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Linking aptamer‐ligand binding and expression platform folding in riboswitches: prospects for mechanistic modeling and design

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The power of riboswitches in regulation of bacterial metabolism derives from coupling of two characteristics: recognition and folding. Riboswitches contain aptamers, which function as biosensors. Upon detection of the signaling molecule, the riboswitch transduces the signal into a genetic decision. The genetic decision is coupled to refolding of the expression platform, which is distinct from, although overlapping with, the aptamer. Early biophysical studies of riboswitches focused on recognition of the ligand by the aptamer‐an important consideration for drug design. A mechanistic understanding of ligand‐induced riboswitch RNA folding can further enhance riboswitch ligand design, and inform efforts to tune and engineer riboswitches with novel properties. X‐ray structures of aptamer/ligand complexes point to mechanisms through which the ligand brings together distal strand segments to form a P1 helix. Transcriptional riboswitches must detect the ligand and form this P1 helix within the timescale of transcription. Depending on the cell's metabolic state and cellular environmental conditions, the folding and genetic outcome may therefore be affected by kinetics of ligand binding, RNA folding, and transcriptional pausing, among other factors. Although some studies of isolated riboswitch aptamers found homogeneous, prefolded conformations, experimental, and theoretical studies point to functional and structural heterogeneity for nascent transcripts. Recently it has been shown that some riboswitch segments, containing the aptamer and partial expression platforms, can form binding‐competent conformers that incorporate an incomplete aptamer secondary structure. Consideration of the free energy landscape for riboswitch RNA folding suggests models for how these conformers may act as transition states—facilitating rapid, ligand‐mediated aptamer folding. WIREs RNA 2015, 6:631–650. doi: 10.1002/wrna.1300 This article is categorized under: RNA Structure and Dynamics > Influence of RNA Structure in Biological Systems RNA Interactions with Proteins and Other Molecules > Small Molecule–RNA Interactions Regulatory RNAs/RNAi/Riboswitches > Riboswitches
Ligand contacts constrain the placement of P1 helix‐forming strands, favoring helix formation. Distances between P1 helix strands or their flanking junctions upon ligand binding in angstroms are highlighted. Maximum and minimum distances between atoms on the distal P1 helix‐forming strand segments (or adjacent ‘junction’ segments) that contact the ligand, either directly or via water molecule or magnesium ion, are indicated as dashed lines. As ligand binding constrains the distances between nonadjacent strands, it facilitates strand association and P1 helix formation. In panels a–f, the ligand is colored by element, red sticks are the closing base pair of P1 helix, blue sticks/ ribbon are J1/2, orange sticks/ ribbon are J3/1 (but J6/1 in FMN riboswitch), red ribbons are P1 helix strands, gray sphere is a water molecule, and green sphere is a magnesium ion. (a) Adenine riboswitch, (b) Guanine riboswitch, (c) FMN riboswitch, (d) c‐di‐GMP riboswitch, (e) SAM III riboswitch, and (f) Lysine riboswitch.
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Riboswitch ligands bring P1 helix strands together. (a) Schematic of P1 helix riboswitch showing three domains of the aptamer and overlapping expression domain. (b) Position of ligand above P1 helices within structures of ligand/aptamer complexes from P1 helix‐regulated riboswitches. The backbone of the riboswitch is shown as tube with the P1 helix highlighted in red, and junctions connecting P1 strands are in orange. The ligand is shown as sphere, and nucleotides that have contacts with the ligand in either the P1 helix or junctions connecting P1 strands are shown as stick. Tertiary structural motifs, such as pseudoknot (PK), kissing loop (KL), helix‐loop (HL) contacts, and nonadjacent nucleotide stack (‘high‐five’ stack, HF) are highlighted in blue. Protein databank (PDB) entries used in this figure are: TPP riboswitch (2cky), FMN riboswitch (3f2q), S‐adenosyl homocysteine (SAH) riboswitch (3npq), Lysine riboswitch (3dil), c‐di‐guanine mononucleotide phosphate (GMP) riboswitch (3irw), Adenine riboswitch (1y26), Guanine riboswitch (1y27), SAM‐III riboswitch (3e5c), and SAM‐I riboswitch (2gis). These riboswitches are classified into three types based on the relative position of ligand to the P1 helix. For the largest group, the ligand clearly stabilizes the closing base pair of the P1 helix through direct contact (middle and lower panels), or indirectly via contacts with linking strands (FMN riboswitch). In one case (SAH), the P4 helix plays the functional role of P1 and shows a similar pattern of contacts with the ligand. Only TPP lacks obvious stabilizing contacts with the P1 helix.
