A nascent riboswitch helix orchestrates robust transcriptional regulation through signal integration.

Widespread manganese-sensing transcriptional riboswitches effect the dependable gene regulation needed for bacterial manganese homeostasis in changing environments. Riboswitches - like most structured RNAs - are believed to fold co-transcriptionally, subject to both ligand binding and transcription events; yet how these processes are orchestrated for robust regulation is poorly understood. Through a combination of single-molecule and bulk approaches, we discover how a single Mn2+ ion and the transcribing RNA polymerase (RNAP), paused immediately downstream by a DNA template sequence, are coordinated by the bridging switch helix P1.1 in the representative Lactococcus lactis riboswitch. This coordination achieves a heretofore-overlooked semi-docked global conformation of the nascent RNA, P1.1 base pair stabilization, transcription factor NusA ejection, and RNAP pause extension, thereby enforcing transcription readthrough. Our work demonstrates how a central, adaptable RNA helix functions analogous to a molecular fulcrum of a first-class lever system to integrate disparate signals for finely balanced gene expression control.

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.

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.

Flipping the script: Understanding riboswitches from an alternative perspective.

Riboswitches are broadly distributed regulatory elements most frequently found in the 5'-leader sequence of bacterial mRNAs that regulate gene expression in response to the binding of a small molecule effector. The occupancy status of the ligand-binding aptamer domain manipulates downstream information in the message that instructs the expression machinery. Currently, there are over 55 validated riboswitch classes, where each class is defined based on the identity of the ligand it binds and/or sequence and structure conservation patterns within the aptamer domain. This classification reflects an "aptamer-centric" perspective that dominates our understanding of riboswitches. In this review, we propose a conceptual framework that groups riboswitches based on the mechanism by which RNA manipulates information directly instructing the expression machinery. This scheme does not replace the established aptamer domain-based classification of riboswitches but rather serves to facilitate hypothesis-driven investigation of riboswitch regulatory mechanisms. Based on current bioinformatic, structural, and biochemical studies of a broad spectrum of riboswitches, we propose three major mechanistic groups: (1) "direct occlusion", (2) "interdomain docking", and (3) "strand exchange". We discuss the defining features of each group, present representative examples of riboswitches from each group, and illustrate how these RNAs couple small molecule binding to gene regulation. While mechanistic studies of the occlusion and docking groups have yielded compelling models for how these riboswitches function, much less is known about strand exchange processes. To conclude, we outline the limitations of our mechanism-based conceptual framework and discuss how critical information within riboswitch expression platforms can inform gene regulation.

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.

T7 phage-assisted evolution of riboswitches using error-prone replication and dual selection.

Leveraging riboswitches, non-coding mRNA fragments pivotal to gene regulation, poses a challenge in effectively selecting and enriching these functional genetic sensors, which can toggle between ON and OFF states in response to their cognate inducers. Here, we show our engineered phage T7, enabling the evolution of a theophylline riboswitch. We have replaced T7's DNA polymerase with a transcription factor controlled by a theophylline riboswitch and have created two types of host environments to propagate the engineered phage. Both types host an error-prone T7 DNA polymerase regulated by a T7 promoter along with another critical gene-either cmk or pifA, depending on the host type. The cmk gene is necessary for T7 replication and is used in the first host type for selection in the riboswitch's ON state. Conversely, the second host type incorporates the pifA gene, leading to abortive T7 infections and used for selection in the riboswitch's OFF state. This dual-selection system, termed T7AE, was then applied to a library of 65,536 engineered T7 phages, each carrying randomized riboswitch variants. Through successive passage in both host types with and without theophylline, we observed an enrichment of phages encoding functional riboswitches that conferred a fitness advantage to the phage in both hosts. The T7AE technique thereby opens new pathways for the evolution and advancement of gene switches, including non-coding RNA-based switches, setting the stage for significant strides in synthetic biology.

A spermidine riboswitch class in bacteria exploits a close variant of an aptamer for the enzyme cofactor S-adenosylmethionine.

