Riboswitch Folding Research Articles

Sequential structure probing of cotranscriptional RNA folding intermediates.

Cotranscriptional RNA folding pathways typically involve the sequential formation of folding intermediates. Existing methods for cotranscriptional RNA structure probing map the structure of nascent RNA in the context of a terminally arrested transcription elongation complex. Consequently, the rearrangement of RNA structures as nucleotides are added to the transcript can be inferred but is not assessed directly. To address this limitation, we have developed linked- m ultipoint T ranscription E longation C omplex RNA structure prob ing (TECprobe-LM), which assesses the cotranscriptional rearrangement of RNA structures by sequentially positioning E. coli RNAP at two or more points within a DNA template so that nascent RNA can be chemically probed. We validated TECprobe-LM by measuring known folding events that occur within the E. coli signal recognition particle RNA, Clostridium beijerinckii pfl ZTP riboswitch, and Bacillus cereus crcB fluoride riboswitch folding pathways. Our findings establish TECprobe-LM as a strategy for detecting cotranscriptional RNA folding events directly using chemical probing.

Thermodynamic compensation to temperature extremes in B. subtilis vs T. maritima lysine riboswitches

T. maritima and B. subtilis are bacteria that inhabit significantly different thermal environments, ∼80 vs. ∼40°C, yet employ similar lysine riboswitches to aid in the transcriptional regulation of the genes involved in the synthesis and transport of amino acids. Despite notable differences in G-C basepair frequency and primary sequence, the aptamer moieties of each riboswitch have striking similarities in tertiary structure, with several conserved motifs and long-range interactions. To explore genetic adaptation in extreme thermal environments, we compare the kinetic and thermodynamic behaviors in T. maritima and B. subtilis lysine riboswitches via single-molecule fluorescence resonance energy transfer analysis. Kinetic studies reveal that riboswitch folding rates increase with lysine concentration while the unfolding rates are independent of lysine. This indicates that both riboswitches bind lysine through an induced-fit (“bind-then-fold”) mechanism, with lysine binding necessarily preceding conformational changes. Temperature-dependent van’t Hoff studies reveal qualitative similarities in the thermodynamic landscapes for both riboswitches in which progression from the open, lysine-unbound state to both transition states (‡) and closed, lysine-bound conformations is enthalpically favored yet entropically penalized, with comparisons of enthalpic and entropic contributions extrapolated to a common [K+] = 100 mM in quantitative agreement. Finally, temperature-dependent Eyring analysis reveals the TMA and BSU riboswitches to have remarkably similar folding/unfolding rate constants when extrapolated to their respective (40 and 80°C) environmental temperatures. Such behavior suggests a shared strategy for ligand binding and aptamer conformational change in the two riboswitches, based on thermodynamic adaptations in number of G-C basepairs and/or modifications in tertiary structure that stabilize the ligand-unbound conformation to achieve biocompetence under both hyperthermophilic and mesothermophilic conditions.

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.

Mechanism of Fluoride Ion Encapsulation by Magnesium Ions in a Bacterial Riboswitch.

Riboswitches sense various ions in bacteria and activate gene expression to synthesize proteins that help maintain ion homeostasis. The crystal structure of the aptamer domain (AD) of the fluoride riboswitch shows that the F- ion is encapsulated by three Mg2+ ions bound to the ligand-binding domain (LBD) located at the core of the AD. The assembly mechanism of this intricate structure is unknown. To this end, we performed computer simulations using coarse-grained and all-atom RNA models to bridge multiple time scales involved in riboswitch folding and ion binding. We show that F- encapsulation by the Mg2+ ions bound to the riboswitch involves multiple sequential steps. Broadly, two Mg2+ ions initially interact with the phosphate groups of the LBD using water-mediated outer-shell coordination and transition to a direct inner-shell interaction through dehydration to strengthen their interaction with the LBD. We propose that the efficient binding mode of the third Mg2+ and F- is that they form a water-mediated ion pair and bind to the LBD simultaneously to minimize the electrostatic repulsion between three Mg2+ bound to the LBD. The tertiary stacking interactions among the LBD nucleobases alone are insufficient to stabilize the alignment of the phosphate groups to facilitate Mg2+ binding. We show that the stability of the whole assembly is an intricate balance of the interactions among the five phosphate groups, three Mg2+, and the encapsulated F- ion aided by the Mg2+ solvated water. These insights are helpful in the rational design of RNA-based ion sensors and fast-switching logic gates.

