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Riboswitches: small-molecule recognition by gene regulatory RNAs
Riboswitches: small-molecule recognition by gene regulatory RNAs Thomas E Edwards, Daniel J Klein and Adrian R Ferre´-D’Amare´ Riboswitches demonstrate the ability of highly structured RNA molecules to recognize small-molecule metabolites with high specificity and subsequently harness the binding energy for the control of gene expression. Crystal structures have now been determined for the metabolite-binding domains of riboswitches that respond to purines, thiamine pyrophosphate and S-adenosylmethionine, as well as for the glmS ribozyme, a catalytic riboswitch that is activated by the metabolite glucosamine-6-phosphate. In addition to these riboswitch structures, a solution NMR structure has been reported for a ribosensor that regulates heat shock genes in response to changes in temperature. These studies reveal the structural basis of the remarkable selectivity of riboswitches and, in conjunction with biochemical and biophysical measurements, provide a framework for detailed mechanistic understanding of riboswitch-mediated modulation of gene expression. Addresses Division of Basic Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, Seattle, WA 98109-1024, USA Corresponding author: Ferre´-D’Amare´, Adrian R ([email protected])
Current Opinion in Structural Biology 2007, 17:273–279 This review comes from a themed issue on Nucleic acids Edited by Dinshaw J Patel and Eric Westhof Available online 15th June 2007 0959-440X/$ – see front matter # 2007 Elsevier Ltd. All rights reserved. DOI 10.1016/j.sbi.2007.05.004
Introduction Riboswitches are cis-acting RNA elements that control gene expression by directly sensing the levels of specific small-molecule metabolites [1–4]. Here, we review advances in the structural characterization of these remarkable RNAs during the preceding two years. Formally, riboswitches consist of two domains [5]. A metabolite-binding or ‘aptamer’ domain recognizes the cognate small molecule. Binding is signaled to an effector domain or ‘expression platform’ that interfaces with the transcriptional, translational or post-transcriptional RNA modification machinery to modulate gene expression. This formal distinction is less clear-cut at the molecular level. Most known riboswitches are part of untranslated regions (UTRs) of mRNAs that appear to function by fluctuating between different conformations. The metabolite-bound www.sciencedirect.com
conformation of the riboswitch sequesters an adjacent UTR segment into a tightly folded RNA domain. In the metabolite-free conformation, the same segment participates in the control of translation (e.g. the segment interacts with or contains a Shine–Dalgarno sequence) or transcription (e.g. the segment forms a transcriptional terminator or anti-terminator stem). Thus, the aptamer and expression platform are not ‘domains’ that can co-exist, but different structural states of an RNA segment. The distinction also breaks down for the glmS ribozyme-riboswitch, for which metabolite binding activates a latent catalytic activity.
Overview of riboswitch structures Crystal structures have been reported for metabolitebound domains of the 50 -UTRs of bacterial mRNAs containing the guanine [6,7] and the adenine [7] variants of the purine riboswitch. The RNA folds into three helices or paired regions, P1, P2 and P3, which are arranged as an inverted ‘h’ (Figure 1a,b). P1 stacks coaxially under P3, and P2 and P3 pack side-by-side. This overall arrangement, stabilized by the interaction of the terminal loops of P2 and P3, is reminiscent of the structure of the hammerhead ribozyme ([8]; see the review by Scott in this issue). The purine ligand is buried in a solvent-inaccessible pocket at the three-way junction of P1, P2 and P3. The 30 strand of P1 (red in Figure 1a,b) forms part of an alternative secondary structure within the expression platform of the