Natural polyamines such as spermidine and spermine cations have characteristics that make them highly likely to be sensed by riboswitches, such as their general affinity to polyanionic RNA and their broad contributions to cell physiology. Despite previous claims that polyamine riboswitches exist, evidence of their biological functions has remained unconvincing. Here, we report that rare variants of bacterial S-adenosylmethionine-I (SAM-I) riboswitches reject SAM and have adapted to selectively sense spermidine. These spermidine-sensing riboswitch variants are associated with genes whose protein products are directly involved in the production of spermidine and other polyamines. Biochemical and genetic assays demonstrate that representatives of this riboswitch class robustly function as genetic "off" switches, wherein spermidine binding causes premature transcription termination to suppress the expression of polyamine biosynthetic genes. These findings confirm the existence of natural spermidine-sensing riboswitches in bacteria and expand the list of variant riboswitch classes that have adapted to bind different ligands.

Creation of Architecturally Minimal Transcriptionally Activating Riboswitches Responsive to Theophylline Reveals an Unconventional Design Strategy.

Riboswitches are noncoding RNA switches that are largely utilized in bacteria and play a significant role in synthetic biology. Nonetheless, their natural counterparts possess lengthy sequences and intricate structures, posing challenges for their modular integration into complex gene circuits. Consequently, it is imperative to develop simplified synthetic riboswitches that can be effortlessly incorporated into gene circuits. The conventional approach to generate synthetic riboswitches entails tedious library construction and extensive screening, which frequently yields suboptimal performance. To overcome this obstacle, alternative methods are urgently needed. In this study, we created a novel approach to designing a diverse set of transcription-activating riboswitches that exhibit high performance and broad compatibility. The strategy involved starting with a synthetic theophylline RNA aptamer and designing an expression platform that forms a transcriptional terminator in its inactive state but switches to an antiterminator when it is activated. Several sequences were designed, constructed, and subjected to virtual screening, resulting in the identification of two transcription-activating riboswitches. These riboswitches were then engineered to reduce the basal leakage and increase the activation level through extending the hairpin region using a screened random sequence. These architecturally minimal synthetic riboswitches were highly adapted to different constitutive promoters in a modular manner, generating a differentially responsive output to theophylline. As a proof-of-principle, the synthetic riboswitches were applied to rewire a synthetic quorum-sensing circuit (QSC). The reprogrammed QSC successfully modulated the temporal responsive profile against the activation. This strategy is expected to expand the variety of high-performance riboswitches that are responsive to different ligands, thereby further facilitating the design of complex genetic circuits.

Observation of coordinated RNA folding events by systematic cotranscriptional RNA structure probing.

RNA begins to fold as it is transcribed by an RNA polymerase. Consequently, RNA folding is constrained by the direction and rate of transcription. Understanding how RNA folds into secondary and tertiary structures therefore requires methods for determining the structure of cotranscriptional folding intermediates. Cotranscriptional RNA chemical probing methods accomplish this by systematically probing the structure of nascent RNA that is displayed from an RNA polymerase. Here, we describe a concise, high-resolution cotranscriptional RNA chemical probing procedure called variable length Transcription Elongation Complex RNA structure probing (TECprobe-VL). We demonstrate the accuracy and resolution of TECprobe-VL by replicating and extending previous analyses of ZTP and fluoride riboswitch folding and mapping the folding pathway of a ppGpp-sensing riboswitch. In each system, we show that TECprobe-VL identifies coordinated cotranscriptional folding events that mediate transcription antitermination. Our findings establish TECprobe-VL as an accessible method for mapping cotranscriptional RNA folding pathways.

Engineering U1-Based Tetracycline-Inducible Riboswitches to Control Gene Expression in Mammals.

Synthetic riboswitches are promising regulatory devices due to their small size, lack of immunogenicity, and ability to fine-tune gene expression in the absence of exogenous trans-acting factors. Based on a gene inhibitory system developed at our lab, termed U1snRNP interference (U1i), we developed tetracycline (TC)-inducible riboswitches that modulate mRNA polyadenylation through selective U1 snRNP recruitment. First, we engineered different TC-U1i riboswitches, which repress gene expression unless TC is added, leading to inductions of gene expression of 3-to-4-fold. Second, we developed a technique called Systematic Evolution of Riboswitches by Exponential Enrichment (SEREX), to isolate riboswitches with enhanced U1 snRNP binding capacity and activity, achieving inducibilities of up to 8-fold. Interestingly, by multiplexing riboswitches we increased inductions up to 37-fold. Finally, we demonstrated that U1i-based riboswitches are dose-dependent and reversible and can regulate the expression of reporter and endogenous genes in culture cells and mouse models, resulting in attractive systems for gene therapy applications. Our work probes SEREX as a much-needed technology for the in vitro identification of riboswitches capable of regulating gene expression in vivo.