Linking folding dynamics and function of SAM/SAH riboswitches at the single molecule level.

Riboswitches are regulatory elements found in bacterial mRNAs that control downstream gene expression through ligand-induced conformational changes. Here, we used single-molecule FRET to map the conformational landscape of the translational SAM/SAH riboswitch and probe how co-transcriptional ligand-induced conformational changes affect its translation regulation function. Riboswitch folding is highly heterogeneous, suggesting a rugged conformational landscape that allows for sampling of the ligand-bound conformation even in the absence of ligand. The addition of ligand shifts the landscape, favoring the ligand-bound conformation. Mutation studies identified a key structural element, the pseudoknot helix, that is crucial for determining ligand-free conformations and their ligand responsiveness. We also investigated ribosomal binding site accessibility under two scenarios: pre-folding and co-transcriptional folding. The regulatory function of the SAM/SAH riboswitch involves kinetically favoring ligand binding, but co-transcriptional folding reduces this preference with a less compact initial conformation that exposes the Shine-Dalgarno sequence and takes min to redistribute to more compact conformations of the pre-folded riboswitch. Such slow equilibration decreases the effective ligand affinity. Overall, our study provides a deeper understanding of the complex folding process and how the riboswitch adapts its folding pattern in response to ligand, modulates ribosome accessibilityand the role of co-transcriptional folding in these processes.

Structural basis for control of bacterial RNA polymerase pausing by a riboswitch and its ligand.

Folding of nascent transcripts can be modulated by the RNA polymerase (RNAP) that carries out their transcription, and vice versa. A pause of RNAP during transcription of a preQ1 riboswitch (termed que-PEC) is stabilized by a previously characterized template consensus sequence and the ligand-free conformation of the nascent RNA. Ligand binding to the riboswitch induces RNAP pause release and downstream transcription termination; however, the mechanism by which riboswitch folding modulates pausing is unclear. Here, we report single-particle cryo-electron microscopy reconstructions of que-PEC in ligand-free and ligand-bound states. In the absence of preQ1, the RNA transcript is in an unexpected hyper-translocated state, preventing downstream nucleotide incorporation. Strikingly, on ligand binding, the riboswitch rotates around its helical axis, expanding the surrounding RNAP exit channel and repositioning the transcript for elongation. Our study reveals the tight coupling by which nascent RNA structures and their ligands can functionally regulate the macromolecular transcription machinery.

Ionic Cooperativity between Lysine and Potassium in the Lysine Riboswitch: Single-Molecule Kinetic and Thermodynamic Studies.

Functionality in many biological systems, including proteins and nucleic acid structures, including protein and nucleic acid riboswitch structures, can depend on cooperative kinetic behavior between multiple small molecule ligands. In this work, single-molecule FRET data on the Bacillus subtilis lysine riboswitch reveals that affinity for the cognate lysine ligand increases significantly with K+, providing evidence for synergism between lysine/K+ binding to the aptamer and successful folding of the riboswitch. To describe/interpret this more complex kinetic scenario, we explore the conventional 4-state ("square") model for aptamer binding as a function of K+. Extension into this additional dimension generates a novel "cube" model for riboswitch folding dynamics with respect to lysine/K+ binding, revealing that riboswitch folding (kfold) and unfolding (kunfold) rate constants increase and decrease dramatically with K+, respectively. Furthermore, temperature-dependent single-molecule kinetic studies indicate that the presence of K+ entropically enhances the transition state barrier to folding but partially compensates for this by increasing the overall exothermicity for lysine binding. We rationalize this behavior as evidence that K+ facilitates hydrogen bonding between the negatively charged carboxyl group of lysine and the RNA, increasing structural rigidity and lowering entropy in the binding pocket. Finally, we explore the effects of cation size with Na+ and Cs+ studies to demonstrate that K+ is optimally suited for bridging interactions between lysine and the riboswitch aptamer domain. Regulation of lysine production and transport, dictated by the riboswitch's ability to recognize and bind lysine, is therefore intimately tied to the presence of K+ in the binding pocket and is strongly modulated by local cation conditions. The results suggest an increase in lysine riboswitch functionality by sensitivity to additional species in the cellular riboswitch environment.

