Aptamer Domain Research Articles

Adenine protonation enables cyclic-di-GMP binding to cyclic-GAMP sensing riboswitches.

In certain structural or functional contexts, RNA structures can contain protonated nucleotides. However, a direct role for stably protonated nucleotides in ligand binding and ligand recognition has not yet been demonstrated unambiguously. Previous X-ray structures of c-GAMP binding riboswitch aptamer domains in complex with their near-cognate ligand c-di-GMP suggest that an adenine of the riboswitch either forms two hydrogen bonds to a G nucleotide of the ligand in the unusual enol tautomeric form or that the adenine in its N1 protonated form binds the G nucleotide of the ligand in its canonical keto tautomeric state. By using NMR spectroscopy we demonstrate that the c-GAMP riboswitches bind c-di-GMP using a stably protonated adenine in the ligand binding pocket. Thereby, we provide novel insights into the putative biological functions of protonated nucleotides in RNA, which in this case influence the ligand selectivity in a riboswitch.

Conformational Dynamics of thiM Riboswitch To Understand the Gene Regulation Mechanism Using Markov State Modeling and the Residual Fluctuation Network Approach.

Thiamine pyrophosphate (TPP) riboswitch is a cis-regulatory element in the noncoding region of mRNA. The aptamer domain of TPP riboswitch detects the high abundance of coenzyme thiamine pyrophosphate (TPP) and modulates the gene expression for thiamine synthetic gene. The mechanistic understanding in recognition of TPP in aptamer domain and ligand-induced compactness for folding of expression platform are most important to designing novel modulators. To understand the dynamic behavior of TPP riboswitch upon TPP binding, molecular dynamics simulations were performed for 400 ns in both apo and TPP bound forms of thiM riboswitch from E. coli and analyzed in terms of eRMSD-based Markov state modeling and residual fluctuation network. Markov state models show good correlations in transition probability among metastable states from simulated trajectory and generated models. Structural compactness in TPP bound form is observed which is correlated with SAXS experiment. The importance of junction of P4 and P5 is evident during dynamics, which correlates with FRET analysis. The dynamic nature of two sensor forearms is due to the flexible P1 helix, which is its intrinsic property. The transient state in TPP-bound form was observed in the Markov state model, along with stable states. We believe that this transient state is responsible to assist the influx and outflux of ligand molecule by creating a solvent channel around the junction region of P4 and P5 and such a structure was anticipated in FRET analysis. The dynamic nature of riboswitch is dependent on the interaction between residues on distal loops L3 and L5/P3 and junction P4 and P5, J3/2 which stabilize the J2/4. It helps in the transfer of allosteric information between J2/4 and P3/L5 tertiary docking region through the active site residues. Understanding such information flow will benefit in highlighting crucial residues in highly dynamic and kinetic systems. Here, we report the residues and segments in riboswitch that play vital roles in providing stability and this can be exploited in designing inhibitors to regulate the functioning of riboswitches.

High-Yield Spin Labeling of Long RNAs for Electron Paramagnetic Resonance Spectroscopy.

Site-directed spin labeling is a powerful tool for investigating the conformation and dynamics of biomacromolecules such as RNA. Here we introduce a spin labeling strategy based on click chemistry in solution that, in combination with enzymatic ligation, allows highly efficient labeling of complex and long RNAs with short reaction times and suppressed RNA degradation. With this approach, a 34-nucleotide aptamer domain of the preQ1 riboswitch and an 81-nucleotide TPP riboswitch aptamer could be labeled with two labels in several positions. We then show that conformations of the preQ1 aptamer and its dynamics can be monitored in the absence and presence of Mg2+ and a preQ1 ligand by continuous wave electron paramagnetic resonance spectroscopy at room temperature and pulsed electron-electron double resonance spectroscopy (PELDOR or DEER) in the frozen state.

Crowding Shifts the FMN Recognition Mechanism of Riboswitch Aptamer from Conformational Selection to Induced Fit.

