SAM recognition and conformational switching mechanism in the Bacillus subtilis yitJ S box/SAM-I riboswitch

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.     

Folding of the SAM-I riboswitch: A tale with a twist

Riboswitches are ligand-dependent RNA genetic regulators that control gene expression by altering their structures. The elucidation of riboswitch conformational changes before and after ligand recognition is crucial to understand how riboswitches can achieve high ligand binding affinity and discrimination against cellular analogs. The detailed characterization of riboswitch folding pathways suggest that they may use their intrinsic conformational dynamics to sample a large array of structures, some of which being nearly identical to ligand-bound molecules. Some of these structural conformers can be "captured" upon ligand binding, which is crucial for the outcome of gene regulation. Recent studies about the SAM-I riboswitch have revealed unexpected and previously unknown RNA folding mechanisms. For instance, the observed helical twist of the P1 stem upon ligand binding to the SAM-I aptamer adds a new element in the repertoire of RNA strategies for recognition of small metabolites. From an RNA folding perspective, these findings also strongly indicate that the SAM-I riboswitch could achieve ligand recognition by using an optimized combination of conformational capture and induced-fit approaches, a feature that may be shared by other RNA regulatory sequences.     

Identification of a tertiary interaction important for cooperative ligand binding by the glycine riboswitch

The glycine riboswitch has a tandem dual aptamer configuration, where each aptamer is a separate ligand-binding domain, but the aptamers function together to bind glycine cooperatively. We sought to understand the molecular basis of glycine riboswitch cooperativity by comparing sites of tertiary contacts in a series of cooperative and noncooperative glycine riboswitch mutants using hydroxyl radical footprinting, in-line probing, and native gel-shift studies. The results illustrate the importance of a direct or indirect interaction between the P3b hairpin of aptamer 2 and the P1 helix of aptamer 1 in cooperative glycine binding. Furthermore, our data support a model in which glycine binding is sequential; where the binding of glycine to the second aptamer allows tertiary interactions to be made that facilitate binding of a second glycine molecule to the first aptamer. These results provide insight into cooperative ligand binding in RNA macromolecules.     

Tertiary contacts control switching of the SAM-I riboswitch

Riboswitches are non-coding RNAs that control gene expression by sensing small molecules through changes in secondary structure. While secondary structure and ligand interactions are thought to control switching, the exact mechanism of control is unknown. Using a novel two-piece assay that competes the anti-terminator against the aptamer, we directly monitor the process of switching. We find that the stabilization of key tertiary contacts controls both aptamer domain collapse and the switching of the SAM-I riboswitch from the aptamer to the expression platform conformation. Our experiments demonstrate that SAM binding induces structural alterations that indirectly stabilize the aptamer domain, preventing switching toward the expression platform conformer. These results, combined with a variety of structural probing experiments performed in this study, show that the collapse and stabilization of the aptamer domain are cooperative, relying on the sum of key tertiary contacts and the bimodal stability of the kink-turn motif for function. Here, ligand binding serves to shift the equilibrium of aptamer domain structures from a more open toward a more stable collapsed form by stabilizing tertiary interactions. Our data show that the thermodynamic landscape for riboswitch operation is finely balanced to allow large conformational rearrangements to be controlled by small molecule interactions.     

Chemical basis of glycine riboswitch cooperativity

The glycine binding riboswitch forms a unique tandem aptamer structure that binds glycine cooperatively. We employed nucleotide analog interference mapping (NAIM) and mutagenesis to explore the chemical basis of glycine riboswitch cooperativity. Based on the interference pattern, at least two sites appear to facilitate cooperative tertiary interactions, namely, the minor groove of the P1 helix from aptamer 1 and the major groove of the P3a helix from both aptamers. Mutation of these residues altered both the cooperativity and binding affinity of the riboswitch. The data support a model in which the P1 helix of the first aptamer participates in a tertiary interaction important for cooperativity, while nucleotides in the P1 helix of the second aptamer interface with the expression platform. These data have direct analogy to well-characterized mutations in hemoglobin, which provides a framework for considering cooperativity in this RNA-based system.     

