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Ligand binding to 2΄-deoxyguanosine sensing riboswitch in metabolic context.

ABSTRACT: The mfl-riboswitch is a transcriptional off-switch, which down-regulates expression of subunit β of ribonucleotide reductase in Mesoplasma florum upon 2΄-deoxyguanosine binding. We characterized binding of 2΄-deoxyguanosine to the mfl-aptamer domain (WT aptamer) and a sequence-stabilized aptamer (MT aptamer) under in vitro and 'in-cell-like' conditions by isothermal titration calorimetry (ITC) and nuclear magnetic resonance (NMR) spectroscopy. 'In-cell-like' environment was simulated by Bacillus subtilis cell extract, in which both aptamers remained sufficiently stable to detect the resonances of structural elements and ligand binding in 2D NMR experiments. Under 'in-cell-like'-environment, (i) the WT aptamer bound the endogenous metabolite guanosine and (ii) 2΄-deoxyguanosine efficiently displaced guanosine from the WT aptamer. In contrast, MT aptamer exhibited moderate binding to 2΄-deoxyguanosine and weak binding to guanosine. NMR experiments indicated that binding of guanosine was not limited to the aptamer domain of the riboswitch but also the full-length mfl-riboswitch bound guanosine, impacting on the regulation efficiency of the riboswitch and hinting that, in addition to 2΄-deoxyguanosine, guanosine plays a role in riboswitch function in vivo. Reporter gene assays in B. subtilis demonstrated the regulation capacity of the WT aptamer, whereas the MT aptamer with lower affinity to 2΄-deoxyguanosine was not able to regulate gene expression.

SUBMITTER: Kim YB

PROVIDER: S-EPMC5435998 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Structure and dynamics of the deoxyguanosine-sensing riboswitch studied by NMR-spectroscopy.

Project description:The mfl-riboswitch regulates expression of ribonucleotide reductase subunit in Mesoplasma florum by binding to 2'-deoxyguanosine and thereby promoting transcription termination. We characterized the structure of the ligand-bound aptamer domain by NMR spectroscopy and compared the mfl-aptamer to the aptamer domain of the closely related purine-sensing riboswitches. We show that the mfl-aptamer accommodates the extra 2'-deoxyribose unit of the ligand by forming a more relaxed binding pocket than these found in the purine-sensing riboswitches. Tertiary structures of the xpt-aptamer bound to guanine and of the mfl-aptamer bound to 2'-deoxyguanosine exhibit very similar features, although the sequence of the mfl-aptamer contains several alterations compared to the purine-aptamer consensus sequence. These alterations include the truncation of a hairpin loop which is crucial for complex formation in all purine-sensing riboswitches characterized to date. We further defined structural features and ligand binding requirements of the free mfl-aptamer and found that the presence of Mg(2+) is not essential for complex formation, but facilitates ligand binding by promoting pre-organization of key structural motifs in the free aptamer.

| S-EPMC3159443 | biostudies-literature

The structure of a tetrahydrofolate-sensing riboswitch reveals two ligand binding sites in a single aptamer.

Project description:Transport and biosynthesis of folate and its derivatives are frequently controlled by the tetrahydrofolate (THF) riboswitch in Firmicutes. We have solved the crystal structure of the THF riboswitch aptamer in complex with folinic acid, a THF analog. Uniquely, this structure reveals two molecules of folinic acid binding to a single structured domain. These two sites interact with ligand in a similar fashion, primarily through recognition of the reduced pterin moiety. 7-deazaguanine, a soluble analog of guanine, binds the riboswitch with nearly the same affinity as its natural effector. However, 7-deazaguanine effects transcriptional termination to a substantially lesser degree than folinic acid, suggesting that the cellular guanine pool does not act upon the THF riboswitch. Under physiological conditions the ligands display strong cooperative binding, with one of the two sites playing a greater role in eliciting the regulatory response, which suggests that the second site may play another functional role.

| S-EPMC3196276 | biostudies-literature

Variants of the guanine riboswitch class exhibit altered ligand specificities for xanthine, guanine, or 2'-deoxyguanosine.

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Structure and ligand binding of the glutamine-II riboswitch.