In bacteria, the binding between the riboswitch aptamer domain and ligand is regulated by environmental cues, such as low Mg2+ in macrophages during pathogenesis to ensure spatiotemporal expression of virulence genes. Binding was investigated between the flavin mononucleotide (FMN) riboswitch aptamer and its anionic ligand in the presence of molecular crowding agent without Mg2+ ion, which mimics pathogenic conditions. Structural, kinetic, and thermodynamic analyses under the crowding revealed more dynamic conformational rearrangements of the FMN riboswitch aptamer compared to dilute Mg2+ -containing solution. It is hypothesized that under crowding conditions FMN binds through an induced fit mechanism in contrast to the conformational selection mechanism previously demonstrated in dilute Mg2+ solution. Since these two mechanisms involve different conformational intermediates and rate constants, these findings have practical significance in areas such as drug design and RNA engineering.

Crowding Shifts the FMN Recognition Mechanism of Riboswitch Aptamer from Conformational Selection to Induced Fit

AbstractIn bacteria, the binding between the riboswitch aptamer domain and ligand is regulated by environmental cues, such as low Mg2+ in macrophages during pathogenesis to ensure spatiotemporal expression of virulence genes. Binding was investigated between the flavin mononucleotide (FMN) riboswitch aptamer and its anionic ligand in the presence of molecular crowding agent without Mg2+ ion, which mimics pathogenic conditions. Structural, kinetic, and thermodynamic analyses under the crowding revealed more dynamic conformational rearrangements of the FMN riboswitch aptamer compared to dilute Mg2+‐containing solution. It is hypothesized that under crowding conditions FMN binds through an induced fit mechanism in contrast to the conformational selection mechanism previously demonstrated in dilute Mg2+solution. Since these two mechanisms involve different conformational intermediates and rate constants, these findings have practical significance in areas such as drug design and RNA engineering.

Recognition of cyclic-di-GMP by a riboswitch conducts translational repression through masking the ribosome-binding site distant from the aptamer domain.

The riboswitch is a class of RNA-based gene regulatory machinery that is dependent on recognition of its target ligand by RNA tertiary structures. Ligand recognition is achieved by the aptamer domain, and ligand-dependent structural changes of the expression platform then usually mediate termination of transcription or translational initiation. Ligand-dependent structural changes of the aptamer domain and expression platform have been reported for several riboswitches with short (<40 nucleotides) expression platforms. In this study, we characterized structural changes of the Vc2 c-di-GMP riboswitch that represses translation of downstream open reading frames in a ligand-dependent manner. The Vc2 riboswitch has a long (97 nucleotides) expression platform, but its structure and function are largely unknown. Through mutational analysis and chemical probing, we identified its secondary structures that are possibly responsible for switch-OFF and switch-ON states of translational initiation.

High Temporal- and Spatial-Resolution Studies of a Helix-to-Coil Transition that Controls the Switching Mechanism of a Riboswitch

Riboswitches are structural elements that are found in the 5’ untranslated regions of many messenger RNAs (mRNAs) and that undergo ligand-dependent structural rearrangements that regulate transcription, splicing, translation, and/or stability of the corresponding mRNA. Because they are typically found in bacteria, and because the majority of bacterial riboswitches lack counterparts in archaea and eukaryotes, riboswitches remain promising antibacterial drug targets. Architecturally, riboswitches are composed of a ‘switching sequence’ that overlaps with an upstream ligand-binding ‘aptamer domain’ and a downstream ‘expression platform’. Ligand-dependent genetic control is achieved when binding of the ligand to the aptamer domain modulates the base-pairing interactions of the switching sequence such that the expression platform adopts a secondary structure that triggers expression or repression of the mRNA. In the prototypical adenine-responsive riboswitch that regulates transcription of the pbuE gene in Bacillus subtilis, the base-pairing interactions of the switching sequence are modulated subsequent to transcription of the expression platform: in the absence of adenine, the switching sequence pairs with the expression platform to form a transcription terminator hairpin, whereas, in the presence of adenine, the switching sequence is prevented from pairing with the expression platform, allowing transcription to proceed.Using a new and powerful single-molecule biophysical approach employing single-molecule field effect transistors (smFETs), we investigated the dynamics of a helix-to-coil transition that controls the base-pairing interactions of the switching sequence of the adenine-responsive pbuE riboswitch at an unprecedented temporal resolution of 40 μsec per measurement and spatial resolution of a single base pair. Our studies have allowed us to investigate how base-pairing- and adenine-binding interactions that are distal to the switching sequence collaborate to allosterically modulate the dynamics of the helix-to-coil transition and, consequently, control transcription of the pbuE gene.