Ligand-Dependent Volumetric Characterization of Manganese Riboswitch Folding: A High-Pressure Single-Molecule Kinetic Study.

Nanoscopic differences in free volume result in pressure-dependent changes in free energies which can therefore impact folding/unfolding stability of biomolecules. Although such effects are typically insignificant under ambient pressure conditions, they are crucially important for deep ocean marine life, where the hydraulic pressure can be on the kilobar scale. In this work, single molecule FRET spectroscopy is used to study the effects of pressure on both the kinetics and overall thermodynamics for folding/unfolding of the manganese riboswitch. Detailed pressure-dependent analysis of the conformational kinetics allows one to extract precision changes (σ ≲ 4-8 Å3) in free volumes not only between the fully folded/unfolded conformations but also with respect to the folding transition state of the manganese riboswitch. This permits first extraction of a novel "reversible work" free energy (PΔV) landscape, which reveals a monotonic increase in manganese riboswitch volume along the folding coordinate. Furthermore, such a tool permits exploration of pressure-dependent effects on both Mn2+ binding and riboswitch folding, which demonstrate that ligand attachment stabilizes the riboswitch under pressure by decreasing the volume increase upon folding (ΔΔV < 0). Such competition between ligand binding and pressure-induced denaturation dynamics could be of significant evolutionary advantage, compensating for a weakening in riboswitch tertiary structure with pressure-mediated ligand binding and promotion of folding response.

Synergism in the Molecular Crowding of Ligand-Induced Riboswitch Folding: Kinetic/Thermodynamic Insights from Single-Molecule Spectroscopy.

Conformational dynamics in riboswitches involves ligand binding and folding of RNA, each of which can be influenced by excluded volume effects under "crowded" in vivo cellular conditions and thus incompletely characterized by in vitro studies under dilute buffer conditions. In this work, temperature-dependent single-molecule fluorescence resonance energy transfer (FRET) spectroscopy is used to characterize the thermodynamics of (i) cognate ligand and (ii) molecular crowders (PEG, polyethylene glycol) on folding of the B. subtilis LysC lysine riboswitch. With the help of detailed kinetic analysis, we isolate and study the effects of PEG on lysine binding and riboswitch folding steps individually, from which we find that PEG crowding facilitates riboswitch folding primarily via a surprising increase in affinity for the cognate ligand. This is furthermore confirmed by temperature-dependent studies, which reveal that PEG crowding is not purely entropic and instead significantly impacts both enthalpic and entropic contributions to the free energy landscape for folding. The results indicate that PEG molecular crowding/stabilization of the lysine riboswitch is more mechanistically complex and requires extension beyond the conventional picture of purely repulsive solvent-solute steric interactions arising from excluded volume and entropy. Instead, the current experimental FRET data support an alternative multistep mechanism, whereby PEG first entropically crowds the unfolded riboswitch into a "pre-folded" conformation, which in turn greatly increases the ligand binding affinity and thereby enhances the overall equilibrium for riboswitch folding.

Alkaline pH has an unexpected effect on transcriptional pausing during synthesis of the Escherichia coli pH-responsive riboswitch

Riboswitches are 5′-untranslated regions of mRNA that change their conformation in response to ligand binding, allowing post-transcriptional gene regulation. This ligand-based model of riboswitch function has been expanded with the discovery of a “pH-responsive element” (PRE) riboswitch in Escherichia coli. At neutral pH, the PRE folds into a translationally inactive structure with an occluded ribosome-binding sequence, whereas at alkaline pH, the PRE adopts a translationally active structure. This unique riboswitch does not rely on ligand binding in a traditional sense to modulate its alternative folding outcomes. Rather, pH controls riboswitch folding by two possible modes that are yet to be distinguished; pH either regulates the transcription rate of RNA polymerase (RNAP) or acts on the RNA itself. Previous work suggested that RNAP pausing is prolonged by alkaline pH at two sites, stimulating PRE folding into the active structure. To date, there has been no rigorous exploration into how pH influences RNAP pausing kinetics during PRE synthesis. To provide that understanding and distinguish between pH acting on RNAP versus RNA, we investigated RNAP pausing kinetics at key sites for PRE folding under different pH conditions. We find that pH influences RNAP pausing but not in the manner proposed previously. Rather, alkaline pH either decreases or has no effect on RNAP pause longevity, suggesting that the modulation of RNAP pausing is not the sole mechanism by which pH affects PRE folding. These findings invite the possibility that the RNA itself actively participates in the sensing of pH.

