A magnesium-induced triplex pre-organizes the SAM-II riboswitch.
ABSTRACT: Our 13C- and 1H-chemical exchange saturation transfer (CEST) experiments previously revealed a dynamic exchange between partially closed and open conformations of the SAM-II riboswitch in the absence of ligand. Here, all-atom structure-based molecular simulations, with the electrostatic effects of Manning counter-ion condensation and explicit magnesium ions are employed to calculate the folding free energy landscape of the SAM-II riboswitch. We use this analysis to predict that magnesium ions remodel the landscape, shifting the equilibrium away from the extended, partially unfolded state towards a compact, pre-organized conformation that resembles the ligand-bound state. Our CEST and SAXS experiments, at different magnesium ion concentrations, quantitatively confirm our simulation results, demonstrating that magnesium ions induce collapse and pre-organization. Agreement between theory and experiment bolsters microscopic interpretation of our simulations, which shows that triplex formation between helix P2b and loop L1 is highly sensitive to magnesium and plays a key role in pre-organization. Pre-organization of the SAM-II riboswitch allows rapid detection of ligand with high selectivity, which is important for biological function.
Project description:Riboswitch operation involves the complex interplay between the aptamer domain and the expression platform. During transcription, these two domains compete against each other for shared sequence. In this study, we explore the cooperative effects of ligand binding and Magnesium interactions in the SAM-I riboswitch in the context of aptamer collapse and anti-terminator formation. Overall, our studies show the apo-aptamer acts as (i) a pre-organized aptamer competent to bind ligand and undergo structural collapse and (ii) a conformation that is more accessible to anti-terminator formation. We show that both Mg(2+) ions and SAM are required for a collapse transition to occur. We then use competition between the aptamer and expression platform for shared sequence to characterize the stability of the collapsed aptamer. We find that SAM and Mg(2+) interactions in the aptamer are highly cooperative in maintaining switch polarity (i.e. aptamer 'off-state' versus anti-terminator 'on-state'). We further show that the aptamer off-state is preferentially stabilized by Mg(2+) and similar divalent ions. Furthermore, the functional switching assay was used to select for phosphorothioate interference, and identifies potential magnesium chelation sites while characterizing their coordinated role with SAM in aptamer stabilization. In addition, we find that Mg(2+) interactions with the apo-aptamer are required for the full formation of the anti-terminator structure, and that higher concentrations of Mg(2+) (>4 mM) shift the equilibrium toward the anti-terminator on-state even in the presence of SAM.
Project description:The S-adenosylmethionine (SAM)-I riboswitch is a noncoding RNA that regulates the transcription termination process in response to metabolite (SAM) binding. The aptamer portion of the riboswitch may adopt an open or closed state depending on the presence of metabolite. Although the transition between the open and closed states is critical for the switching process, its atomistic details are not well understood. Using atomistic simulations, we calculate the effect of SAM and magnesium ions on the folding free energy landscape of the SAM-I riboswitch. These molecular simulation results are consistent with our previous wetlab experiments and aid in interpreting the SHAPE probing measurements. Here, molecular dynamics simulations explicitly identify target RNA motifs sensitive to magnesium ions and SAM. In the simulations, we observe that, whereas the metabolite mostly stabilizes the P1 and P3 helices, magnesium serves an important role in stabilizing a pseudoknot interaction between the P2 and P4 helices, even at high metabolite concentrations. The pseudoknot stabilization by magnesium, in combination with P1 stabilization by SAM, explains the requirement of both SAM and magnesium to form the fully collapsed metabolite-bound closed state of the SAM-I riboswitch. In the absence of SAM, frequent open-to-closed conformational transitions of the pseudoknot occur, akin to breathing. These pseudoknot fluctuations disrupt the binding site by facilitating fluctuations in the 5'-end of helix P1. Magnesium biases the landscape toward a collapsed state (preorganization) by coordinating pseudoknot and 5'-P1 fluctuations. The cooperation between SAM and magnesium in stabilizing important tertiary interactions elucidates their functional significance in transcription regulation.
Project description:Investigations of most riboswitches remain confined to the ligand-binding aptamer domain. However, during the riboswitch mediated transcription regulation process, the aptamer domain and the expression platform compete for a shared strand. If the expression platform dominates, an anti-terminator helix is formed, and the transcription process is active (ON state). When the aptamer dominates, transcription is terminated (OFF state). Here, we use an expression platform switching experimental assay and structure-based electrostatic simulations to investigate this ON-OFF transition of the full length SAM-I riboswitch and its magnesium concentration dependence. Interestingly, we find the ratio of the OFF population to the ON population to vary non-monotonically as magnesium concentration increases. Upon addition of magnesium, the aptamer domain pre-organizes, populating the OFF state, but only up to an intermediate magnesium concentration level. Higher magnesium concentration preferentially stabilizes the anti-terminator helix, populating the ON state, relatively destabilizing the OFF state. Magnesium mediated aptamer-expression platform domain closure explains this relative destabilization of the OFF state at higher magnesium concentration. Our study reveals the functional potential of magnesium in controlling transcription of its downstream genes and underscores the importance of a narrow concentration regime near the physiological magnesium concentration ranges, striking a balance between the OFF and ON states in bacterial gene regulation.
