loop L4)--all of this for the creation of a specific lysine binding site. We have determined that the interaction P2-L4 is particularly important for the organization of the ligand-binding site and for the riboswitch transcription attenuation control. Moreover, we have observed that a folding synergy between L2-L3 and P2-L4 allows both interactions to fold at lower magnesium ion concentrations. The P2-L4 interaction is also critical for the close juxtaposition involving stems P1 and P5. This is facilitated by the presence of lysine, suggesting an active role of the ligand in the folding transition. We also show that a previously uncharacterized stem-loop located in the expression platform is highly important for the riboswitch activity. Thus, folding elements located in the aptamer and the expression platform both influence the lysine riboswitch gene regulation.

Many bacterial mRNAs are regulated at the transcriptional or translational level by ligand-binding elements called riboswitches. Although they both bind adenine, the adenine riboswitches of Bacillus subtilis and Vibrio vulnificus differ by controlling transcription and translation, respectively. Here, we demonstrate that, beyond the obvious difference in transcriptional and translational modulation, both adenine riboswitches exhibit different ligand binding properties and appear to operate under different regulation regimes (kinetic versus thermodynamic). While the B. subtilis pbuE riboswitch fully depends on co-transcriptional binding of adenine to function, the V. vulnificus add riboswitch can bind to adenine after transcription is completed and still perform translation regulation. Further investigation demonstrates that the rate of transcription is critical for the B. subtilis pbuE riboswitch to perform efficiently, which is in agreement with a co-transcriptional regulation. Our results suggest that the nature of gene regulation control, that is transcription or translation, may have a high importance in riboswitch regulatory mechanisms., Competing Interests: The authors have declared that no competing interests exist.

Therapeutic approaches employing RNA as a tool or as a drug target have recently emerged and have been employed for various applications-ranging from cancer treatment to virus infection. Despite the paucity of its molecular groups compared to proteins, RNA has nevertheless proved to be an excellent choice for researchers who have aspired to develop therapeutic tools. Ribozymes and riboswitches are RNA-based therapeutic tools that are most often employed to knockdown gene expression and to inhibit bacterial infections, respectively. The aim of this review is to summarize recent advances observed in ribozyme- and riboswitch-based therapeutic applications that, in some cases, have reached clinical trials., (Crown Copyright © 2010. Published by Elsevier Ltd. All rights reserved.)

Riboswitches are regulatory elements modulating gene expression in response to specific metabolite binding. It has been recently reported that riboswitch agonists may exhibit antimicrobial properties by binding to the riboswitch domain. Guanine riboswitches are involved in the regulation of transport and biosynthesis of purine metabolites, which are critical for the nucleotides cellular pool. Upon guanine binding, these riboswitches stabilize a 5'-untranslated mRNA structure that causes transcription attenuation of the downstream open reading frame. In principle, any agonistic compound targeting a guanine riboswitch could cause gene repression even when the cell is starved for guanine. Antibiotics binding to riboswitches provide novel antimicrobial compounds that can be rationally designed from riboswitch crystal structures. Using this, we have identified a pyrimidine compound (PC1) binding guanine riboswitches that shows bactericidal activity against a subgroup of bacterial species including well-known nosocomial pathogens. This selective bacterial killing is only achieved when guaA, a gene coding for a GMP synthetase, is under the control of the riboswitch. Among the bacterial strains tested, several clinical strains exhibiting multiple drug resistance were inhibited suggesting that PC1 targets a different metabolic pathway. As a proof of principle, we have used a mouse model to show a direct correlation between the administration of PC1 and the reduction of Staphylococcus aureus infection in mammary glands. This work establishes the possibility of using existing structural knowledge to design novel guanine riboswitch-targeting antibiotics as powerful and selective antimicrobial compounds. Particularly, the finding of this new guanine riboswitch target is crucial as community-acquired bacterial infections have recently started to emerge.

The adenine and guanine riboswitches regulate gene expression in response to their purine ligand. X-ray structures of the aptamer moiety of these riboswitches are characterized by a compact fold in which the ligand forms a Watson-Crick base pair with residue 65. Phylogenetic analyses revealed a strict restriction at position 39 of the aptamer that prevents the G39-C65 and A39-U65 combinations, and mutational studies indicate that aptamers with these sequence combinations are impaired for ligand binding. In order to investigate the rationale for sequence conservation at residue 39, structural characterization of the U65C mutant from Bacillus subtilis pbuE adenine riboswitch aptamer was undertaken. NMR spectroscopy and X-ray crystallography studies demonstrate that the U65C mutant adopts a compact ligand-free structure, in which G39 occupies the ligand-binding site of purine riboswitch aptamers. These studies present a remarkable example of a mutant RNA aptamer that adopts a native-like fold by means of ligand mimicking and explain why this mutant is impaired for ligand binding. Furthermore, this work provides a specific insight into how the natural sequence has evolved through selection of nucleotide identities that contribute to formation of the ligand-bound state, but ensures that the ligand-free state remains in an active conformation.