Calculation of Ion-Dependent RNA Folding Free Energy using Coarse-Grained Simulation

Magnesium ions (Mg2+) are fundamentally important in RNA biology, playing indispensable roles in RNA structure, folding and function. In order to develop a model that treats monovalent ions implicitly while retaining explicit divalent cations we developed a theory based on liquid state integral equation theory that captures both inner shell and outer shell bindings of Mg to phosphate groups. Coupling this approach with our thermodynamically consistent RNA model reproduces Mg2+-RNA free energies for several RNA molecules, ranging from a small pseudoknot to the aptamer domain of adenine riboswitch. The model not only works for the RNA folded state, but also can be used to give accurate Mg2+-RNA thermodynamics for intermediate and unfolded states. In addition, we provide here one of the first attempts to elucidate the full three dimensional distribution of Mg2+ ions around RNA. Comparing Mg2+ with Ca2+ , it is revealed that magnesium binds using inner shell and outer shell equally well while calcium prefers to bind directly with phosphate groups due to the ease of dehydration of the lower charge density cation. Our model provides a comprehensive treatment of Mg2+-RNA interaction in terms of both energetic and structural features, allowing us to study ion-dependent RNA folding.

The multi-state energy landscape of the SAM-I riboswitch: A single-molecule Förster resonance energy transfer spectroscopy study.

RNA (ribonucleic acid) molecules are highly flexible biopolymers fluctuating at physiological temperatures among many different conformations that are represented by minima in a hierarchical conformational free energy landscape. Here we have employed single-molecule FRET (smFRET) to explore the energy landscape of the B. subtilis yitJ SAM-I riboswitch (RS). In this small RNA molecule, specific binding of an S-adenosyl-L-methionine (SAM) ligand in the aptamer domain regulates gene expression by inducing structural changes in another domain, the expression platform, causing transcription termination by the RNA polymerase. We have measured smFRET histograms over wide ranges of Mg2+ concentration for three RS variants that were specifically labeled with fluorescent dyes on different sites. In the analysis, different conformations are associated with discrete Gaussian model distributions, which are typically fairly broad on the FRET efficiency scale and thus can be extremely challenging to unravel due to their mutual overlap. Our earlier work on two SAM-I RS variants revealed four major conformations. By introducing a global fitting procedure which models both the Mg2+ concentration dependencies of the fractional populations and the average FRET efficiencies of the individual FRET distributions according to Mg2+ binding isotherms, we were able to consistently describe the histogram data of both variants at all studied Mg2+ concentrations. With the third FRET-labeled variant, however, we found significant deviations when applying the four-state model to the data. This can arise because the different FRET labeling of the new variant allows two states to be distinguished that were previously not separable due to overlap. Indeed, the resulting five-state model presented here consistently describes the smFRET histograms of all three variants as well as their variations with Mg2+ concentration. We also performed a triangulation of the donor position for two of the constructs to explore how the expression platform is oriented with respect to the aptamer.

Folding behaviors of purine riboswitch aptamers

Most riboswitches are characterized by two components, an aptamer domain that folds into a unique ligand binding pocket to interact with the ligand, and an expression platform that converts folding changes in the aptamer into changes in gene expression. Using the recently developed systematic helix-based computational method, we theoretically studied the refolding and co-transcriptional folding behaviors of the purine riboswitch aptamers from Bacillus subtilis xpt-pbuX guanine riboswitch and Vibrio vulnificus add adenine riboswitch. Despite several intermediate structures persisting a short time during the transcription, helices P2, P3 and P1 fold in turn for both aptamers. Although some misfolded structures are observed during the refolding process, the RNAs can fold into the ligand binding pocket structure containing helices P2, P3 and P1 within a few seconds, suggesting the aptamer domains are highly evolved. The purine riboswitch aptamers can quickly fold into the ligand binding pocket structure even at a high transcription speed, possibly because formation of this structure is the necessary prerequisite for the riboswitch to bind its ligand and then regulate relevant gene expression.