Variable sequences outside the SAM-binding core critically influence the conformational dynamics of the SAM-III/SMK box riboswitch

The S_MK box (SAM-III) translational riboswitches were identified in S-adenosyl-l-methionine (SAM) synthetase metK genes in members of Lactobacillales. This riboswitch switches between two alternative conformations in response to intracellular SAM concentration and controls metK expression at the level of translation initiation. We previously reported the crystal structure of the SAM-bound S_MK box riboswitch. In this study, we combined selective 2′-hydroxyl acylation analyzed by primer extension chemical probing with mutagenesis to probe the ligand-induced conformational switching mechanism. We revealed that while the majority of the apo S_MK box RNA molecules exist in an alternatively base-paired (ON) conformation, a subset of them pre-organize into a SAM-bound-like (READY) conformation, which, upon SAM exposure, is selectively stabilized into the SAM-bound (OFF) conformation through an induced-fit mechanism. Mutagenesis showed that the ON state is only slightly more stable than the READY state, as several single-nucleotide substitutions in a hypervariable region outside the SAM-binding core can alter the folding landscape to favor the READY state. Such S_MK variants display a “constitutively OFF” behavior both in vitro and in vivo. Time-resolved and temperature-dependent selective 2′-hydroxyl acylation analyzed by primer extension analyses revealed adaptation of the S_MK box RNA to its mesothermal working environment. The latter analysis revealed that the SAM-bound S_MK box RNA follows a two-step folding/unfolding process.     

Molecular insights into the ligand-controlled organization of the SAM-I riboswitch

S-adenosylmethionine (SAM) riboswitches are widespread in bacteria, and up to five different SAM riboswitch families have been reported, highlighting the relevance of SAM regulation. On the basis of crystallographic and biochemical data, it has been postulated, but never demonstrated, that ligand recognition by SAM riboswitches involves key conformational changes in the RNA architecture. We show here that the aptamer follows a two-step hierarchical folding selectively induced by metal ions and ligand binding, each of them leading to the formation of one of the two helical stacks observed in the crystal structure. Moreover, we find that the anti-antiterminator P1 stem is rotated along its helical axis upon ligand binding, a mechanistic feature that could be common to other riboswitches. We also show that the nonconserved P4 helical domain is used as an auxiliary element to enhance the ligand-binding affinity. This work provides the first comprehensive characterization, to our knowledge, of a ligand-controlled riboswitch folding pathway.     

RNA tertiary interactions in a riboswitch stabilize the structure of a kink turn

The kink turn is a widespread RNA motif that introduces an acute kink into the axis of duplex RNA, typically comprising a bulge followed by a G?A and A?G pairs. The kinked conformation is stabilized by metal ions, or the binding of proteins including L7Ae. We now demonstrate a third mechanism for the stabilization of k-turn structure, involving tertiary interactions within a larger RNA structure. The SAM-I riboswitch contains an essential standard k-turn sequence that kinks a helix so that its terminal loop can make?a?long-range interaction. We find that some sequence variations in the k-turn within the riboswitch do not prevent SAM binding, despite preventing the folding of the k-turn in isolation. Furthermore, two crystal structures show that the sequence-variant k-turns are conventionally folded within the riboswitch. This study shows that the folded structure of the k-turn can be stabilized by tertiary interactions within a larger RNA structure.     

Conformational changes in the expression domain of the Escherichia coli thiM riboswitch

The thiM riboswitch contains an aptamer domain that adaptively binds the coenzyme thiamine pyrophosphate (TPP). The binding of TPP to the aptamer domain induces structural rearrangements that are relayed to a second domain, the so-called expression domain, thereby interfering with gene expression. The recently solved crystal structures of the aptamer domains of the thiM riboswitches in complex with TPP revealed how TPP stabilizes secondary and tertiary structures in the RNA ligand complex. To understand the global modes of reorganization between the two domains upon metabolite binding the structure of the entire riboswitch in presence and absence of TPP needs to be determined. Here we report the secondary structure of the entire thiM riboswitch from Escherichia coli in its TPP-free form and its transition into the TPP-bound variant, thereby depicting domains of the riboswitch that serve as communication links between the aptamer and the expression domain. Furthermore, structural probing provides an explanation for the lack of genetic control exerted by a riboswitch variant with mutations in the expression domain that still binds TPP.     

Rational design of SAM analogues targeting SAM-II riboswitch aptamer

Riboswitches are noncoding RNA elements embedded in 5′-untranslated region of many bacterial mRNAs regulating gene expression in response to essential metabolites. They are unique from other RNA targets because they have evolved to form specific structural receptors for the purpose of binding small molecular metabolites suggesting that structure-based rational drug design approach may be used in designing metabolite mimics targeting riboswitches. We have developed a fluorescence binding assay for SAM-II riboswitch aptamer and identified an S-adenosylmethionine (SAM) analogue that selectively binds to SAM-II riboswitch aptamer with comparable binding affinity to its native metabolite using structure-based design approach.     