Affinity-Based Profiling of the Flavin Mononucleotide Riboswitch.

Riboswitches are structural RNA elements that control gene expression. These naturally occurring RNA sensors are of continued interest as antibiotic targets, molecular sensors, and functional elements of synthetic circuits. Here, we describe affinity-based profiling of the flavin mononucleotide (FMN) riboswitch to characterize ligand binding and structural folding. We designed and synthesized photoreactive ligands and used them for photoaffinity labeling. We showed selective labeling of the FMN riboswitch and used this covalent interaction to quantitatively measure ligand binding, which we demonstrate with the naturally occurring antibiotic roseoflavin. We measured conditional riboswitch folding as a function of temperature and cation concentration. Furthermore, combining photoaffinity labeling with reverse transcription revealed ligand binding sites within the aptamer domain with single-nucleotide resolution. The photoaffinity probe was applied to cellular extracts of Bacillus subtilis to demonstrate conditional folding of the endogenous low-abundant ribD FMN riboswitch in biologically derived samples using quantitative PCR. Lastly, binding of the riboswitch-targeting antibiotic roseoflavin to the FMN riboswitch was measured in live bacteria using the photoaffinity probe.

Structural and mechanistic study of a pH-responsive riboswitch

Riboswitches are structured, 5'-untranslated pieces of a messenger RNA that control whether the downstream transcript gets translated into protein. Recently, a “pH-responsive element” (PRE) riboswitch was uncovered at the alx gene locus in E. coli. At neutral pH, the PRE folds into a translationally inactive structure with an occluded ribosome binding sequence, while at alkaline pH, the PRE adopts a translationally active structure. In this unique riboswitch system, pH governs riboswitch folding by regulating the transcription rate of RNA polymerase (RNAP).

Effect of ligand binding on riboswitch folding: Theory and simulations.

The effect of ligand binding on the conformational transitions of the add A-riboswitch in cellular environments is investigated theoretically within the framework of the generalized Langevin equation combined with steered molecular dynamics simulations. Results for the transition path time distribution provide an estimate of the transit times, which are difficult to determine experimentally. The time for the conformational transitions of the riboswitch aptamer is longer for the ligand bound state as compared to that of the unbound one. The transition path time of the riboswitch follows a counterintuitive trend as it decreases with an increase in the barrier height. The mean transition path time of either transitions of the riboswitch in the ligand bound/unbound state increases with an increase in the complexity of the surrounding environment due to the caging effect. The results of the probability density function, transition path time distribution, and mean transition path time obtained from the theory qualitatively agree with those obtained from the simulations and with earlier experimental and theoretical studies.

A translational riboswitch coordinates nascent transcription-translation coupling.

Bacterial messenger RNA (mRNA) synthesis by RNA polymerase (RNAP) and first-round translation by the ribosome are often coupled to regulate gene expression, yet how coupling is established and maintained is ill understood. Here, we develop biochemical and single-molecule fluorescence approaches to probe the dynamics of RNAP-ribosome interactions on an mRNA with a translational preQ1-sensing riboswitch in its 5' untranslated region. Binding of preQ1 leads to the occlusion of the ribosome binding site (RBS), inhibiting translation initiation. We demonstrate that RNAP poised within the mRNA leader region promotes ribosomal 30S subunit binding, antagonizing preQ1-induced RBS occlusion, and that the RNAP-30S bridging transcription factors NusG and RfaH distinctly enhance 30S recruitment and retention, respectively. We further find that, while 30S-mRNA interaction significantly impedes RNAP in the absence of translation, an actively translating ribosome promotes productive transcription. A model emerges wherein mRNA structure and transcription factors coordinate to dynamically modulate the efficiency of transcription-translation coupling.

