Engineering and In Vivo Applications of Riboswitches.

Riboswitches are common gene regulatory units mostly found in bacteria that are capable of altering gene expression in response to a small molecule. These structured RNA elements consist of two modular subunits: an aptamer domain that binds with high specificity and affinity to a target ligand and an expression platform that transduces ligand binding to a gene expression output. Significant progress has been made in engineering novel aptamer domains for new small molecule inducers of gene expression. Modified expression platforms have also been optimized to function when fused with both natural and synthetic aptamer domains. As this field expands, the use of these privileged scaffolds has permitted the development of tools such as RNA-based fluorescent biosensors. In this review, we summarize the methods that have been developed to engineer new riboswitches and highlight applications of natural and synthetic riboswitches in enzyme and strain engineering, in controlling gene expression and cellular physiology, and in real-time imaging of cellular metabolites and signals.

Widespread Protection of RNA Cleavage Sites by a Riboswitch Aptamer that Folds as a Compact Obstacle to Scanning by RNase E.

Riboswitches are thought generally to function by modulating transcription elongation or translation initiation. In rare instances, ligand binding to a riboswitch has been found to alter the rate of RNA degradation by directly stimulating or inhibiting nearby cleavage. Here, we show that guanidine-induced pseudoknot formation by the aptamer domain of a guanidine III riboswitch from Legionella pneumophila has a different effect, stabilizing mRNA by protecting distal cleavage sites en masse from ribonuclease attack. It does so by creating a coaxially base-paired obstacle that impedes scanning from a monophosphorylated 5' end to those sites by the regulatory endonuclease RNase E. Ligand binding by other riboswitch aptamers peripheral to the path traveled by RNase E does not inhibit distal cleavage. These findings reveal that a riboswitch aptamer can function independently of any overlapping expression platform to regulate gene expression by acting directly to prolong mRNA longevity in response to ligand binding.

A decade of riboswitches.

Riboswitches were discovered in 2002 in bacteria as RNA-based intracellular sensors of vitamin derivatives. During the last decade, naturally occurring RNA sensor elements have been found to bind a range of small metabolites and ions and to exert regulatory control of transcription, translation, splicing, and RNA stability. Extensive biochemical, structural, and genetic studies have established the basic principles underpinning riboswitch function in all three kingdoms of life with implications for developing antibiotics, designing new molecular sensors, and integrating riboswitches into synthetic circuits.

Riboswitch control of aminoglycoside antibiotic resistance.

The majority of riboswitches are regulatory RNAs that regulate gene expression by binding small-molecule metabolites. Here we report the discovery of an aminoglycoside-binding riboswitch that is widely distributed among antibiotic-resistant bacterial pathogens. This riboswitch is present in the leader RNA of the resistance genes that encode the aminoglycoside acetyl transferase (AAC) and aminoglycoside adenyl transferase (AAD) enzymes that confer resistance to aminoglycoside antibiotics through modification of the drugs. We show that expression of the AAC and AAD resistance genes is regulated by aminoglycoside binding to a secondary structure in their 5' leader RNA. Reporter gene expression, direct measurements of drug RNA binding, chemical probing, and UV crosslinking combined with mutational analysis demonstrate that the leader RNA functions as an aminoglycoside-sensing riboswitch in which drug binding to the leader RNA leads to the induction of aminoglycosides antibiotic resistance.

Discovering riboswitches: the past and the future.

Riboswitches are structured noncoding RNA domains used by many bacteria to monitor the concentrations of target ligands and regulate gene expression accordingly. In the past 20 years over 55 distinct classes of natural riboswitches have been discovered that selectively sense small molecules or elemental ions, and thousands more are predicted to exist. Evidence suggests that some riboswitches might be direct descendants of the RNA-based sensors and switches that were likely present in ancient organisms before the evolutionary emergence of proteins. We provide an overview of the current state of riboswitch research, focusing primarily on the discovery of riboswitches, and speculate on the major challenges facing researchers in the field.

Molecular dynamics in multidimensional space explains how mutations affect the association path of neomycin to a riboswitch.

