A structural intermediate pre-organizes the add adenine riboswitch for ligand recognition

Riboswitches are RNA sequences that regulate gene expression by undergoing structural changes upon the specific binding of cellular metabolites. Crystal structures of purine-sensing riboswitches have revealed an intricate network of interactions surrounding the ligand in the bound complex. The mechanistic details about how the aptamer folding pathway is involved in the formation of the metabolite binding site have been previously shown to be highly important for the riboswitch regulatory activity. Here, a combination of single-molecule FRET and SHAPE assays have been used to characterize the folding pathway of the adenine riboswitch from Vibrio vulnificus. Experimental evidences suggest a folding process characterized by the presence of a structural intermediate involved in ligand recognition. This intermediate state acts as an open conformation to ensure ligand accessibility to the aptamer and folds into a structure nearly identical to the ligand-bound complex through a series of structural changes. This study demonstrates that the add riboswitch relies on the folding of a structural intermediate that pre-organizes the aptamer global structure and the ligand binding site to allow efficient metabolite sensing and riboswitch genetic regulation.     

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.     

Structural distinctions between NAD+ riboswitch domains 1 and 2 determine differential folding and ligand binding

Riboswitches are important gene regulatory elements frequently encountered in bacterial mRNAs. The recently discovered nadA riboswitch contains two similar, tandemly arrayed aptamer domains, with the first domain possessing high affinity for nicotinamide adenine dinucleotide (NAD⁺). The second domain which comprises the ribosomal binding site in a putative regulatory helix, however, has withdrawn from detection of ligand-induced structural modulation thus far, and therefore, the identity of the cognate ligand and the regulation mechanism have remained unclear. Here, we report crystal structures of both riboswitch domains, each bound to NAD⁺. Furthermore, we demonstrate that ligand binding to domain 2 requires significantly higher concentrations of NAD⁺ (or ADP retaining analogs) compared to domain 1. Using a fluorescence spectroscopic approach, we further shed light on the structural features which are responsible for the different ligand affinities, and describe the Mg²⁺-dependent, distinct folding and pre-organization of their binding pockets. Finally, we speculate about possible scenarios for nadA RNA gene regulation as a putative two-concentration sensor module for a time-controlled signal that is primed and stalled by the gene regulation machinery at low ligand concentrations (domain 1), and finally triggers repression of translation as soon as high ligand concentrations are reached in the cell (domain 2).     

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.     

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.     

Ligand binding by the tandem glycine riboswitch depends on aptamer dimerization but not double ligand occupancy

The glycine riboswitch predominantly exists as a tandem structure, with two adjacent, homologous ligand-binding domains (aptamers), followed by a single expression platform. The recent identification of a leader helix, the inclusion of which eliminates cooperativity between the aptamers, has reopened the debate over the purpose of the tandem structure of the glycine riboswitch. An equilibrium dialysis-based assay was combined with binding-site mutations to monitor glycine binding in each ligand-binding site independently to understand the role of each aptamer in glycine binding and riboswitch tertiary interactions. A series of mutations disrupting the dimer interface was used to probe how dimerization impacts ligand binding by the tandem glycine riboswitch. While the wild-type tandem riboswitch binds two glycine equivalents, one for each aptamer, both individual aptamers are capable of binding glycine when the other aptamer is unoccupied. Intriguingly, glycine binding by aptamer-1 is more sensitive to dimerization than glycine binding by aptamer-2 in the context of the tandem riboswitch. However, monomeric aptamer-2 shows dramatically weakened glycine-binding affinity. In addition, dimerization of the two aptamers in trans is dependent on glycine binding in at least one aptamer. We propose a revised model for tandem riboswitch function that is consistent with these results, wherein ligand binding in aptamer-1 is linked to aptamer dimerization and stabilizes the P1 stem of aptamer-2, which controls the expression platform.     

The yjdF riboswitch candidate regulates gene expression by binding diverse azaaromatic compounds

The yjdF motif RNA is an orphan riboswitch candidate that almost exclusively associates with the yjdF protein-coding gene in many bacteria. The function of the YjdF protein is unknown, which has made speculation regarding the natural ligand for this putative riboswitch unusually challenging. By using a structure-probing assay for ligand binding, we found that a surprisingly broad diversity of nitrogen-containing aromatic heterocycles, or “azaaromatics,” trigger near-identical changes in the structures adopted by representative yjdF motif RNAs. Regions of the RNA that undergo ligand-induced structural modulation reside primarily in portions of the putative aptamer region that are highly conserved in nucleotide sequence, as is typical for riboswitches. Some azaaromatic molecules are bound by the RNA with nanomolar dissociation constants, and a subset of these ligands activate riboswitch-mediated gene expression in cells. Furthermore, genetic elements most commonly adjacent to the yjdF motif RNA or to the yjdF protein-coding region are homologous to protein regulators implicated in mitigating the toxic effects of diverse phenolic acids or polycyclic compounds. Although the precise type of natural ligand sensed by yjdF motif RNAs remains unknown, our findings suggest that this riboswitch class might serve as part of a genetic response system to toxic or signaling compounds with chemical structures similar to azaaromatics.     