metabolitefree conformational state of the 50 -UTR. Direct interactions between the 30 strand of the P1 ‘switch helix’ and both the purine and the J2/3 interhelical junction explain the ligand dependence of P1 formation. The structures of the metabolite-binding domain of the thiamine pyrophosphate (TPP) riboswitch from the eukaryote Arabidopsis thaliana [9] and the bacterium Escherichia coli [10,11] are essentially identical. Like the purine riboswitch, the metabolite-bound TPP riboswitch adopts a compact, inverted-h architecture with two parallel sets of coaxially stacked helices (P1-P2-P3 and P4-P5) joined by a three-way junction (Figure 1c,d). An A-minor interaction between L5 and P3, functionally analogous to the loop– loop interaction of the purine riboswitch, stabilizes this arrangement. Unlike the purine riboswitch, TPP is not buried at the three-way helical junction; instead, TPP bridges the two parallel helical stacks. This places TPP 25 A˚ away from the P1 switch helix, suggesting that metabolite binding only indirectly stabilizes P1. Three distinct classes of S-adenosylmethionine (SAM) riboswitches have been identified [12–14]. The crystal Current Opinion in Structural Biology 2007, 17:273–279
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Figure 1
Crystal structures of riboswitch–effector complexes, and schematic depiction of conserved primary and secondary structures. (a,b) Purine riboswitch (PDB code 1U8D), (c,d) TPP riboswitch (PDB code 2GDI), (e,f) SAM riboswitch (PDB code 2GIS) and (g,h) glmS ribozyme-riboswitch (PDB code 2H0Z). The 30 half of the P1 helices, which participate in the genetic switch event, is colored red, and tertiary interactions thought to be important in folding and RNA–metabolite complex stability are green. Bound metabolites are shown in blue; PK and KT denote pseudoknots and K-turns, respectively; ‘var’ indicates an RNA segment of phylogenetically variable length and composition. Filled spheres indicate that the sequence is not conserved, but the Watson–Crick pairing is.
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structure of the metabolite-binding domain of the first class to be discovered (the SAM-I riboswitch) reveals an architecture that is distinctly different from the inverted-h fold of the purine and TPP riboswitches (Figure 1e,f) [15]. The SAM-I riboswitch contains two stacks (P1-P4 and P2a-P3) that, rather than packing sideby-side, cross at an angle of 708. A pseudoknot coupled to a kink-turn [16] atop P2 appears to stabilize this fold. The SAM-binding site is located at the interface of the minor grooves of P1 and P3, and has features reminiscent of the metabolite-binding sites of both the TPP and the purine riboswitches. Like TPP, SAM bridges two helical stacks. Like the purine riboswitches, SAM makes van der Waals contact with the 30 strand of the P1 switch helix. In many Gram-positive bacteria, the glmS ribozyme-riboswitch is part of the 50 -UTR of the mRNA that encodes glucosamine-6-phosphate (GlcN6P) synthetase [17]. This riboswitch has a self-cleavage activity that becomes activated when it binds GlcN6P. The structure of the glmS ribozyme [18,19] consists of three parallel helical stacks (Figure 1g,h). A doubly pseudoknotted core (P1-P2-P2.1P2.2) is buttressed by a peripheral RNA domain (P4-P4.1). The solvent-exposed GlcN6P-binding pocket is composed of two highly distorted major grooves and abuts the site of self-cleavage, reflecting the coenzyme function of GlcN6P (see the review by Scott in this issue). Among currently characterized riboswitches, the glmS ribozyme is unique because it adopts its active structure in the absence of its metabolite ligand [18,20]. As other ribozymes, such as the natural hairpin ribozyme [21,22] and the in vitro selected Diels–Alderase [23], also assemble rigid active sites, this disparity might reflect the different constraints under which ribozymes and RNAs that function by alternative folding evolved.