Riboswitches are messenger RNA (mRNA) fragments binding specific small molecules to regulate gene expression. A synthetic N1 riboswitch, inserted into yeast mRNA controls the translation of a reporter gene in response to neomycin. However, its regulatory activity is sensitive to single-point RNA mutations, even those distant from the neomycin binding site. While the association paths of neomycin to N1 and its variants remain unknown, recent fluorescence kinetic experiments indicate a two-step process driven by conformational selection. This raises the question of which step is affected by mutations. To address this, we performed all-atom two-dimensional replica-exchange molecular dynamics simulations for N1 and U14C, U14C[Formula: see text], U15A, and A17G mutants, ensuring extensive conformational sampling of both RNA and neomycin. The obtained neomycin association and binding paths, along with multidimensional free-energy profiles, revealed a two-step binding mechanism, consisting of conformational selection and induced fit. Neomycin binds to a preformed N1 conformation upon identifying a stable upper stem and U-turn motif in the riboswitch hairpin. However, the positioning of neomycin in the binding site occurs at different RNA-neomycin distances for each mutant, which may explain their different regulatory activities. The subsequent induced fit arises from the interactions of the neomycin's N3 amino group with RNA, causing the G9 backbone to rearrange. In the A17G mutant, the critical C6-A17/G17 stacking forms at a closer RNA-neomycin distance compared to N1. These findings together with estimated binding free energies coincide with experiments and elucidate why the A17G mutation decreases and U15A enhances N1 activity in response to neomycin.

Direct and indirect control of Rho-dependent transcription termination by the Escherichia coli lysC riboswitch.

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.

Magnesium ions mitigate metastable states in the regulatory landscape of mRNA elements.

Residing in the 5' untranslated region of the mRNA, the 2'-deoxyguanosine (2'-dG) riboswitch mRNA element adopts an alternative structure upon binding of the 2'-dG molecule, which terminates transcription. RNA conformations are generally strongly affected by positively charged metal ions (especially Mg2+). We have quantitatively explored the combined effect of ligand (2'-dG) and Mg2+ binding on the energy landscape of the aptamer domain of the 2'-dG riboswitch with both explicit solvent all-atom molecular dynamics simulations (99 µsec aggregate sampling for the study) and selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) experiments. We show that both ligand and Mg2+ are required for the stabilization of the aptamer domain; however, the two factors act with different modalities. The addition of Mg2+ remodels the energy landscape and reduces its frustration by the formation of additional contacts. In contrast, the binding of 2'-dG eliminates the metastable states by nucleating a compact core for the aptamer domain. Mg2+ ions and ligand binding are required to stabilize the least stable helix, P1 (which needs to unfold to activate the transcription platform), and the riboswitch core formed by the backbone of the P2 and P3 helices. Mg2+ and ligand also facilitate a more compact structure in the three-way junction region.

Rock, scissors, paper: How RNA structure informs function.

RNA can fold back on itself to adopt a wide range of structures. These range from relatively simple hairpins to intricate 3D folds and can be accompanied by regulatory interactions with both metabolites and macromolecules. The last 50 yr have witnessed elucidation of an astonishing array of RNA structures including transfer RNAs, ribozymes, riboswitches, the ribosome, the spliceosome, and most recently entire RNA structuromes. These advances in RNA structural biology have deepened insight into fundamental biological processes including gene editing, transcription, translation, and structure-based detection and response to temperature and other environmental signals. These discoveries reveal that RNA can be relatively static, like a rock; that it can have catalytic functions of cutting bonds, like scissors; and that it can adopt myriad functional shapes, like paper. We relate these extraordinary discoveries in the biology of RNA structure to the plant way of life. We trace plant-specific discovery of ribozymes and riboswitches, alternative splicing, organellar ribosomes, thermometers, whole-transcriptome structuromes and pan-structuromes, and conclude that plants have a special set of RNA structures that confer unique types of gene regulation. We finish with a consideration of future directions for the RNA structure-function field.

Functionally uncoupled transcription-translation in Bacillus subtilis.