Project description:Although S-adenosylhomocysteine (SAH), a metabolic by-product of S-adenosylmethionine (SAM), differs from SAM only by a single methyl group and an overall positive charge, SAH binds the SAM-II riboswitch with more than 1000-fold less affinity than SAM. Using atomistic molecular dynamics simulations, we investigated the molecular basis of such high selectivity in ligand recognition by SAM-II riboswitch. The biosynthesis of SAM exclusively generates the (S,S) stereoisomer, and (S,S)-SAM can spontaneously convert to the (R,S) form. We, therefore, also examined the effects of (R,S)-SAM binding to SAM-II and its potential biological function. We find that the unfavorable loss in entropy in SAM-II binding is greater for (S,S)- and (R,S)-SAM than SAH, which is compensated by stabilizing electrostatic interactions with the riboswitch. The positively charged sulfonium moiety on SAM acts as the crucial anchor point responsible for the formation of key ionic interactions as it fits favorably in the negatively charged binding pocket. In contrast, SAH, with its lone pair of electrons on the sulfur, experiences repulsion in the binding pocket of SAM-II and is enthalpically destabilized. In the presence of SAH, similar to the unbound riboswitch, the pseudoknot structure of SAM-II is not completely formed, thus exposing the Shine-Dalgarno sequence. Unlike SAM, this may further facilitate ribosomal assembly and translation initiation. Our analysis of the conformational ensemble sampled by SAM-II in the absence of ligands and when bound to SAM or SAH reveals that ligand binding follows a combination of conformational selection and induced-fit mechanisms.
Project description:Riboswitches that sense S-adenosylmethionine (SAM) are widely distributed throughout a variety of bacterial lineages. Four classes of SAM-binding riboswitches have been reported to date, constituting the most diverse collection of riboswitch classes that sense the same compound. Three of these classes, termed SAM-I, SAM-II, and SAM-III represent unique structures that form distinct binding pockets for the ligand. SAM-IV riboswitches carry different conserved sequence and structural features compared to other SAM riboswitches, but nucleotides and substructures corresponding to the ligand binding pocket are identical to SAM-I aptamers. In this article, we describe a fifth class of SAM binding aptamer, which we have termed SAM-V. SAM-V was discovered by analyzing GC-rich intergenic regions preceding metabolic genes in the marine alpha-proteobacterium "Candidatus Pelagibacter ubique." Although the motif is nearly unrepresented in cultured bacteria whose genomes have been completely sequenced, SAM-V is prevalent in marine metagenomic sequences. The consensus sequence and structure of SAM-V show some similarities to that of the SAM-II riboswitch, and it is likely that the two aptamers form similar ligand binding pockets. In addition, we identified numerous examples of a tandem SAM-II/SAM-V aptamer architecture. In this arrangement, the SAM-II aptamer is always positioned 5' of the SAM-V aptamer and the SAM-II aptamer is followed by a predicted intrinsic transcription terminator stem. The SAM-V aptamer, however, appears to use a ribosome binding site occlusion mechanism for genetic regulation. This tandem riboswitch arrangement exhibits an architecture that can potentially control both the transcriptional and translational stages of gene expression.
Project description:Riboswitches are regulatory RNAs that control downstream gene expression in response to direct association with intracellular metabolites or metals. Typically, riboswitch aptamer domains bind to a single small-molecule metabolite. In contrast, an X-ray crystallographic structural model for the M-box riboswitch aptamer revealed the absence of an organic metabolite ligand but the presence of at least six tightly associated magnesiums. This observation agrees well with the proposed role of the M-box riboswitch in functioning as a sensor of intracellular magnesium, although additional nonspecific metal interactions are also undoubtedly required for these purposes. To gain greater functional insight into the metalloregulatory capabilities of M-box RNAs, we sought to determine whether all or a subset of the RNA-chelated magnesium ions were required for riboswitch function. To accomplish this task, each magnesium-binding site was simultaneously yet individually perturbed through random incorporation of phosphorothioate nucleotide analogues, and RNA molecules were investigated for their ability to fold in varying levels of magnesium. These data revealed that all of the magnesium ions observed in the structural model are important for magnesium-dependent tertiary structure formation. Additionally, these functional data revealed a new core of potential metal-binding sites that are likely to assist formation of key tertiary interactions and were previously unobserved in the structural model. It is clear from these data that M-box RNAs require specific binding of a network of metal ions for partial fulfillment of their metalloregulatory functions.