Structural and dynamic mechanisms for coupled folding and tRNA recognition of a translational T-box riboswitch.

T-box riboswitches are unique riboregulators where gene regulation is mediated through interactions between two highly structured RNAs. Despite extensive structural insights, how RNA-RNA interactions drive the folding and structural transitions of T-box to achieve functional conformations remains unclear. Here, by combining SAXS, single-molecule FRET and computational modeling, we elaborate the folding energy landscape of a translational T-box aptamer consisting of stems I, II and IIA/B, which Mg2+-induced global folding and tRNA binding are cooperatively coupled. smFRET measurements reveal that high Mg2+ stabilizes IIA/B and its stacking on II, which drives the pre-docking of I and II into a competent conformation, subsequent tRNA binding promotes docking of I and II to form a high-affinity tRNA binding groove, of which the essentiality of IIA/B and S-turn in II is substantiated with mutational analysis. We highlight a delicate balance among Mg2+, the intra- and intermolecular RNA-RNA interactions in modulating RNA folding and function.

Single-molecule FRET observes opposing effects of urea and TMAO on structurally similar meso- and thermophilic riboswitch RNAs.

Bacteria live in a broad range of environmental temperatures that require adaptations of their RNA sequences to maintain function. Riboswitches are regulatory RNAs that change conformation upon typically binding metabolite ligands to control bacterial gene expression. The paradigmatic small class-I preQ1 riboswitches from the mesophile Bacillus subtilis (Bsu) and the thermophile Thermoanaerobacter tengcongensis (Tte) adopt similar pseudoknot structures when bound to preQ1. Here, we use UV-melting analysis combined with single-molecule detected chemical denaturation by urea to compare the thermodynamic and kinetic folding properties of the two riboswitches, and the urea-countering effects of trimethylamine N-oxide (TMAO). Our results show that, first, the Tte riboswitch is more thermotolerant than the Bsu riboswitch, despite only subtle sequence differences. Second, using single-molecule FRET, we find that urea destabilizes the folded pseudoknot structure of both riboswitches, yet has a lower impact on the unfolding kinetics of the thermodynamically less stable Bsu riboswitch. Third, our analysis shows that TMAO counteracts urea denaturation and promotes folding of both the riboswitches, albeit with a smaller effect on the more stable Tte riboswitch. Together, these findings elucidate how subtle sequence adaptations in a thermophilic bacterium can stabilize a common RNA structure when a new ecological niche is conquered.

An RNA thermometer in the chloroplast genome of Chlamydomonas facilitates temperature-controlled gene expression.

Riboregulators such as riboswitches and RNA thermometers provide simple, protein-independent tools to control gene expression at the post-transcriptional level. In bacteria, RNA thermometers regulate protein synthesis in response to temperature shifts. Thermometers outside of the bacterial world are rare, and in organellar genomes, no RNA thermometers have been identified to date. Here we report the discovery of an RNA thermometer in a chloroplast gene of the unicellular green alga Chlamydomonas reinhardtii. The thermometer, residing in the 5' untranslated region of the psaA messenger RNA forms a hairpin-type secondary structure that masks the Shine-Dalgarno sequence at 25°C. At 40°C, melting of the secondary structure increases accessibility of the Shine-Dalgarno sequence to initiating ribosomes, thus enhancing protein synthesis. By targeted nucleotide substitutions and transfer of the thermometer into Escherichia coli, we show that the secondary structure is necessary and sufficient to confer the thermometer properties. We also demonstrate that the thermometer provides a valuable tool for inducible transgene expression from the Chlamydomonas plastid genome, in that a simple temperature shift of the algal culture can greatly increase recombinant protein yields.

Genetic dissection of regulation by a repressing and novel activating corrinoid riboswitch enables engineering of synthetic riboswitches.

