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.     

Riboswitch structure: an internal residue mimicking the purine ligand

The adenine and guanine riboswitches regulate gene expression in response to their purine ligand. X-ray structures of the aptamer moiety of these riboswitches are characterized by a compact fold in which the ligand forms a Watson–Crick base pair with residue 65. Phylogenetic analyses revealed a strict restriction at position 39 of the aptamer that prevents the G39–C65 and A39–U65 combinations, and mutational studies indicate that aptamers with these sequence combinations are impaired for ligand binding. In order to investigate the rationale for sequence conservation at residue 39, structural characterization of the U65C mutant from Bacillus subtilis pbuE adenine riboswitch aptamer was undertaken. NMR spectroscopy and X-ray crystallography studies demonstrate that the U65C mutant adopts a compact ligand-free structure, in which G39 occupies the ligand-binding site of purine riboswitch aptamers. These studies present a remarkable example of a mutant RNA aptamer that adopts a native-like fold by means of ligand mimicking and explain why this mutant is impaired for ligand binding. Furthermore, this work provides a specific insight into how the natural sequence has evolved through selection of nucleotide identities that contribute to formation of the ligand-bound state, but ensures that the ligand-free state remains in an active conformation.     

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.     

Single-molecule force spectroscopy of the add adenine riboswitch relates folding to regulatory mechanism

Riboswitches regulate gene expression via ligand binding to an aptamer domain which induces conformational changes in a regulatory expression platform. By unfolding and refolding single add adenine riboswitch molecules in an optical trap, an integrated picture of the folding was developed and related to the regulatory mechanism. Force-extension curves (FECs) and constant-force folding trajectories measured on the aptamer alone revealed multiple partially-folded states, including several misfolded states not on the native folding pathway. All states were correlated to key structural components and interactions within hierarchical folding pathways. FECs of the full-length riboswitch revealed that the thermodynamically stable conformation switches upon ligand binding from a structure repressing translation to one permitting it. Along with rapid equilibration of the two structures in the absence of adenine, these results support a thermodynamically-controlled regulatory mechanism, in contrast with the kinetic control of the closely-related pbuE adenine riboswitch. Comparison of the folding of these riboswitches revealed many similarities arising from shared structural features but also essential differences related to their different regulatory mechanisms.     

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.     

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.     

Structural insights into the interactions of xpt riboswitch with novel guanine analogues: a molecular dynamics simulation study

Ligand recognition in purine riboswitches is a complex process requiring different levels of conformational changes. Recent efforts in the area of purine riboswitch research have focused on ligand analogue binding studies. In the case of the guanine xanthine phosphoribosyl transferase (xpt) riboswitch, synthetic analogues that resemble guanine have the potential to tightly bind and subsequently influence the genetic expression of xpt mRNA in prokaryotes. We have carried out 25 ns Molecular Dynamics (MD) simulation studies of the aptamer domain of the xpt G-riboswitch in four different states: guanine riboswitch in free form, riboswitch bound with its cognate ligand guanine, and with two guanine analogues SJ1 and SJ2. Our work reveals novel interactions of SJ1 and SJ2 ligands with the binding core residues of the riboswitch. The ligands proposed in this work bind to the riboswitch with greater overall stability and lower root mean square deviations and fluctuations compared to guanine ligand. Reporter gene assay data demonstrate that the ligand analogues, upon binding to the RNA, lower the genetic expression of the guanine riboswitch. Our work has important implications for future ligand design and binding studies in the exciting field of riboswitches.     

Molecular sensing by the aptamer domain of the FMN riboswitch: a general model for ligand binding by conformational selection

Understanding the nature of the free state of riboswitch aptamers is important for illuminating common themes in gene regulation by riboswitches. Prior evidence indicated the flavin mononucleotide (FMN)-binding riboswitch aptamer adopted a ‘bound-like’ structure in absence of FMN, suggesting only local conformational changes upon ligand binding. In the scope of pinpointing the general nature of such changes at the nucleotide level, we performed SHAPE mapping experiments using the aptamer domain of two phylogenetic variants, both in absence and in presence of FMN. We also solved the crystal structures of one of these domains both free (3.3 Å resolution) and bound to FMN (2.95 Å resolution). Our comparative study reveals that structural rearrangements occurring upon binding are restricted to a few of the joining regions that form the binding pocket in both RNAs. This type of binding event with minimal structural perturbations is reminiscent of binding events by conformational selection encountered in other riboswitches and various RNAs.     

Ligand-induced conformational capture of a synthetic tetracycline riboswitch revealed by pulse EPR

RNA aptamers are in vitro-selected binding domains that recognize their respective ligand with high affinity and specificity. They are characterized by complex three-dimensional conformations providing preformed binding pockets that undergo conformational changes upon ligand binding. Small molecule-binding aptamers have been exploited as synthetic riboswitches for conditional gene expression in various organisms. In the present study, double electron-electron resonance (DEER) spectroscopy combined with site-directed spin labeling was used to elucidate the conformational transition of a tetracycline aptamer upon ligand binding. Different sites were selected for post-synthetic introduction of either the (1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl) methanethiosulfonate by reaction with a 4-thiouridine modified RNA or of 4-isocyanato-2,6-tetramethylpiperidyl-N-oxid spin label by reaction with 2'-aminouridine modified RNA. The results of the DEER experiments indicate the presence of a thermodynamic equilibrium between two aptamer conformations in the free state and capture of one conformation upon tetracycline binding.     