The two helical stacks of the thi-box riboswitch separately recognize the aminopyrimidine and pyrophosphate moieties of TPP. J3/2 of the ‘pyrimidine sensor helix’ (the P1P2-P3 stack) adopts a canonical T-loop fold [9,10,11]. Binding of the aminopyrimidine ring of TPP to G40 of this T-loop (Figure 2b) mimics a tertiary interaction between the D- and T-loops in the classic L-shaped fold of tRNA. The aminopyrimidine of TPP and G40 replace G18 and C55, respectively (purines and pyrimidines are reversed between the riboswitch and the tRNA). Mimicry of an allRNA structure by an exogenous small molecule is reminiscent of ATP binding by an in vitro selected aptamer RNA, whereby the ATP completes a GNRA tetraloop [27,28]. Rather than directly binding to the negatively charged pyrophosphate of TPP, the ‘pyrophosphate sensor helix’ (the P4-P5 stack) of the riboswitch coordinates the pyrophosphate mostly through two solvated divalent metals ions (the exception is G78) [9,10,11,29]. Thus, the riboswitch effectively binds a positively charged TPP– cation complex. Structures of the TPP riboswitch bound to three metabolite analogs suggest that the pyrimidine sensor helix is largely preformed, whereas the pyrophosphate sensor helix becomes organized concomitant with binding of the TPP–cation complex [11]. The SAM-I riboswitch sandwiches its ligand between two parallel helices [15]. P1 recognizes the ribose–sulfur backbone of SAM primarily through van der Waals contacts (Figure 2c). By contrast, the P3 helix binds the adenine ring and the amino acid by making several hydrogen bonds and stacking interactions. Interestingly, the RNA does not directly recognize the methionine e-methyl group. Rather, the positively charged sulfur atom makes a favorable electrostatic interaction with the partial negative charge on O2 of U7. This might explain the enhanced binding of SAM compared to S-adenosylhomocysteine and other non-positively charged analogs [12,30].
Ligand recognition by riboswitches The purine riboswitch recognizes its ligand almost exclusively through hydrogen-bonding interactions that satisfy nearly all possible acceptors and donors of the purine (Figure 2a). Purine riboswitch structures have been solved bound to 2,6-diaminopurine [24] and 2,4,6-triaminopyrimidine [25], in addition to the biological activators hypoxanthine [6], guanine [7] and adenine [7]. The purine ligand is primarily recognized by residue 74 of the riboswitch, a pyrimidine, through Watson–Crick pairing [6,7,26]. In addition, U51 and the 20 -OH of U22 hydrogen bond to the N3/N9 edge (corresponding to the sugar edge of nucleotides) and the N7, respectively, of the purine. Reliance on Watson–Crick (as opposed to Hoogsteen) pairing for recognition enables the same RNA scaffold to regulate either adenine or guanine metabolism by having U74 or C74, respectively. Gilbert et al. [24] noted that the purine ligand makes poor stacking interactions and proposed that this enhances the discriminatory role of Watson–Crick pairing with residue 74. www.sciencedirect.com
Recognition of GlcN6P, a simple phosphorylated hexosamine sugar, by the glmS ribozyme-riboswitch presents a challenge for RNA that is distinct from those posed by purines, TPP and SAM, all of which have nucleotide-like substructures. Crystal structures of the glmS ribozymeriboswitch were solved bound to glucose-6-phosphate (Glc6P), an isosteric competitive inhibitor (antagonist) of GlcN6P [18], and to the authentic activator GlcN6P ([19]; DJ Klein and AR Ferre´-D’Amare´, unpublished), revealing that GlcN6P and Glc6P are equivalently positioned in the glmS ribozyme-riboswitch active site. The sugar hydroxyl groups hydrogen bond to G1, C2, A50 and G65 (Figure 2d). The amine of GlcN6P hydrogen bonds to a water molecule, U51 and the 50 oxygen of G1; the last is the leaving group of the transesterification reaction catalyzed by the ribozyme. As in the TPP riboswitch, the phosphate of the metabolite interacts with the RNA through two solvated divalent metal ions ([19]; DJ Klein and AR Ferre´-D’Amare´, unpublished). TPP, SAM and Current Opinion in Structural Biology 2007, 17:273–279
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Figure 2
GlcN6P all present negatively charged moieties to the N1/N2 face of a guanine residue (G78, G11 and G1, respectively). This face of guanine has previously been shown to be an anion-binding site [31].