Magnesium Mediated Fluoride Binding Anchors the Docked Conformation of the Fluoride Riboswitch

Riboswitches are structured non-coding RNA elements typically found in the 5' untranslated regions of bacterial mRNAs. They regulate gene expression, most often at the level of transcription and translation, through the binding of specific ligands that induce changes in the riboswitch structure. Fluoride riboswitches are a family of transcriptional riboswitches that are present in many bacteria and archaea; regulate transcription by preventing the formation of an intrinsic terminator in presence of fluoride. Previous studies have shed light on its structure and the importance of the transcriptional machinery for riboswitch folding, a deep conformational analysis at different points during transcription are needed to fully understand the impact of ligand-binding during transcription. Therefore, we explored the conformational dynamics of the Bacillus cereus crcB fluoride riboswitch at the single-molecule level for both isolated RNA and active transcription elongation complexes. We resolved three conformational states: two undocked and one pseudoknot-structured docked state, and determined their fractional population and interconversion rates. The first undocked state was observed at early stage of transcription; while the other two states, a docked and an undocked state, appear when the aptamer is fully transcribed by RNA polymerase. The riboswitch folds into a transient dynamic docked conformation in presence of magnesium, which upon further binding of fluoride folds into a stably docked conformation. This feature is lost upon disrupting a long-range base pair A40-U48. In active transcription elongation complexes, RNA polymerase fine-tunes the activation energy and promotes the riboswitch to fold into the docked conformation. Further, we showed that ligand binding at short transcript lengths is an early step on the pathway to the docked conformation. These results demonstrate a multi-step mechanism of magnesium- and ligand-dependent conformational transitions that enable regulation of downstream gene expression.

Sequential Folding of the Nickel/Cobalt Riboswitch Is Facilitated by a Conformational Intermediate: Insights from Single-Molecule Kinetics and Thermodynamics.

The present work presents the first single-molecule fluorescence resonant energy transfer (smFRET) studies of the nickel/cobalt (NiCo) riboswitch, with temperature-dependent, single-molecule confocal microscopy to provide comprehensive kinetic and thermodynamic information on folding into a biochemically competent structure. The results indicate that the NiCo riboswitch first folds into a more compact "prefolded" conformation, with a preorganized binding pocket partially stabilized under physiological conditions by noncognate monovalent/divalent cations. Such a prefolded intermediate then has opportunity to fold further into a tightly ligand-bound structure, in response to the cognate ligands, Ni2+ or Co2+, with submicromolar affinities. Such stepwise ligand-induced folding represents a particularly clean example of a conformational selection ("fold-then-bind") mechanism, whereby a configuration dynamically accessible by thermal fluctuation is stabilized into the final folded state by ligand association. In addition, we observe a strong positive cooperativity in the ligand-induced folding kinetics with respect to both Ni2+ and Co2+ ligands. This provides maximal sensitivity in the riboswitch conformational response near [Ni2+] or [Co2+] ≈ Kd, which facilitates more accurate biochemical probing of the cell environment and therefore bioregulation of gene expression. Temperature-dependent kinetics at the single-molecule level has also been explored, which permits free energies to be deconstructed into enthalpic and entropic components along the folding coordinate. In the absence of the cognate ligand, a predominantly enthalpic barrier between the unfolded riboswitch (U) and the prefolded intermediate (I) suggests a rearrangement of the hydrogen bonding network, whereas in the presence of the cognate ligand, a large entropic penalty (-TΔS0 > 0) in forming the folded riboswitch conformation (F) is almost perfectly counterbalanced by an equivalent enthalpic gain (ΔH0 < 0) to yield ΔG0 ≈ 0. The thermodynamic results are therefore consistent with a simple physical picture of riboswitch folding, whereby association of the cognate ligand is strongly stabilized by Coulombic attraction while forming an entropically more ordered structure around the binding site.

Single-Molecule FRET Kinetics of the Mn2+ Riboswitch: Evidence for Allosteric Mg2+ Control of "Induced-Fit" vs "Conformational Selection" Folding Pathways.