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Schematic illustration of ‘conformational collapse’ of the SAM‐I riboswitch due to interaction of SAM with a hotspot G residue (highlighted in red) in junction J12. In the absence of SAM, the G residue in question is predicted to base pair with over 30 partners in alternative riboswitch conformers (two of the lowest energy conformers are illustrated). SAM makes extensive contacts with the G residue, blocking alternative base pairings and alternative conformations. Residues 3′ of the aptamer are shown in blue. The schematic represents predicted riboswitch folding during the ‘sensing window’ when antiterminator, but not terminator transcription have been completed.
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Schematic illustration of classical kinetic trapping model for ligand‐induced riboswitch aptamer folding under kinetic control. Transcription is initiated (panel a) upstream of the start site (AUG). In the absence of ligand (b) the nascent transcript forms a series of hairpin loops from adjacent strand segments, including a secondary structure element in the expression platform. This element may be an antiterminator (for a negatively regulated transcriptional riboswitch, as shown) or a terminator (positively regulated transcriptional riboswitch) or it may sequester or expose an SD sequence (positively or negatively regulated translational riboswitch), respectively. This model assumes that the distal strand segments constituting the P1 helix can anneal before segments involved in competing downstream helices are fully transcribed. If there is sufficient ligand to bind to and fix aptamer folding (c) before the completion of expression platform transcription then aptamer formation prevents antiterminator formation, which in turn enables terminator formation further downstream. If the transcription complex reaches the start site before the terminator folds (d) transcription could proceed in the presence of the ligand.
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Schematic illustration of models for how ligand binding may perturb riboswitch folding thermodynamically or kinetically, as they would appear on the free energy landscape (FEL). (a) In the absence of ligand, the FEL represents the free energy linked to each possible secondary and tertiary structure fold of the RNA. The horizontal axes represent the multidimensional RNA conformational space. (b) In the presence of ligand, the FEL represents the free energy of the system of RNA + ligand. According to the ‘conformational capture’ model, the free energy of a single RNA conformation is bound by the ligand, leading to a dip in free energy corresponding to the binding affinity, while the relative free energy of other conformers is unaffected. (c) If the ligand also binds, with reduced affinity, to a subset of conformers (called ‘binding competent’), then the corresponding regions of conformational space also display a more modest dip in free energy. If the binding‐competent region includes ‘transition states’ in the aptamer folding pathway, then the aptamer formation will be facilitated kinetically as well as thermodynamically.
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Schematic models for kinetic control of riboswitch folding and gene expression outcome during transcription, illustrating the role of multiple competing conformations. Top panel, the P1 helix does not form and nucleate sufficient P1 base pairs for ligand binding until other binding‐competent intermediates form during the ‘decision window’, when the expression platform is being transcribed. The bottom panel illustrates a modified picture of kinetic trapping. The aptamer folds as it is transcribed‐the ligand binds and fixes the aptamer fold, blocking the nucleation of the competing helix or blocking its formation via branch migration. Depending on whether the riboswitch is a positive or negative regulator, an additional terminator helix may form further downstream in the absence of the competing ‘antiterminator’ helix. These two models should not be viewed as mutually exclusive. In fact, varying combinations of the two are likely in different ribsowitches, and even when a single riboswitch is transcribed in different cellular environments.
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Alternative folds of SAM‐I riboswitch in the absence of ligand. (a) Putative secondary structure of B. subtilis yitJ SAM‐I riboswitch 251 yitJ construct from Winkler et al. (left) and 3P1_10AT yitJ construct from Boyapati et al. (right) with P1, P2, P3, P4, and anti P4 helices. Residues in cyan are involved in the formation of the pseudoknot. Residues indicated in green are required for formation of the Anti‐P4 or the P4 helix in 251 yitJ and 3P1_10AT yitJ, respectively. (b) Inline probing data of 251 yitJ (left) and 3P1_10AT yitJ (right). The lanes NR, OH, and T1 indicate no reaction, alkaline hydrolysis, and RNase T1, respectively. Residues protected by pseudo knot interaction in the presence of SAM in both the constructs are indicated by a cyan box, green boxed bands correspond to green boxed residues in panel a. (b, left panel: Reprinted with permission from Ref . Copyright 2003 Macmillan)
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Regulatory RNAs/RNAi/Riboswitches > Riboswitches
Regulatory RNAs/RNAi/Riboswitches
RNA Interactions with Proteins and Other Molecules > Small Molecule–RNA Interactions
RNA Structure and Dynamics > Influence of RNA Structure in Biological Systems

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