Insights into metalloregulation by M-box riboswitch RNAs via structural analysis of manganese-bound complexes

The M-box riboswitch couples intracellular magnesium levels to expression of bacterial metal transport genes. Structural analyses on other riboswitch RNA classes, which typically respond to a small organic metabolite, have revealed that ligand recognition occurs through a combination of base-stacking, electrostatic, and hydrogen-bonding interactions. In contrast, the M-box RNA triggers a change in gene expression upon association with an undefined population of metals, rather than responding to only a single ligand. Prior biophysical experimentation suggested that divalent ions associate with the M-box RNA to promote a compacted tertiary conformation, resulting in sequestration of a short sequence tract otherwise required for downstream gene expression. Electrostatic shielding from loosely associated metals is undoubtedly an important influence during this metal-mediated compaction pathway. However, it is also likely that a subset of divalent ions specifically occupies cation binding sites and promotes proper positioning of functional groups for tertiary structure stabilization. To better elucidate the role of these metal binding sites, we resolved a manganese-chelated M-box RNA complex to 1.86 Å by X-ray crystallography. These data support the presence of at least eight well-ordered cation binding pockets, including several sites that had been predicted by biochemical studies but were not observed in prior structural analysis. Overall, these data support the presence of three metal-binding cores within the M-box RNA that facilitate a network of long-range interactions within the metal-bound, compacted conformation.     

Structure and mechanism of a metal-sensing regulatory RNA

Organisms maintain the correct balance of intracellular metals primarily through metal-sensing proteins that control transport and storage of the target ion(s). Here, we reveal the basis of metal sensing and genetic control by a metalloregulatory RNA. Our data demonstrate that a previously uncharacterized orphan riboswitch, renamed the "M-box," is a divalent metal-sensing RNA involved in Mg(2+) homeostasis. A combination of genetic, biochemical, and biophysical techniques demonstrate that Mg(2+) induces a compacted tertiary architecture for M-box RNAs that regulates the accessibility of nucleotides involved in genetic control. Molecular details are provided by crystallographic structure determination of a Mg(2+)-bound M-box RNA. Given the distribution of this RNA element, it may constitute a common mode for bacterial metal ion regulation, and its discovery suggests the possibility of additional RNA-based metal sensors in modern and primordial organisms.     

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.     

Structural transitions and thermodynamics of a glycine-dependent riboswitch from Vibrio cholerae

Riboswitches are complex folded RNA domains found in noncoding regions of mRNA that regulate gene expression upon small molecule binding. Recently, Breaker and coworkers reported a tandem aptamer riboswitch (VCI-II) that binds glycine cooperatively. Here, we use hydroxyl radical footprinting and small-angle X-ray scattering (SAXS) to study the conformations of this tandem aptamer as a function of Mg(2+) and glycine concentration. We fit a simple three-state thermodynamic model that describes the energetic coupling between magnesium-induced folding and glycine binding. Furthermore, we characterize the structural conformations of each of the three states: In low salt with no magnesium present, the VCI-II construct has an extended overall conformation, presumably representing unfolded structures. Addition of millimolar concentrations of Mg(2+) in the absence of glycine leads to a significant compaction and partial folding as judged by hydroxyl radical protections. In the presence of millimolar Mg(2+) concentrations, the tandem aptamer binds glycine cooperatively. The glycine binding transition involves a further compaction, additional tertiary packing interactions and further uptake of magnesium ions relative to the state in high Mg(2+) but no glycine. Employing density reconstruction algorithms, we obtain low resolution 3-D structures for all three states from the SAXS measurements. These data provide a first glimpse into the structural conformations of the VCI-II aptamer, establish rigorous constraints for further modeling, and provide a framework for future mechanistic studies.     

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.     

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.     

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.     

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.     