Computational Methods for Modeling Aptamers and Designing Riboswitches.

Riboswitches, which are located within certain noncoding RNA region perform functions as genetic “switches”, regulating when and where genes are expressed in response to certain ligands. Understanding the numerous functions of riboswitches requires computation models to predict structures and structural changes of the aptamer domains. Although aptamers often form a complex structure, computational approaches, such as RNAComposer and Rosetta, have already been applied to model the tertiary (three-dimensional (3D)) structure for several aptamers. As structural changes in aptamers must be achieved within the certain time window for effective regulation, kinetics is another key point for understanding aptamer function in riboswitch-mediated gene regulation. The coarse-grained self-organized polymer (SOP) model using Langevin dynamics simulation has been successfully developed to investigate folding kinetics of aptamers, while their co-transcriptional folding kinetics can be modeled by the helix-based computational method and BarMap approach. Based on the known aptamers, the web server Riboswitch Calculator and other theoretical methods provide a new tool to design synthetic riboswitches. This review will represent an overview of these computational methods for modeling structure and kinetics of riboswitch aptamers and for designing riboswitches.

Structural Insight into the Unbound State of the DNA Analogue of the PreQ1 Riboswitch: A Thermodynamic Approach.

The preQ1 riboswitch aptamer domain is very dynamic in its unbound state with the ability to form multiple structures: a hairpin, kissing hairpins, and pseudoknot-like structure. The aim of this study is to determine whether the DNA analogue (PreQ1) is able to form structures similar to that of the reported RNA aptamer. Using a thermodynamic approach, we report on structural determination using differential scanning calorimetry under different salt conditions. Further analysis of the primary sequence allowed us to design modified molecules to determine what potential structures are forming in this single-stranded DNA analogue. We found, in a 16 mM Na+ solution, PreQ1 has three transitions with TM values of 14.8, 19.4, and 26.2 °C and a total ΔH of -44.7 kcal/mol. With the increase in salt concentration to 116 mM, there are TM values of 22.3, 28.7, and 38.9 °C and a ΔH of -69.1 kcal/mol, while at 216 mM, the three transitions have TM values of 24.4, 31.6, and 42.9 °C with a total ΔH of -71.5 kcal/mol. Therefore, the increase in enthalpy is due to the formation of additional base-pair stacks. The modified molecules, which would inhibit pseudoknot formation, kissing hairpins, and internal loop interactions, were fully characterized and compared to the native DNA analogue. The analysis of the enthalpy and differential binding of counterions allows us to conclude this single-stranded DNA analogue under physiological conditions is not forming a pseudoknot-like structure. Instead, two potential structures, Compact-Hairpin and Kissing-Complex, are more likely and could be in equilibrium.

Tertiary Interactions in the Unbound Guanine-Sensing Riboswitch Focus Functional Conformational Variability on the Binding Site

Riboswitches are genetic regulatory elements mainly found in bacteria, which regulate gene expression based on the availability of a ligand. Purine-sensing riboswitches, including the guanine-sensing riboswitch (Gsw), possess tertiary interactions connecting the L2 and L3 loops. These interactions are important for ligand binding to the aptamer. However, atomic-level structural knowledge about the unbound state and how the tertiary interactions influence the conformational heterogeneity of the aptamer is still scarce. We performed replica exchange molecular dynamics simulations of the aptamer domain of wild-type Gsw and a G37A/C61U mutant, which exhibits destabilized tertiary interactions, at different Mg2+ concentrations with an aggregate simulation time of ∼16 μs, and subsequently obtained free-energy landscapes. Our data provide evidence that suggests that the unbound state of wild-type Gsw is conformationally rather homogeneous from a global viewpoint, yet the ligand binding site shows functionally necessary mobility required for ligand binding. For the mutant, the data suggest a heterogeneous ensemble, in particular without Mg2+. Hence, the tertiary interactions focus functional conformational variability on the binding site region of wild-type Gsw. Our data allow speculating that already the weakening of the tertiary interactions by two hydrogen bonds shifts the kinetics of folding from downhill folding without traps or intermediate states for wild-type Gsw to a folding including intermediates and misfolded structures for the mutant. A slowed-down folding of the aptamer might favor a decision during transcriptional regulation for the off-path, even if the ligand binds.