IMPORTANCE: In addition to proteins, microbes can use structured RNAs such as riboswitches for the important task of regulating gene expression. Riboswitches control gene expression by changing their structure in response to binding a small molecule and are widespread among bacteria. Here we determine the mechanism of regulation in a riboswitch that responds to corrinoids-a family of coenzymes related to vitamin B12. We report the alternative RNA secondary structures that couple corrinoid sensing with response in a repressing and novel activating corrinoid riboswitch. We then applied this knowledge to flipping the regulatory sign by constructing synthetic riboswitches that activate expression to a higher level than the natural one. In the process, we observed patterns in which sequence, in addition to structure, impacts function in paired RNA regions. The synthetic riboswitches we describe here have potential applications as biosensors.

8-oxoguanine riboswitches in bacteria detect and respond to oxidative DNA damage.

Riboswitches rely on structured aptamer domains to selectively sense their target ligands and regulate gene expression. However, some riboswitch aptamers in bacteria carry mutations in their otherwise strictly conserved binding pockets that change ligand specificities. The aptamer domain of a riboswitch class originally found to selectively sense guanine forms a three-stem junction that has since been observed to exploit numerous alterations in its ligand-binding pocket. These rare variants have modified their ligand specificities to sense other purines or purine derivatives, including adenine, 2'-deoxyguanosine (three classes), and xanthine. Herein, we report the characteristics of a rare variant that is narrowly distributed in the Paenibacillaceae family of bacteria. Known representatives are always associated with genes encoding 8-oxoguanine deaminase. As predicted from this gene association, these variant riboswitches tightly bind 8-oxoguanine (8-oxoG), strongly discriminate against other purine derivatives, and function as genetic "ON" switches. Following exposure of cells to certain oxidative stresses, a representative 8-oxoG riboswitch activates gene expression, likely caused by the accumulation of 8-oxoG due to oxidative damage to G nucleobases in DNA, RNA, and the nucleotide pool. Furthermore, an engineered version of the variant aptamer was prepared that exhibits specificity for 8-oxoadenine, further demonstrating that RNA aptamers can acquire mutations that expand their ability to detect and respond to oxidative damage.

Elimination of editing plasmid mediated by theophylline riboswitch in Zymomonas mobilis.

Zymomonas mobilis is regarded as a potential chassis for the production of platform chemicals. Genome editing using the CRISPR-Cas system could meet the need for gene modification in metabolic engineering. However, the low curing efficiency of CRISPR editing plasmid is a common bottleneck in Z. mobilis. In this study, we utilized a theophylline-dependent riboswitch to regulate the expression of the replicase gene of the editing plasmid, thereby promoting the elimination of exogeneous plasmid. The riboswitch D (RSD) with rigorous regulatory ability was identified as the optimal candidate by comparing the transformation efficiency of four theophylline riboswitch-based backbone editing plasmids, and the optimal theophylline concentration for inducing RSD was determined to be 2 mM. A highly effective method for eliminating the editing plasmid, cells with RSD-based editing plasmid which were cultured in liquid and solid RM media in alternating passages at 37 °C without shaking, was established by testing the curing efficiency of backbone editing plasmids pMini and pMini-RSD in RM medium with or without theophylline at 30 °C or 37 °C. Finally, the RSD-based editing plasmid was applied to genome editing, resulting in an increase of more than 10% in plasmid elimination efficiency compared to that of pMini-based editing plasmid. KEY POINTS: • An effective strategy for curing CRISPR editing plasmid has been established in Z. mobilis. • Elimination efficiency of the CRISPR editing plasmid was enhanced by 10% to 20% under the regulation of theophylline-dependent riboswitch RSD.

Rational design of eukaryotic riboswitches that up-regulate IRES-mediated translation initiation with high switching efficiency through a kinetic trapping mechanism in vitro.

In general, riboswitches functioning through a cotranscriptional kinetic trapping mechanism (kt-riboswitches) show higher switching efficiencies in response to practical concentrations of their ligand molecules than eq-riboswitches, which function by an equilibrium mechanism. However, the former have been much more difficult to design due to their more complex mechanism. We here successfully developed a rational strategy for constructing eukaryotic kt-riboswitches that ligand-dependently enhance translation initiation mediated by an internal ribosome entry site (IRES). This was achieved both by utilizing some predicted structural features of a highly efficient bacterial kt-riboswitch identified through screening and by completely decoupling an aptamer domain from the IRES. Three kt-riboswitches optimized through this strategy, each responding to a different ligand, exhibited three- to sevenfold higher induction ratios (up to ∼90) than previously optimized eq-riboswitches regulating the same IRES-mediated translation in wheat germ extract. Because the IRES used functions well in various eukaryotic expression systems, these types of kt-riboswitches are expected to serve as major eukaryotic gene regulators based on RNA. In addition, the present strategy could be applied to the rational construction of other types of kt-riboswitches, including those functioning in bacterial expression systems.