Tetracycline Determines the Conformation of Its Aptamer at Physiological Magnesium Concentrations

Synthetic riboswitches are versatile tools for the study and manipulation of biological systems. Yet, the underlying mechanisms governing its structural properties and regulation under physiological conditions are poorly studied. We performed spectroscopic and calorimetric experiments to explore the folding kinetics and thermodynamics of the tetracycline-binding aptamer, which can be employed as synthetic riboswitch, in the range of physiological magnesium concentrations. The dissociation constant of the ligand-aptamer complex was found to strongly depend on the magnesium concentration. At physiological magnesium concentrations, tetracycline induces a significant conformational shift from a compact, but heterogeneous intermediate state toward the completely formed set of tertiary interactions defining the regulation-competent structure. Thus, the switching functionality of the tetracycline-binding aptamer appears to include both a conformational rearrangement toward the regulation-competent structure and its thermodynamic stabilization.     

Bacterial gene regulation: metal ion sensing by proteins or RNA

Until recently, metal sensing in bacteria seemed to be accomplished exclusively by metalloregulatory proteins; however, a surprising new finding is that a metal ion itself can act as a riboswitch ligand to shut down gene expression. Interestingly, this ion is Mg(2+), known to be required for a wide variety of cellular functions and for correct folding of RNAs. It remains to be discovered whether other ion-dependent riboswitches exist, which would open up a new dimension for regulatory RNAs.     

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.     

A Structural Basis for the Recognition of 2′-Deoxyguanosine by the Purine Riboswitch

Riboswitches are noncoding RNA elements that are commonly found in the 5′-untranslated region of bacterial mRNA. Binding of a small-molecule metabolite to the riboswitch aptamer domain guides the folding of the downstream sequence into one of two mutually exclusive secondary structures that directs gene expression. The purine riboswitch family, which regulates aspects of purine biosynthesis and transport, contains three distinct classes that specifically recognize guanine/hypoxanthine, adenine, or 2′-deoxyguanosine (dG). Structural analysis of the guanine and adenine classes revealed a binding pocket that almost completely buries the nucleobase within the core of the folded RNA. Thus, it is somewhat surprising that this family of RNA elements also recognizes dG. We have used a combination of structural and biochemical techniques to understand how the guanine riboswitch could be converted into a dG binder and the structural basis for dG recognition. These studies reveal that a limited number of sequence changes to a guanine-sensing RNA are required to cause a specificity switch from guanine to 2′-deoxyguanosine, and to impart an altered structure for accommodating the additional deoxyribose sugar moiety.     

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.     

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 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.     

Modeling the noncovalent interactions at the metabolite binding site in purine riboswitches

We present gas phase quantum chemical studies on the metabolite binding interactions in two important purine riboswitches, the adenine and guanine riboswitches, at the B3LYP/6-31G(d,p) level of theory. In order to gain insights into the strucutral basis of their discriminative abilities of regulating gene expression, the structural properties and binding energies for the gas phase optimized geometries of the metabolite bound binding pocket are analyzed and compared with their respective crystal geometries. Kitaura-Morokuma analysis has been carried out to calculate and decompose the interaction energy into various components. NBO and AIM analysis has been carried out to understand the strength and nature of binding of the individual aptamer bases with their respective purine metabolites. The Y74 base, U in case of adenine riboswitch and C in case of guanine riboswitch constitutes the only differentiating element between the two binding pockets. As expected, with W:W cis G:C74 interaction contributing more than 50% of the total binding energy, the interaction energy for metabolite binding as calculated for guanine (-46.43 Kcal/mol) is nearly double compared to the corresponding value for that of adenine (-24.73 Kcal/mol) in the crystal context. Variations in the optimized geometries for different models and comparison of relative contribution to metabolite binding involving four conserved bases reveal the possible role of U47:U51 W:H trans pair in the conformational transition of the riboswitch from the metabolite free to metabolite bound state. Our results are also indicative of significant contributions from stacking and magnesium ion interactions toward cooperativity effects in metabolite recognition.