Project description:We have determined the structure of the glutamine-II riboswitch ligand binding domain using X-ray crystallography. The structure was solved using a novel combination of homology modeling and molecular replacement. The structure comprises three coaxial helical domains, the central one of which is a pseudoknot with partial triplex character. The major groove of this helix provides the binding site for L-glutamine, which is extensively hydrogen bonded to the RNA. Atomic mutation of the RNA at the ligand binding site leads to loss of binding shown by isothermal titration calorimetry, explaining the specificity of the riboswitch. A metal ion also plays an important role in ligand binding. This is directly bonded to a glutamine carboxylate oxygen atom, and its remaining inner-sphere water molecules make hydrogen bonding interactions with the RNA.

| S-EPMC6698751 | biostudies-literature

Structure and ligand binding of the SAM-V riboswitch.

Project description:SAM-V is one of the class of riboswitches that bind S-adenosylmethione, regulating gene expression by controlling translation. We have solved the crystal structure of the metY SAM-V riboswitch bound to its SAM ligand at 2.5 Å resolution. The RNA folds as an H-type pseudoknot, with a major-groove triple helix in which resides the SAM ligand binding site. The bound SAM adopts an elongated conformation aligned with the axis of the triple helix, and is held at either end by hydrogen bonding to the adenine and the amino acid moieties. The central sulfonium cation makes electrostatic interactions with an U:A.U base triple, so conferring specificity. We propose a model in which SAM binding leads to association of the triplex third strand that stabilizes a short helix and occludes the ribosome binding site. Thus the new structure explains both ligand specificity and the mechanism of genetic control.

| S-EPMC6061858 | biostudies-literature

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

Project description: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.

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Structural Basis for Ligand Binding to the Guanidine-I Riboswitch.

Project description:The guanidine-I riboswitch is a conserved RNA element with approximately 2,000 known examples across four phyla of bacteria. It exists upstream of nitrogen metabolism and multidrug resistance transporter genes and alters expression through the specific recognition of a free guanidinium cation. Here we report the structure of a guanidine riboswitch aptamer from Sulfobacillus acidophilus at 2.7 Å resolution. Helices P1, P1a, P1b, and P2 form a coaxial stack that acts as a scaffold for ligand binding. A previously unidentified P3 helix docks into P1a to form the guanidinium binding pocket, which is completely enclosed. Every functional group of the ligand is recognized through hydrogen bonding to guanine bases and phosphate oxygens. Guanidinium binding is further stabilized through cation-? interactions with guanine bases. This allows the riboswitch to recognize guanidinium while excluding other bacterial metabolites with a guanidino group, including the amino acid arginine.

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Structural basis for ligand binding to the guanidine-II riboswitch.

Project description:The guanidine-II riboswitch, also known as mini-ykkC, is a conserved mRNA element with more than 800 examples in bacteria. It consists of two stem-loops capped by identical, conserved tetraloops that are separated by a linker region of variable length and sequence. Like the guanidine-I riboswitch, it controls the expression of guanidine carboxylases and SugE-like genes. The guanidine-II riboswitch specifically binds free guanidinium cations and functions as a translationally controlled on-switch. Here we report the structure of a P2 stem-loop from the Pseudomonas aeruginosa guanidine-II riboswitch aptamer bound to guanidine at 1.57 Å resolution. The hairpins dimerize via the conserved tetraloop, which also contains the binding pocket. Two guanidinium molecules bind near the dimerization interface, one in each tetraloop. The guanidinium cation is engaged in extensive hydrogen bonding to the RNA. Contacts include the Hoogsteen face of a guanine base and three nonbridging phosphate oxygens. Cation-π interactions and ionic interactions also stabilize ligand binding. The guanidine-II riboswitch utilizes the same recognition strategies as the guanidine-I riboswitch while adopting an entirely different and much smaller RNA fold.

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A transient conformation facilitates ligand binding to the adenine riboswitch.

Project description:RNAs adopt various conformations to perform different functions in cells. Incapable of acquiring intermediates, the key initiations of ligand recognition in the adenine riboswitch have not been characterized. In this work, stopped-flow fluorescence was used to track structural switches in the full-length adenine riboswitch in real time. We used PLOR (position-selective labeling of RNA) to incorporate fluorophores into desired positions in the RNA. The switching sequence P1 responded to adenine more rapidly than helix P4 and the binding pocket, followed by stabilization of the binding pocket, P4, and annealing of P1. Moreover, a transient intermediate consisting of an unwound P1 was detected during adenine binding. These events were observed in both the WT riboswitch and a functional mutant. The findings provide insight into the conformational changes of the riboswitch RNA triggered by a ligand.

| S-EPMC8652005 | biostudies-literature

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