Tight coupling of transcription and translation is considered a defining feature of bacterial gene expression1,2. The pioneering ribosome can both physically associate and kinetically coordinate with RNA polymerase (RNAP)3-11, forming a signal-integration hub for co-transcriptional regulation that includes translation-based attenuation12,13 and RNA quality control2. However, it remains unclear whether transcription-translation coupling-together with its broad functional consequences-is indeed a fundamental characteristic of bacteria other than Escherichia coli. Here we show that RNAPs outpace pioneering ribosomes in the Gram-positive model bacterium Bacillus subtilis, and that this 'runaway transcription' creates alternative rules for both global RNA surveillance and translational control of nascent RNA. In particular, uncoupled RNAPs in B. subtilis explain the diminished role of Rho-dependent transcription termination, as well as the prevalence of mRNA leaders that use riboswitches and RNA-binding proteins. More broadly, we identified widespread genomic signatures of runaway transcription in distinct phyla across the bacterial domain. Our results show that coupled RNAP-ribosome movement is not a general hallmark of bacteria. Instead, translation-coupled transcription and runaway transcription constitute two principal modes of gene expression that determine genome-specific regulatory mechanisms in prokaryotes.

Ligand Modulates Cross-Coupling between Riboswitch Folding and Transcriptional Pausing.

Numerous classes of riboswitches have been found to regulate bacterial gene expression in response to physiological cues, offering new paths to antibacterial drugs. As common studies of isolated riboswitches lack the functional context of the transcription machinery, we here combine single-molecule, biochemical, and simulation approaches to investigate the coupling between co-transcriptional folding of the pseudoknot-structured preQ1 riboswitch and RNA polymerase (RNAP) pausing. We show that pausing at a site immediately downstream of the riboswitch requires a ligand-free pseudoknot in the nascent RNA, a precisely spaced sequence resembling the pause consensus, and electrostatic and steric interactions with the RNAP exit channel. While interactions with RNAP stabilize the native fold of the riboswitch, binding of the ligand signals RNAP release from the pause. Our results demonstrate that the nascent riboswitch and its ligand actively modulate the function of RNAP and vice versa, a paradigm likely to apply to other cellular RNA transcripts.

Structure-based characterization and compound identification of the wild-type THF class-II riboswitch.

Riboswitches are conserved regulatory RNA elements participating in various metabolic pathways. Recently, a novel RNA motif known as the folE RNA motif was discovered upstream of folE genes. It specifically senses tetrahydrofolate (THF) and is therefore termed THF-II riboswitch. To unravel the ligand recognition mechanism of this newly discovered riboswitch and decipher the underlying principles governing its tertiary folding, we determined both the free-form and bound-form THF-II riboswitch in the wild-type sequences. Combining structural information and isothermal titration calorimetry (ITC) binding assays on structure-based mutants, we successfully elucidated the significant long-range interactions governing the function of THF-II riboswitch and identified additional compounds, including alternative natural metabolites and potential lead compounds for drug discovery, that interact with THF-II riboswitch. Our structural research on the ligand recognition mechanism of the THF-II riboswitch not only paves the way for identification of compounds targeting riboswitches, but also facilitates the exploration of THF analogs in diverse biological contexts or for therapeutic applications.

Ribocentre-switch: a database of riboswitches.

Riboswitches are regulatory elements found in the untranslated regions (UTRs) of certain mRNA molecules. They typically comprise two distinct domains: an aptamer domain that can bind to specific small molecules, and an expression platform that controls gene expression. Riboswitches work by undergoing a conformational change upon binding to their specific ligand, thus activating or repressing the genes downstream. This mechanism allows gene expression regulation in response to metabolites or small molecules. To systematically summarise riboswitch structures and their related ligand binding functions, we present Ribocentre-switch, a comprehensive database of riboswitches, including the information as follows: sequences, structures, functions, ligand binding pockets and biological applications. It encompasses 56 riboswitches and 26 orphan riboswitches from over 430 references, with a total of 89 591 sequences. It serves as a good resource for comparing different riboswitches and facilitating the identification of potential riboswitch candidates. Therefore, it may facilitate the understanding of RNA structural conformational changes in response to ligand signaling. The database is publicly available at https://riboswitch.ribocentre.org.

A novel regulatory interplay between atypical B12 riboswitches and uORF translation in Mycobacterium tuberculosis.