Direct observation of tRNA-chaperoned folding of a dynamic mRNA ensemble.

T-box riboswitches are multi-domain noncoding RNAs that surveil individual amino acid availabilities in most Gram-positive bacteria. T-boxes directly bind specific tRNAs, query their aminoacylation status to detect starvation, and feedback control the transcription or translation of downstream amino-acid metabolic genes. Most T-boxes rapidly recruit their cognate tRNA ligands through an intricate three-way stem I-stem II-tRNA interaction, whose establishment is not understood. Using single-molecule FRET, SAXS, and time-resolved fluorescence, we find that the free T-box RNA assumes a broad distribution of open, semi-open, and closed conformations that only slowly interconvert. tRNA directly binds all three conformers with distinct kinetics, triggers nearly instantaneous collapses of the open conformations, and returns the T-box RNA to their pre-binding conformations upon dissociation. This scissors-like dynamic behavior is enabled by a hinge-like pseudoknot domain which poises the T-box for rapid tRNA-induced domain closure. This study reveals tRNA-chaperoned folding of flexible, multi-domain mRNAs through a Venus flytrap-like mechanism.

Characterization and designing of an SAM riboswitch to establish a high-throughput screening platform for SAM overproduction in Saccharomyces cerevisiae.

S-adenosyl- l-methionine (SAM) is a high-value compound widely used in the treatment of various diseases. SAM can be produced through fermentation, but further enhancing the microbial production of SAM requires novel high-throughput screening methods for rapid detection and screening of mutant libraries. In this work, an SAM-OFF riboswitch capable of responding to the SAM concentration was obtained and a high-throughput platform for screening SAM overproducers was established. SAM synthase was engineered by semirational design and directed evolution, which resulted in the SAM2S203F,W164R,T251S,Y285F,S365R mutant with almost twice higher catalytic activity than the parental enzyme. The best mutant was then introduced into Saccharomyces cerevisiae BY4741, and the resulting strain BSM8 produced a sevenfold higher SAM titer in shake-flask fermentation, reaching 1.25 g L-1 . This work provides a reference for designing biosensors to dynamically detect metabolite concentrations for high-throughput screening and the construction of effective microbial cell factories.

Exploring L-isoleucine riboswitches for enhancing 4-hydroxyisoleucine production in Corynebacterium glutamicum.

OBJECTIVES: To explore an L-isoleucine (Ile)-induced biosensor for down-regulation of Ile synthesis pathway and enhancement of 4-hydroxyisoleucine (4-HIL) production in Corynebacterium glutamicum SN01. RESULTS: Four Ile-induced riboswitches (IleRSN) with different strength were screened from mutation library based on TPP riboswitch. Firstly, IleRSN were integrated into the chromosome of strain SN01 immediately upstream of ilvA gene. The 4-HIL titer of strains carrying PtacM-driven IleRS1 or IleRS3 (14.09 ± 1.07, 15.20 ± 0.93 g 4-HIL L-1) were similar with control strain S-D5I (15.73 ± 2.66 g 4-HIL L-1). Then, another copy of IleRS3-ilvA was integrated downstream of the chromosomal cg0963 gene in SN01-derived strain D-RS with down-regulated L-lysine (Lys) biosynthesis. The Ile supply and 4-HIL titer increased in ilvA two-copy strains KIRSA-3-D5I and KIRSA-3-9I, and Ile concentration was maintained less than 35 mmol L-1 under the control of IleRS3 during fermentation. The resulting strain KIRSA-3-9I produced 22.46 ± 0.96 g 4-HIL L-1. CONCLUSION: The screened IleRS was effective in the dynamic down-regulation of Ile synthesis pathway in C. glutamicum, and IleRSN with different strength can be applied in various conditions.