Project description:Riboswitches are promising targets for the design of novel antibiotics and engineering of portable genetic regulatory elements. There is evidence that variability in riboswitch properties allows tuning of expression for genes involved in different stages of biosynthetic pathways by mechanisms that are not currently understood. Here, we explore the mechanism for tuning of S-adenosyl methionine (SAM)-I riboswitch folding. Most SAM-I riboswitches function at the transcriptional level by sensing the cognate ligand SAM. SAM-I riboswitches orchestrate the biosynthetic pathways of cysteine, methionine, SAM, and so forth. We use base-pair probability predictions to examine the secondary-structure folding landscape of several SAM-I riboswitch sequences. We predict different folding behaviors for different SAM-I riboswitch sequences. We identify several "decoy" base-pairing interactions involving 5' riboswitch residues that can compete with the formation of a P1 helix, a component of the ligand-bound "transcription OFF" state, in the absence of SAM. We hypothesize that blockage of these interactions through SAM contacts contributes to stabilization of the OFF state in the presence of ligand. We also probe folding patterns for a SAM-I riboswitch RNA using constructs with different 3' truncation points experimentally. Folding was monitored through fluorescence, susceptibility to base-catalyzed cleavage, nuclear magnetic resonance, and indirectly through SAM binding. We identify key decision windows at which SAM can affect the folding pathway towards the OFF state. The presence of decoy conformations and differential sensitivities to SAM at different transcript lengths is crucial for SAM-I riboswitches to modulate gene expression in the context of global cellular metabolism.
Project description:PreQ1 riboswitches regulate the synthesis of the hypermodified tRNA base queuosine by sensing the pyrrolopyrimidine metabolite preQ1. Here, we use single-molecule FRET to interrogate the structural dynamics of apo and preQ1-bound states of the preQ1-II riboswitch from Lactobacillus rhamnosus. We find that the apo-form of the riboswitch spontaneously samples multiple conformations. Magnesium ions and preQ1 stabilize conformations that sequester the ribosome-binding site of the mRNA within the pseudoknotted structure, thus inhibiting translation initiation. Our results reveal that folding of the preQ1-II riboswitch is complex and provide evidence favoring a conformational selection model of effector binding by riboswitches of this class.
Project description:Riboswitches are metabolite-sensing, conserved domains located in non-coding regions of mRNA that are central to regulation of gene expression. Here we report the first three-dimensional structure of the recently discovered S-adenosyl-L-methionine responsive SAM-VI riboswitch. SAM-VI adopts a unique fold and ligand pocket that are distinct from all other known SAM riboswitch classes. The ligand binds to the junctional region with its adenine tightly intercalated and Hoogsteen base-paired. Furthermore, we reveal the ligand discrimination mode of SAM-VI by additional X-ray structures of this riboswitch bound to S-adenosyl-L-homocysteine and a synthetic ligand mimic, in combination with isothermal titration calorimetry and fluorescence spectroscopy to explore binding thermodynamics and kinetics. The structure is further evaluated by analysis of ligand binding to SAM-VI mutants. It thus provides a thorough basis for developing synthetic SAM cofactors for applications in chemical and synthetic RNA biology.
Project description:S-box (SAM-I) riboswitches are a widespread class of riboswitches involved in the regulation of sulfur metabolism in Gram-positive bacteria. We report here the 3.0-Å crystal structure of the aptamer domain of the Bacillus subtilis yitJ S-box (SAM-I) riboswitch bound to S-adenosyl-L-methionine (SAM). The RNA folds into two sets of helical stacks spatially arranged by tertiary interactions including a K-turn and a pseudoknot at a four-way junction. The tertiary structure is further stabilized by metal coordination, extensive ribose zipper interactions, and SAM-mediated tertiary interactions. Despite structural differences in the peripheral regions, the SAM-binding core of the B. subtilis yitJ riboswitch is virtually superimposable with the previously determined Thermoanaerobacter tengcongensis yitJ riboswitch structure, suggesting that a highly conserved ligand-recognition mechanism is utilized by all S-box riboswitches. SHAPE (selective 2'-hydroxyl acylation analyzed by primer extension) chemical probing analysis further revealed that the alternative base-pairing element in the expression platform controls the conformational switching process. In the absence of SAM, the apo yitJ aptamer domain folds predominantly into a pre-binding conformation that resembles, but is not identical with, the SAM-bound state. We propose that SAM enters the ligand-binding site through the "J1/2-J3/4" gate and "locks" down the SAM-bound conformation through an induced-fit mechanism. Temperature-dependent SHAPE revealed that the tertiary interaction-stabilized SAM-binding core is extremely stable, likely due to the cooperative RNA folding behavior. Mutational studies revealed that certain modifications in the SAM-binding region result in loss of SAM binding and constitutive termination, which suggests that these mutations lock the RNA into a form that resembles the SAM-bound form in the absence of SAM.