Aptamers and riboswitches The discovery of RNA ‘aptamers’ that bind small molecules through in vitro selection (reviewed in [32]) preceded the discovery of their naturally occurring counterparts, riboswitches. We compared RNA and ligand solvent accessibilities for several aptamer–ligand (reviewed in [33]) and riboswitch–effector structures. Riboswitches bury 90 6% (mean standard deviation) of the accessible surface of their effectors. Aptamers, by contrast, bury 71 14% of the solvent-accessible surface of their cognate ligand, consistent with an in vitro selection methodology in which the ligand is tethered to beads. Furthermore, riboswitches tend to have more compact structures than aptamers, as indicated by solvent-accessible surface areas of 156 5 A˚2/nt for riboswitches and 171 8 A˚2/nt for aptamers (end correction as in [34]). Despite these differences and the different evolutionary origins of aptamers and riboswitches, the binding energy correlates linearly with the buried surface area of the ligand (Figure 3). Such a relationship might be expected on theoretical grounds [35]. This relationship implies that the vitamin B12 riboswitch (Kd 0.3 mM [36]) and the tetracycline aptamer (Kd 0.8 nM [37]) can achieve their known affinity by burying 40% (of 1200 A˚2) and nearly all (600 A˚2), respectively, of the surface area of their ligands. Exceptions to the trend are RNAs that bind the smallest ligands, such as the purine riboswitches and the theophylline aptamer, which completely envelop the small molecule. For these ligands, higher affinity must result from other factors, such as RNA–RNA interactions. Our analysis implies that the glycine riboswitch will also be an outlier, consistent with the cooperative binding exhibited by this RNA [38]. Structural and biochemical analyses of several GTP aptamers of varying degrees of complexity show that increased RNA complexity correlates with higher affinity [39]. However, higher affinity does not result in higher specificity [40]. These studies suggest that higher affinity is derived from additional RNA–RNA interactions that stabilize the global RNA fold and that explicit selection is needed to achieve higher specificity. Compared to aptamers, riboswitch structures are stabilized by a variety of peripheral RNA contacts (Figure 1) and these cellular regulators presumably have undergone stringent natural selection, thus accounting for their higher affinities and selectivity. RNA–ligand interaction in the (a) purine, (b) thi-box, (c) SAM-I and (d) glmS ribozyme riboswitches. Color scheme as in Figure 1, with the addition of metal ions (green spheres) and water molecules (red spheres). Dotted lines denote hydrogen bonds; solid lines denote inner-sphere metal ion coordination. In (a), C74 is colored with nitrogens in blue and oxygens in red to illustrate hydrogen bonding with hypoxanthine (HX). Current Opinion in Structural Biology 2007, 17:273–279
Riboswitch folding and regulation of gene expression Although chemical probing has been used to investigate the small-molecule-dependent alternative folding of some full-length riboswitches (reviewed in [1]), most biochemical and biophysical studies of riboswitches have www.sciencedirect.com
Metabolite recognition by riboswitches Edwards, Klein and Ferre´-D’Amare´ 277
Figure 3
thus far focused on isolated metabolite-binding domains. Published studies include ensemble and single-molecule fluorescence resonance energy transfer (FRET) characterization of the adenine riboswitch (demonstrating the presence of Mg2+-dependent folding intermediates [41]), solution NMR experiments on the Mg2+ and effector dependence of the terminal loop–loop interaction in the purine riboswitches [42], and small-angle X-ray scattering analysis of the shape of the glycine riboswitch in the unliganded, Mg2+-induced folded and effector-bound states [43]. With the exception of the glmS ribozyme, riboswitchmodulated gene regulation results from the sequestration of a switch segment (red in Figure 1) into either a metabolite-bound or a metabolite-free conformational fold. For riboswitches that exert genetic control at the transcriptional level, co-transcriptional mRNA folding is an essential component of the mechanism of action [44] and must be considered alongside the thermodynamics and kinetics of ligand binding [24,45], as well as the relative speed of transcription [46]. In a study of the flavin mononucleotide (FMN) riboswitch, it was shown that the speed of transcription relative to the kinetics of FMN binding precludes this RNA from reaching thermodynamic equilibrium before the point at which a genetic decision must be made [46]. Consequently, metabolite concentrations needed to trigger this riboswitch in vivo must be considerably higher than the in vitro dissociation constant measured with previously transcribed RNA. These studies also implicate transcriptional pause sites as critical components of riboswitch-mediated regulation. Several ribosensors have been described that function by means other than direct metabolite binding. These include non-coding mRNA elements that respond to the presence of uncharged tRNAs [47], the concentration of Mg2+ [48] and temperature [49]. A series of ‘RNA thermometers’ in the 50 -UTRs of heat shock response genes unfold at elevated temperatures, releasing a Shine–Dalgarno sequence sequestered at lower temperatures (Figure 4a). A solution NMR structure of the lower temperature state has been determined, in which several residues form noncanonical base pairs (Figure 4b) [49]. The imino proton resonances of these residues disappear at elevated temperatures, indicating that these pairs melt. The coupling of folding with the control of gene expression is a feature shared by these ribosensors and riboswitches.