Molecular dynamics simulation study of the binding of purine bases to the aptamer domain of the guanine sensing riboswitch

Riboswitches are a novel class of genetic control elements that function through the direct interaction of small metabolite molecules with structured RNA elements. The ligand is bound with high specificity and affinity to its RNA target and induces conformational changes of the RNA''s secondary and tertiary structure upon binding. To elucidate the molecular basis of the remarkable ligand selectivity and affinity of one of these riboswitches, extensive all-atom molecular dynamics simulations in explicit solvent (≈1 μs total simulation length) of the aptamer domain of the guanine sensing riboswitch are performed. The conformational dynamics is studied when the system is bound to its cognate ligand guanine as well as bound to the non-cognate ligand adenine and in its free form. The simulations indicate that residue U51 in the aptamer domain functions as a general docking platform for purine bases, whereas the interactions between C74 and the ligand are crucial for ligand selectivity. These findings either suggest a two-step ligand recognition process, including a general purine binding step and a subsequent selection of the cognate ligand, or hint at different initial interactions of cognate and noncognate ligands with residues of the ligand binding pocket. To explore possible pathways of complex dissociation, various nonequilibrium simulations are performed which account for the first steps of ligand unbinding. The results delineate the minimal set of conformational changes needed for ligand release, suggest two possible pathways for the dissociation reaction, and underline the importance of long-range tertiary contacts for locking the ligand in the complex.     

Ligand-dependent folding of the three-way junction in the purine riboswitch

Riboswitches are highly structured cis-acting elements located in the 5'-untranslated region of messenger RNAs that directly bind small molecule metabolites to regulate gene expression. Structural and biochemical studies have revealed riboswitches experience significant ligand-dependent conformational changes that are coupled to regulation. To monitor the coupling of ligand binding and RNA folding within the aptamer domain of the purine riboswitch, we have chemically probed the RNA with N-methylisatoic anhydride (NMIA) over a broad temperature range. Analysis of the temperature-dependent reactivity of the RNA in the presence and absence of hypoxanthine reveals that a limited set of nucleotides within the binding pocket change their conformation in response to ligand binding. Our data demonstrate that a distal loop-loop interaction serves to restrict the conformational freedom of a significant portion of the three-way junction, thereby promoting ligand binding under physiological conditions.     

MD simulations of ligand-bound and ligand-free aptamer: Molecular level insights into the binding and switching mechanism of the add A-riboswitch

Riboswitches are structural cis-acting genetic regulatory elements in 5′ UTRs of mRNAs, consisting of an aptamer domain that regulates the behavior of an expression platform in response to its recognition of, and binding to, specific ligands. While our understanding of the ligand-bound structure of the aptamer domain of the adenine riboswitches is based on crystal structure data and is well characterized, understanding of the structure and dynamics of the ligand-free aptamer is limited to indirect inferences from physicochemical probing experiments. Here we report the results of 15-nsec-long explicit-solvent molecular dynamics simulations of the add A-riboswitch crystal structure (1Y26), both in the adenine-bound (CLOSED) state and in the adenine-free (OPEN) state. Root-mean-square deviation, root-mean-square fluctuation, dynamic cross-correlation, and backbone torsion angle analyses are carried out on the two trajectories. These, along with solvent accessible surface area analysis of the two average structures, are benchmarked against available experimental data and are shown to constitute the basis for obtaining reliable insights into the molecular level details of the binding and switching mechanism. Our analysis reveals the interaction network responsible for, and conformational changes associated with, the communication between the binding pocket and the expression platform. It further highlights the significance of a, hitherto unreported, noncanonical W:H trans base pairing between A73 and A24, in the OPEN state, and also helps us to propose a possibly crucial role of U51 in the context of ligand binding and ligand discrimination.     

Riboswitches that sense S-adenosylmethionine and S-adenosylhomocysteine.

Numerous riboswitches have been discovered that specifically recognize metabolites and modulate gene expression. Each riboswitch class is defined either by the consensus sequence and structural features of its metabolite-binding aptamer domain, or by the distinct metabolite that the aptamer recognizes. Several distinct classes of riboswitches that respond to S-adenosylmethionine (SAM or AdoMet) have been discovered. Representatives of these classes have been shown to strongly discriminate against S-adenosylhomocystenine (SAH or AdoHcy), which is the metabolic byproduct produced when SAM is used as a cofactor for methylation reactions. However, a distinct class of riboswitches that selectively binds SAH, and strongly discriminates against SAM, also has been discovered. Herein we compare the features of SAM and SAH riboswitches, which help showcase the enormous structural diversity that RNA can harness to form precision genetic switches for compounds that are critical for fundamental metabolic processes.     

Folding of the SAM aptamer is determined by the formation of a K-turn-dependent pseudoknot

The S-adenosylmethionine (SAM) riboswitch is one of the most recurrent riboswitches found in bacteria and has three known different natural aptamers. The Bacillus subtilis yitJ SAM riboswitch aptamer is organized around a four-way junction which is characterized by the presence of a pseudoknot and a K-turn motif. By replacing the adenine involved in a Watson-Crick base pair at position 138 in the core region of the aptamer with the fluorescent analogue 2-aminopurine (2AP), we show that the ligand-induced reorganization of the aptamer strongly attenuates 2AP fluorescence. The fluorescence quenching process is specific to SAM on the basis of the observation that the structural analogue S-adenosylhomocysteine does not promote a similar effect. We find that the pseudoknot is important for the reorganization of the core domain and that the K-turn motif also has a marked influence on the core domain reorganization, most probably through its important role in pseudoknot formation. Finally, we show that SAM riboswitch ligand binding is facilitated by the L7Ae K-turn binding protein, which suggests that K-turn motifs may be protein anchor sites used by riboswitches to promote RNA folding.