Gene expression in bacteria is often regulated dynamically by conformational changes in a riboswitch upon ligand binding, a detailed understanding of which is very much in its infancy. For example, the manganese riboswitch is a widespread RNA motif that conformationally responds in regulating bacterial gene expression to micromolar levels of its eponymous ligand, Mn2+, but the mechanistic pathways are poorly understood. In this work, we quantitatively explore the dynamic folding behavior of the manganese riboswitch by single-molecule fluorescence resonance energy transfer spectroscopy as a function of cation/ligand conditions. From the detailed analysis of the kinetics, the Mn2+ is shown to fold the riboswitch by a "bind-then-fold" (i.e., "induced-fit", IF) mechanism, whereby the ligand binds first and then promotes folding. On the other hand, the data also clearly reveal the presence of a folded yet ligand-free structure predominating due to the addition of physiological Mg2+ to a nonselective metal ion binding site. Of particular kinetic interest, such a Mg2+ "prefolded" conformation of the riboswitch is shown to exhibit a significantly increased affinity for Mn2+ and further stabilization by subsequent binding of the ligand, thereby promoting efficient riboswitch folding by a "fold-then-bind" (i.e., "conformational selection", CS) mechanism. Our results not only demonstrate Mg2+-controlled switching between IF and CS riboswitch folding pathways but also suggest a novel heterotropic allosteric control in the manganese riboswitch activity co-regulated by Mg2+ binding.

Single Molecule Snapshots of Riboswitch Conformational Change and RNA Switch Based Biosensing on a Nanopore Maglet Device

Riboswitches are a class of biosensory-regulatory segments of messenger-RNAs. Their binding with specific cellular ligands e.g. metabolic intermediates, can induce a conformational change in riboswitch's tertiary structure, ultimately programming the production of the proteins encoded by the mRNA. Precision tools to explore ligand-programmed riboswitch folding/unfolding mechanism are important in both developing riboswitch-targeted antibiotic intervention and RNA switch-based biosensors. Our nanopore weak-interaction spectroscopy can snapshot a single RNA riboswitch's folding/unfolding pathway at single base-pair resolution. Different from thermodynamic approaches and force microscopies (e.g. AFM and optic tweeze), the unique feature of nanopore is unfolding along the backbone of RNA-Vectorial Unfolding. This tool enables us to discriminate RNA unfolding pathway in both 3’>5’ and 5’>3’ directions, dissect the non-canonical triple-strand conformations and interactions that control the RNA stability, and many interconnected folding intermediates. Based on these findings, we further develop a nanopore maglet automatic machine that can “lock” a single riboswitch (preQ1) molecule in the nanopore. The locked single RNA can be electrically driven to repeatedly move back and forth to transform among different conformations: partial folding→ligand binding →fully folding→Unfolding. We can probe the folding change of riboswitch upon ligand (preQ1) binding, explore riboswitch's induced fit and/or conformational selection folding mechanism, and study how designed drug compounds regulate the riboswitch folding pathway. Finally, this nanopore molecular machine can be utilized as RNA switch-based biosensor for detection a variety of disease-derived ligands and proteins.

Detailed Kinetics of RNA Folding Pathways and Thermodynamic Origins of Crowding by Single-Molecule FRET

Specific three dimensional folding is required for RNA function while RNA can be a highly dynamic system that actively samples different configurations under thermal fluctuations. For instance, in gene regulation of RNA riboswitch, the prompt structural transition in response to ligand is crucial for triggering transcriptional termination, making the time scale of folding and ligand response exceedingly relevant to RNA functions. In single-molecule FRET (smFRET) experiments, the conformational changes of RNA are visualized by the time-dependent energy transfer to determine folding and unfolding rates. Such kinetic information allows us to construct a model for manganese riboswitch folding and explore the detailed folding mechanisms, where Mg2+ binding is found to pre-fold the RNA, followed by ligand (Mn2+) association to further stabilize the folded structure. Moreover, the thermodynamic origins can be obtained from temperature dependent folding. The manganese riboswitch folding is found unexpectedly promoted by heat and the process is endothermic with net entropy gain, much contradictory to the conventional idea of folding into a more order state. In molecular crowding study where we try to understand the influence of fundamental excluded volume on RNA structures in highly concentrated intracellular environments, the chemically inert crowders are shown to entropically favor the more compact folded structure of RNA; a simple physical picture of osmotic pressure is employed to explain the crowder size dependence. Lastly, a hydraulic pressure generator is used to apply pressure (up to 5 kbar) to RNA sample through capillary. The free volume change (pΔV) during RNA folding may be related to structural response under crowding conditions.

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.