Small-molecule binding by aptamers and riboswitches. (a) The solvent-accessible surface area of the ligand that is buried when in complex with aptamers or riboswitches correlates linearly with affinity, with a binding energy of 19 cal/mol per A˚2 of buried area. Red and blue represent riboswitches and aptamers, respectively; squares and circles denote structures solved by X-ray crystallography or NMR, www.sciencedirect.com
respectively; 95% prediction intervals are shown as dashed lines. The purine and theophylline complexes were excluded from the analysis. (b) Solvent-accessible surfaces of riboswitch- or aptamerbound ligands. Panels show the absolute solvent-accessible area of each ligand atom (dark blue 0–1 A˚2, light blue 1–10 A˚2, green 10–20 A˚2, yellow 20–30 A˚2, orange 30–40 A˚2, red 40–60 A˚2) mapped onto the molecular surface overlaying it. Buried surface areas were calculated using a 1.4 A˚ radius probe [50]. Aptamer structures are reviewed in [32]. Current Opinion in Structural Biology 2007, 17:273–279
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Figure 4
Foundation. TEE and DJK contributed equally to the writing of this review.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
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Secondary structure (a) and NMR structure (PDB code 2GIO) (b) of the ROSE (repressor of heat-shock gene expression) ribosensor, which functions as an RNA thermometer. Upon increase in temperature (D), a helix primarily consisting of non-canonical Watson–Crick pairings melts to liberate a Shine–Dalgarno sequence (depicted in gray boxes). Except for line drawings, figures were prepared with PyMol [51].
Conclusions The recent determination of the structures of the metabolite-binding domains of riboswitches opens the way to multiple research avenues that take advantage of highresolution information. These include analysis of the molecular recognition mechanisms employed by riboswitches and design of artificial effectors, elucidation of the mechanism of action of the coenzyme GlcN6P in the glmS ribozyme and, most generally, coupling of ligandinduced folding of RNA to the cellular processes of transcription, post-transcriptional RNA processing and translation.
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Current Opinion in Structural Biology 2007, 17:273–279
Current Opinion in Structural Biology 2007, 17:273–279 This review comes from a themed issue on Nucleic acids Edited by Dinshaw J Patel and Eric Westhof Available online 15th June 2007 0959-440X/$ – see front matter # 2007 Elsevier Ltd. All rights reserved. DOI 10.1016/j.sbi.2007.05.004
Introduction Riboswitches are cis-acting RNA elements that control gene expression by directly sensing the levels of specific small-molecule metabolites [1–4]. Here, we review advances in the structural characterization of these remarkable RNAs during the preceding two years. Formally, riboswitches consist of two domains [5]. A metabolite-binding or ‘aptamer’ domain recognizes the cognate small molecule. Binding is signaled to an effector domain or ‘expression platform’ that interfaces with the transcriptional, translational or post-transcriptional RNA modification machinery to modulate gene expression. This formal distinction is less clear-cut at the molecular level. Most known riboswitches are part of untranslated regions (UTRs) of mRNAs that appear to function by fluctuating between different conformations. The metabolite-bound www.sciencedirect.com
conformation of the riboswitch sequesters an adjacent UTR segment into a tightly folded RNA domain. In the metabolite-free conformation, the same segment participates in the control of translation (e.g. the segment interacts with or contains a Shine–Dalgarno sequence) or transcription (e.g. the segment forms a transcriptional terminator or anti-terminator stem). Thus, the aptamer and expression platform are not ‘domains’ that can co-exist, but different structural states of an RNA segment. The distinction also breaks down for the glmS ribozyme-riboswitch, for which metabolite binding activates a latent catalytic activity.