Vitamin B12 is an essential cofactor in all domains of life and B12-sensing riboswitches are some of the most widely distributed riboswitches. Mycobacterium tuberculosis, the causative agent of tuberculosis, harbours two B12-sensing riboswitches. One controls expression of metE, encoding a B12-independent methionine synthase, the other controls expression of ppe2 of uncertain function. Here, we analysed ligand sensing, secondary structure and gene expression control of the metE and ppe2 riboswitches. Our results provide the first evidence of B12 binding by these riboswitches and show that they exhibit different preferences for individual isoforms of B12, use distinct regulatory and structural elements and act as translational OFF switches. Based on our results, we propose that the ppe2 switch represents a new variant of Class IIb B12-sensing riboswitches. Moreover, we have identified short translated open reading frames (uORFs) upstream of metE and ppe2, which modulate the expression of their downstream genes. Translation of the metE uORF suppresses MetE expression, while translation of the ppe2 uORF is essential for PPE2 expression. Our findings reveal an unexpected regulatory interplay between B12-sensing riboswitches and the translational machinery, highlighting a new level of cis-regulatory complexity in M. tuberculosis. Attention to such mechanisms will be critical in designing next-level intervention strategies.

Co-transcriptional folding of the glmS ribozyme enables a rapid response to metabolite.

The glmS ribozyme riboswitch, located in the 5' untranslated region of the Bacillus subtilis glmS messenger RNA (mRNA), regulates cell wall biosynthesis through ligand-induced self-cleavage and decay of the glmS mRNA. Although self-cleavage of the refolded glmS ribozyme has been studied extensively, it is not known how early the ribozyme folds and self-cleaves during transcription. Here, we combine single-molecule fluorescence with kinetic modeling to show that self-cleavage can occur during transcription before the ribozyme is fully synthesized. Moreover, co-transcriptional folding of the RNA at a physiological elongation rate allows the ribozyme catalytic core to react without the downstream peripheral stability domain. Dimethyl sulfate footprinting further revealed how slow sequential folding favors formation of the native core structure through fraying of misfolded helices and nucleation of a native pseudoknot. Ribozyme self-cleavage at an early stage of transcription may benefit glmS regulation in B. subtilis, as it exposes the mRNA to exoribonuclease before translation of the open reading frame can begin. Our results emphasize the importance of co-transcriptional folding of RNA tertiary structure for cis-regulation of mRNA stability.

A sensitive and scalable fluorescence anisotropy single stranded RNA targeting approach for monitoring riboswitch conformational states.

The capacity of riboswitches to undergo conformational changes in response to binding their native ligands is closely tied to their functional roles and is an attractive target for antimicrobial drug design. Here, we established a probe-based fluorescence anisotropy assay to monitor riboswitch conformational switching with high sensitivity and throughput. Using the Bacillus subtillis yitJ S-Box (SAM-I), Fusobacterium nucleatum impX RFN element of (FMN) and class-I cyclic-di-GMP from Vibrio cholerae riboswitches as model systems, we developed short fluorescent DNA probes that specifically recognize either ligand-free or -bound riboswitch conformational states. We showed that increasing concentrations of native ligands cause measurable and reproducible changes in fluorescence anisotropy that correlate with riboswitch conformational changes observed by native gel analysis. Furthermore, we applied our assay to several ligand analogues and confirmed that it can discriminate between ligands that bind, triggering the native conformational change, from those that bind without causing the conformational change. This new platform opens the possibility of high-throughput screening compound libraries to identify potential new antibiotics that specifically target functional conformational changes in riboswitches.

The discovery of novel noncoding RNAs in 50 bacterial genomes.

Structured noncoding RNAs (ncRNAs) contribute to many important cellular processes involving chemical catalysis, molecular recognition and gene regulation. Few ncRNA classes are broadly distributed among organisms from all three domains of life, but the list of rarer classes that exhibit surprisingly diverse functions is growing. We previously developed a computational pipeline that enables the near-comprehensive identification of structured ncRNAs expressed from individual bacterial genomes. The regions between protein coding genes are first sorted based on length and the fraction of guanosine and cytidine nucleotides. Long, GC-rich intergenic regions are then examined for sequence and structural similarity to other bacterial genomes. Herein, we describe the implementation of this pipeline on 50 bacterial genomes from varied phyla. More than 4700 candidate intergenic regions with the desired characteristics were identified, which yielded 44 novel riboswitch candidates and numerous other putative ncRNA motifs. Although experimental validation studies have yet to be conducted, this rate of riboswitch candidate discovery is consistent with predictions that many hundreds of novel riboswitch classes remain to be discovered among the bacterial species whose genomes have already been sequenced. Thus, many thousands of additional novel ncRNA classes likely remain to be discovered in the bacterial domain of life.