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.     

Dissecting the influence of Mg2+ on 3D architecture and ligand-binding of the guanine-sensing riboswitch aptamer domain

Long-range tertiary interactions determine the three-dimensional structure of a number of metabolite-binding riboswitch RNA elements and were found to be important for their regulatory function. For the guanine-sensing riboswitch of the Bacillus subtilis xpt-pbuX operon, our previous NMR-spectroscopic studies indicated pre-formation of long-range tertiary contacts in the ligand-free state of its aptamer domain. Loss of the structural pre-organization in a mutant of this RNA (G37A/C61U) resulted in the requirement of Mg²⁺ for ligand binding. Here, we investigate structural and stability aspects of the wild-type aptamer domain (Gsw) and the G37A/C61U-mutant (Gsw^loop) of the guanine-sensing riboswitch and their Mg²⁺-induced folding characteristics to dissect the role of long-range tertiary interactions, the link between pre-formation of structural elements and ligand-binding properties and the functional stability. Destabilization of the long-range interactions as a result of the introduced mutations for Gsw^loop or the increase in temperature for both Gsw and Gsw^loop involves pronounced alterations of the conformational ensemble characteristics of the ligand-free state of the riboswitch. The increased flexibility of the conformational ensemble can, however, be compensated by Mg²⁺. We propose that reduction of conformational dynamics in remote regions of the riboswitch aptamer domain is the minimal pre-requisite to pre-organize the core region for specific ligand binding.     

What defines a synthetic riboswitch? – Conformational dynamics of ciprofloxacin aptamers with similar binding affinities but varying regulatory potentials

Among the many in vitro-selected aptamers derived from SELEX protocols, only a small fraction has the potential to be applied for synthetic riboswitch engineering. Here, we present a comparative study of the binding properties of three different aptamers that bind to ciprofloxacin with similar K_D values, yet only two of them can be applied as riboswitches. We used the inherent ligand fluorescence that is quenched upon binding as the reporter signal in fluorescence titration and in time-resolved stopped-flow experiments. Thus, we were able to demonstrate differences in the binding kinetics of regulating and non-regulating aptamers. All aptamers studied underwent a two-step binding mechanism that suggests an initial association step followed by a reorganization of the aptamer to accommodate the ligand. We show that increasing regulatory potential is correlated with a decreasing back-reaction rate of the second binding step, thus resulting in a virtually irreversible last binding step of regulating aptamers. We suggest that a highly favoured structural adaption of the RNA to the ligand during the final binding step is essential for turning an aptamer into a riboswitch. In addition, our results provide an explanation for the fact that so few aptamers with regulating capacity have been found to date. Based on our data, we propose an adjustment of the selection protocol for efficient riboswitch detection.     

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.     

The k-junction motif in RNA structure

The k-junction is a structural motif in RNA comprising a three-way helical junction based upon kink turn (k-turn) architecture. A computer program written to examine relative helical orientation identified the three-way junction of the Arabidopsis TPP riboswitch as an elaborated k-turn. The Escherichia coli TPP riboswitch contains a related k-junction, and analysis of >11 000 sequences shows that the structure is common to these riboswitches. The k-junction exhibits all the key features of an N1-class k-turn, including the standard cross-strand hydrogen bonds. The third helix of the junction is coaxially aligned with the C (canonical) helix, while the k-turn loop forms the turn into the NC (non-canonical) helix. Analysis of ligand binding by ITC and global folding by gel electrophoresis demonstrates the importance of the k-turn nucleotides. Clearly the basic elements of k-turn structure are structurally well suited to generate a three-way helical junction, retaining all the key features and interactions of the k-turn.     

Metal-ion binding and metal-ion induced folding of the adenine-sensing riboswitch aptamer domain

Divalent cations are important in the folding and stabilization of complex RNA structures. The adenine-sensing riboswitch controls the expression of mRNAs for proteins involved in purine metabolism by directly sensing intracellular adenine levels. Adenine binds with high affinity and specificity to the ligand binding or aptamer domain of the adenine-sensing riboswitch. The X-ray structure of this domain in complex with adenine revealed an intricate RNA-fold consisting of a three-helix junction stabilized by long-range base-pairing interactions and identified five binding sites for hexahydrated Mg²⁺-ions. Furthermore, a role for Mg²⁺-ions in the ligand-induced folding of this RNA was suggested. Here, we describe the interaction of divalent cations with the RNA–adenine complex in solution as studied by high-resolution NMR spectroscopy. Paramagnetic line broadening, chemical shift mapping and intermolecular nuclear Overhauser effects (NOEs) indicate the presence of at least three binding sites for divalent cations. Two of them are similar to those in the X-ray structure. The third site, which is important for the folding of this RNA, has not been observed previously. The ligand-free state of the RNA is conformationally heterogeneous and contains base-pairing patterns detrimental to ligand binding in the absence of Mg²⁺, but becomes partially pre-organized for ligand binding in the presence of Mg²⁺. Compared to the highly similar guanine-sensing riboswitch, the folding pathway for the adenine-sensing riboswitch aptamer domain is more complex and the influence of Mg²⁺ is more pronounced.