RNA-Based Fluorescent Biosensors for Detecting Metabolites in vitro and in Living Cells.

Genetically encoded sensors are important tools for measuring metabolites and other small molecules in vitro and in live cells. Until recently, genetically encoded sensors exclusively comprised fluorescent proteins that undergo changes in Förster resonance energy transfer upon binding a target analyte. However, recently a new class of fluorescent sensor has been developed composed of RNA. These RNA-based sensors rely on Spinach and other RNA mimics of green fluorescent protein. In each case, the RNA-based sensors contain an analyte-binding aptamer domain which transduces binding of the analyte into a conformational change in Spinach. Two types of sensors have been developed: allosteric Spinach sensors and Spinach riboswitches. Allosteric Spinach sensors exhibit metabolite-induced folding and subsequent fluorescence. Spinach riboswitches are naturally occurring riboswitches that have been modified to contain the Spinach aptamer. The resulting RNA is a fluorogenic riboswitch, and produces fluorescence upon binding its cognate analyte. We describe the development of this new technology, its uses, and future directions to facilitate the use of this assay technology in mammalian cells and in high-throughput applications.

Design and implementation of a synthetic pre-miR switch for controlling miRNA biogenesis in mammals.

Synthetic RNA-based systems have increasingly been used for the regulation of eukaryotic gene expression. Due to their structural properties, riboregulators provide a convenient basis for the development of ligand-dependent controllable systems. Here, we demonstrate reversible conditional control of miRNA biogenesis with an aptamer domain as a sensing unit connected to a natural miRNA precursor for the first time. For the design of the pre-miR switch, we replaced the natural terminal loop with the TetR aptamer. Thus, the TetR aptamer was positioned close to the Dicer cleavage sites, which allowed sterical control over pre-miR processing by Dicer. Our design proved to be highly versatile, allowing us to regulate the biogenesis of three structurally different miRNAs: miR-126, -34a and -199a. Dicer cleavage was inhibited up to 143-fold via co-expression of the TetR protein, yet could be completely restored upon addition of doxycycline. Moreover, we showed the functionality of the pre-miR switches for gene regulation through the interaction of the respective miRNA with its specific target sequence. Our designed device is capable of robust and reversible control of miRNA abundance. Thus, we offer a novel investigational tool for functional miRNA analysis.

Structure and function of c-di-GMP riboswitches

Cyclic diguanosine monophosphate (c-di-GMP) is a ubiquitous nucleotide second messenger present in a wide variety of bacteria. It regulates many important bacterial physiological functions such as biofilm formation, motility, adhesion, virulence and extracellular polysaccharide synthesis. It binds with many different proteins or RNA receptors, one of which is called riboswitch that is usually located at the 5'-untranslational region (5'-UTR) in some mRNA. Riboswitch usually comprises a specific ligand-binding (sensor) domain (named aptamer domain, AD), as well as a variable domain, termed expression platform (EP), to regulate expression of downstream coding sequences. When a specific metabolite concentration exceeds its threshold level, it will bind to its cognate riboswitch receptor to induce a conformational change of 5'-UTR, leading to modulation of downstream gene expression. Two classes of c-di-GMP-binding riboswitches (c-di-GMP-Ⅰ and c-di-GMP-Ⅱ) have been discovered that bind with this second messenger with high affinity to regulate diverse downstream genes, underscoring the importance of this unique RNA receptor in this pathway. Class Ⅰ c-di-GMP riboswitches are present in a wide variety of bacteria, and are most common in the phyla Firmicutes and Proteobacteria, while class Ⅱ c-di-GMP riboswitches typically function as allosteric ribozymes, binding to c-di-GMP to induce folding changes at atypical splicing site junctions to modulate downstream gene expression. This review introduces the discovery, classification, function, and also the affected downstream genes of c-di-GMP riboswitches.