Towards parsimonious generative modeling of RNA families.

Generative probabilistic models emerge as a new paradigm in data-driven, evolution-informed design of biomolecular sequences. This paper introduces a novel approach, called Edge Activation Direct Coupling Analysis (eaDCA), tailored to the characteristics of RNA sequences, with a strong emphasis on simplicity, efficiency, and interpretability. eaDCA explicitly constructs sparse coevolutionary models for RNA families, achieving performance levels comparable to more complex methods while utilizing a significantly lower number of parameters. Our approach demonstrates efficiency in generating artificial RNA sequences that closely resemble their natural counterparts in both statistical analyses and SHAPE-MaP experiments, and in predicting the effect of mutations. Notably, eaDCA provides a unique feature: estimating the number of potential functional sequences within a given RNA family. For example, in the case of cyclic di-AMP riboswitches (RF00379), our analysis suggests the existence of approximately 1039 functional nucleotide sequences. While huge compared to the known <4000 natural sequences, this number represents only a tiny fraction of the vast pool of nearly 1082 possible nucleotide sequences of the same length (136 nucleotides). These results underscore the promise of sparse and interpretable generative models, such as eaDCA, in enhancing our understanding of the expansive RNA sequence space.

FRET-guided modeling of nucleic acids.

The functional diversity of RNAs is encoded in their innate conformational heterogeneity. The combination of single-molecule spectroscopy and computational modeling offers new attractive opportunities to map structural transitions within nucleic acid ensembles. Here, we describe a framework to harmonize single-molecule Förster resonance energy transfer (FRET) measurements with molecular dynamics simulations and de novo structure prediction. Using either all-atom or implicit fluorophore modeling, we recreate FRET experiments in silico, visualize the underlying structural dynamics and quantify the reaction coordinates. Using multiple accessible-contact volumes as a post hoc scoring method for fragment assembly in Rosetta, we demonstrate that FRET can be used to filter a de novo RNA structure prediction ensemble by refuting models that are not compatible with in vitro FRET measurement. We benchmark our FRET-assisted modeling approach on double-labeled DNA strands and validate it against an intrinsically dynamic manganese(II)-binding riboswitch. We show that a FRET coordinate describing the assembly of a four-way junction allows our pipeline to recapitulate the global fold of the riboswitch displayed by the crystal structure. We conclude that computational fluorescence spectroscopy facilitates the interpretability of dynamic structural ensembles and improves the mechanistic understanding of nucleic acid interactions.

The effect of pseudoknot base pairing on cotranscriptional structural switching of the fluoride riboswitch.

A central question in biology is how RNA sequence changes influence dynamic conformational changes during cotranscriptional folding. Here we investigated this question through the study of transcriptional fluoride riboswitches, non-coding RNAs that sense the fluoride anion through the coordinated folding and rearrangement of a pseudoknotted aptamer domain and a downstream intrinsic terminator expression platform. Using a combination of Escherichia coli RNA polymerase in vitro transcription and cellular gene expression assays, we characterized the function of mesophilic and thermophilic fluoride riboswitch variants. We showed that only variants containing the mesophilic pseudoknot function at 37°C. We next systematically varied the pseudoknot sequence and found that a single wobble base pair is critical for function. Characterizing thermophilic variants at 65°C through Thermus aquaticus RNA polymerase in vitro transcription showed the importance of this wobble pair for function even at elevated temperatures. Finally, we performed all-atom molecular dynamics simulations which supported the experimental findings, visualized the RNA structure switching process, and provided insight into the important role of magnesium ions. Together these studies provide deeper insights into the role of riboswitch sequence in influencing folding and function that will be important for understanding of RNA-based gene regulation and for synthetic biology applications.