Molecular dynamics simulation on the allosteric analysis of the c‐di‐GMP class I riboswitch induced by ligand binding

Riboswitches are RNA molecules that regulate gene expression using conformation change, affected by binding of small molecule ligands. Although a number of ligand‐bound aptamer complex structures have been solved, it is important to know ligand‐free conformations of the aptamers in order to understand the mechanism of specific binding by ligands. In this paper, we use dynamics simulations on a series of models to characterize the ligand‐free and ligand‐bound aptamer domain of the c‐di‐GMP class I (GEMM‐I) riboswitch. The results revealed that the ligand‐free aptamer has a stable state with a folded P2 and P3 helix, an unfolded P1 helix and open binding pocket. The first Mg ions binding to the aptamer is structurally favorable for the successive c‐di‐GMP binding. The P1 helix forms when c‐di‐GMP is successive bound. Three key junctions J1/2, J2/3 and J1/3 in the GEMM‐I riboswitch contributing to the formation of P1 helix have been found. The binding of the c‐di‐GMP ligand to the GEMM‐I riboswitch induces the riboswitch's regulation through the direct allosteric communication network in GEMM‐I riboswitch from the c‐di‐GMP binding sites in the J1/2 and J1/3 junctions to the P1 helix, the indirect ones from those in the J2/3 and P2 communicating to P1 helix via the J1/2 and J1/3 media.     

Nonlocal Helix Formation Is Key to Understanding S-Adenosylmethionine-1 Riboswitch Function

Riboswitches are noncoding RNAs that regulate gene expression in response to changing concentrations of specific metabolites. Switching activity is affected by the interplay between the aptamer domain and expression platform of the riboswitch. The aptamer domain binds the metabolite, locking the riboswitch in a ligand-bound conformation. In absence of the metabolite, the expression platform forms an alternative secondary structure by sequestering the 3′ end of a nonlocal helix called P1. We use all-atom structure-based simulations to characterize the folding, unfolding, and metabolite binding of the aptamer domain of the S-adenosylmethionine-1 (SAM-1) riboswitch. Our results suggest that folding of the nonlocal helix (P1) is rate-limiting in aptamer domain formation. Interestingly, SAM assists folding of the P1 helix by reducing the associated free energy barrier. Because the 3′ end of the P1 helix is sequestered by an alternative helix in the absence of metabolites, this observed ligand-control of P1 formation provides a mechanistic explanation of expression platform regulation.     

Ligand-induced stabilization of the aptamer terminal helix in the add adenine riboswitch

Riboswitches are structured mRNA elements that modulate gene expression. They undergo conformational changes triggered by highly specific interactions with sensed metabolites. Among the structural rearrangements engaged by riboswitches, the forming and melting of the aptamer terminal helix, the so-called P1 stem, is essential for genetic control. The structural mechanisms by which this conformational change is modulated upon ligand binding mostly remain to be elucidated. Here, we used pulling molecular dynamics simulations to study the thermodynamics of the P1 stem in the add adenine riboswitch. The P1 ligand-dependent stabilization was quantified in terms of free energy and compared with thermodynamic data. This comparison suggests a model for the aptamer folding in which direct P1-ligand interactions play a minor role on the conformational switch when compared with those related to the ligand-induced aptamer preorganization.     

Evidence for a second class of S-adenosylmethionine riboswitches and other regulatory RNA motifs in alpha-proteobacteria

Background

Riboswitches are RNA elements in the 5' untranslated leaders of bacterial mRNAs that directly sense the levels of specific metabolites with a structurally conserved aptamer domain to regulate expression of downstream genes. Riboswitches are most common in the genomes of low GC Gram-positive bacteria (for example, Bacillus subtilis contains examples of all known riboswitches), and some riboswitch classes seem to be restricted to this group.

Results

We used comparative sequence analysis and structural probing to identify five RNA elements (serC, speF, suhB, ybhL, and metA) that reside in the intergenic regions of Agrobacterium tumefaciens and many other α-proteobacteria. One of these, the metA motif, is found upstream of methionine biosynthesis genes and binds S-adenosylmethionine (SAM). This natural aptamer most likely functions as a SAM riboswitch (SAM-II) with a consensus sequence and structure that is distinct from the class of SAM riboswitches (SAM-I) predominantly found in Gram-positive bacteria. The minimal functional SAM-II aptamer consists of fewer than 70 nucleotides, which form a single stem and a pseudoknot. Despite its simple architecture and lower affinity for SAM, the SAM-II aptamer strongly discriminates against related compounds.

Conclusion

SAM-II is the only metabolite-binding riboswitch class identified so far that is not found in Gram-positive bacteria, and its existence demonstrates that biological systems can use multiple RNA structures to sense a single chemical compound. The two SAM riboswitches might be 'RNA World' relics that were selectively retained in certain bacterial lineages or new motifs that have emerged since the divergence of the major bacterial groups.