Real-time crystallographic studies of the adenine riboswitch using an X-ray free-electron laser.

Structures of the four reaction states of the adenine riboswitch aptamer domain, including a transient intermediate state were solved by serial femtosecond crystallography. The structures not only demonstrate the use of X-ray free-electron lasers for RNA crystallography but have also proven that transient states can be determined in real time by mix-and-inject crystallography. These results illustrate the structural basis for the ligand-induced conformational changes associated with the molecular 'switch'.

An excited state underlies gene regulation of a transcriptional riboswitch.

Riboswitches control gene expression through ligand-dependent structural rearrangements of the sensing aptamer domain. However, we found that the Bacillus cereus fluoride riboswitch aptamer adopts identical tertiary structures in solution with and without ligand. Using chemical exchange saturation transfer (CEST) NMR spectroscopy, we revealed that the structured ligand-free aptamer transiently accesses a low-populated (~1%) and short-lived (~3 ms) excited conformational state that unravels a conserved ‘linchpin’ base pair to signal transcription termination. Upon fluoride binding, this highly localized fleeting process is allosterically suppressed to activate transcription. We demonstrated that this mechanism confers effective fluoride-dependent gene activation over a wide range of transcription rates, which is essential for robust toxicity response across diverse cellular conditions. These results unveil a novel switching mechanism that employs ligand-dependent suppression of an aptamer excited state to coordinate regulatory conformational transitions rather than adopting distinct aptamer ground-state tertiary architectures, exemplifying a new mode of ligand-dependent RNA regulation.

Structures of riboswitch RNA reaction states by mix-and-inject XFEL serial crystallography

Riboswitches are structural RNA elements that are generally located in the 5' untranslated region of messenger RNA. During regulation of gene expression, ligand binding to the aptamer domain of a riboswitch triggers a signal to the downstream expression platform. A complete understanding of the structural basis of this mechanism requires the ability to study structural changes over time. Here we use femtosecond X-ray free electron laser (XFEL) pulses to obtain structural measurements from crystals so small that diffusion of a ligand can be timed to initiate a reaction before diffraction. We demonstrate this approach by determining four structures of the adenine riboswitch aptamer domain during the course of a reaction, involving two unbound apo structures, one ligand-bound intermediate, and the final ligand-bound conformation. These structures support a reaction mechanism model with at least four states and illustrate the structural basis of signal transmission. The three-way junction and the P1 switch helix of the two apo conformers are notably different from those in the ligand-bound conformation. Our time-resolved crystallographic measurements with a 10-second delay captured the structure of an intermediate with changes in the binding pocket that accommodate the ligand. With at least a 10-minute delay, the RNA molecules were fully converted to the ligand-bound state, in which the substantial conformational changes resulted in conversion of the space group. Such notable changes in crystallo highlight the important opportunities that micro- and nanocrystals may offer in these and similar time-resolved diffraction studies. Together, these results demonstrate the potential of 'mix-and-inject' time-resolved serial crystallography to study biochemically important interactions between biomacromolecules and ligands, including those that involve large conformational changes.

Pausing guides RNA folding to populate transiently stable RNA structures for riboswitch-based transcription regulation.

In bacteria, the regulation of gene expression by cis-acting transcriptional riboswitches located in the 5'-untranslated regions of messenger RNA requires the temporal synchronization of RNA synthesis and ligand binding-dependent conformational refolding. Ligand binding to the aptamer domain of the riboswitch induces premature termination of the mRNA synthesis of ligand-associated genes due to the coupled formation of 3'-structural elements acting as terminators. To date, there has been no high resolution structural description of the concerted process of synthesis and ligand-induced restructuring of the regulatory RNA element. Here, we show that for the guanine-sensing xpt-pbuX riboswitch from Bacillus subtilis, the conformation of the full-length transcripts is static: it exclusively populates the functional off-state but cannot switch to the on-state, regardless of the presence or absence of ligand. We show that only the combined matching of transcription rates and ligand binding enables transcription intermediates to undergo ligand-dependent conformational refolding.