Overview of riboswitch structures Crystal structures have been reported for metabolitebound domains of the 50 -UTRs of bacterial mRNAs containing the guanine [6,7] and the adenine [7] variants of the purine riboswitch. The RNA folds into three helices or paired regions, P1, P2 and P3, which are arranged as an inverted ‘h’ (Figure 1a,b). P1 stacks coaxially under P3, and P2 and P3 pack side-by-side. This overall arrangement, stabilized by the interaction of the terminal loops of P2 and P3, is reminiscent of the structure of the hammerhead ribozyme ([8]; see the review by Scott in this issue). The purine ligand is buried in a solvent-inaccessible pocket at the three-way junction of P1, P2 and P3. The 30 strand of P1 (red in Figure 1a,b) forms part of an alternative secondary structure within the expression platform of the metabolitefree conformational state of the 50 -UTR. Direct interactions between the 30 strand of the P1 ‘switch helix’ and both the purine and the J2/3 interhelical junction explain the ligand dependence of P1 formation. The structures of the metabolite-binding domain of the thiamine pyrophosphate (TPP) riboswitch from the eukaryote Arabidopsis thaliana [9] and the bacterium Escherichia coli [10,11] are essentially identical. Like the purine riboswitch, the metabolite-bound TPP riboswitch adopts a compact, inverted-h architecture with two parallel sets of coaxially stacked helices (P1-P2-P3 and P4-P5) joined by a three-way junction (Figure 1c,d). An A-minor interaction between L5 and P3, functionally analogous to the loop– loop interaction of the purine riboswitch, stabilizes this arrangement. Unlike the purine riboswitch, TPP is not buried at the three-way helical junction; instead, TPP bridges the two parallel helical stacks. This places TPP 25 A˚ away from the P1 switch helix, suggesting that metabolite binding only indirectly stabilizes P1. Three distinct classes of S-adenosylmethionine (SAM) riboswitches have been identified [12–14]. The crystal Current Opinion in Structural Biology 2007, 17:273–279
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Figure 1
Crystal structures of riboswitch–effector complexes, and schematic depiction of conserved primary and secondary structures. (a,b) Purine riboswitch (PDB code 1U8D), (c,d) TPP riboswitch (PDB code 2GDI), (e,f) SAM riboswitch (PDB code 2GIS) and (g,h) glmS ribozyme-riboswitch (PDB code 2H0Z). The 30 half of the P1 helices, which participate in the genetic switch event, is colored red, and tertiary interactions thought to be important in folding and RNA–metabolite complex stability are green. Bound metabolites are shown in blue; PK and KT denote pseudoknots and K-turns, respectively; ‘var’ indicates an RNA segment of phylogenetically variable length and composition. Filled spheres indicate that the sequence is not conserved, but the Watson–Crick pairing is.
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structure of the metabolite-binding domain of the first class to be discovered (the SAM-I riboswitch) reveals an architecture that is distinctly different from the inverted-h fold of the purine and TPP riboswitches (Figure 1e,f) [15]. The SAM-I riboswitch contains two stacks (P1-P4 and P2a-P3) that, rather than packing sideby-side, cross at an angle of 708. A pseudoknot coupled to a kink-turn [16] atop P2 appears to stabilize this fold. The SAM-binding site is located at the interface of the minor grooves of P1 and P3, and has features reminiscent of the metabolite-binding sites of both the TPP and the purine riboswitches. Like TPP, SAM bridges two helical stacks. Like the purine riboswitches, SAM makes van der Waals contact with the 30 strand of the P1 switch helix. In many Gram-positive bacteria, the glmS ribozyme-riboswitch is part of the 50 -UTR of the mRNA that encodes glucosamine-6-phosphate (GlcN6P) synthetase [17]. This riboswitch has a self-cleavage activity that becomes activated when it binds GlcN6P. The structure of the glmS ribozyme [18,19] consists of three parallel helical stacks (Figure 1g,h). A doubly pseudoknotted core (P1-P2-P2.1P2.2) is buttressed by a peripheral RNA domain (P4-P4.1). The solvent-exposed GlcN6P-binding pocket is composed of two highly distorted major grooves and abuts the site of self-cleavage, reflecting the coenzyme function of GlcN6P (see the review by Scott in this issue). Among currently characterized riboswitches, the glmS ribozyme is unique because it adopts its active structure in the absence of its metabolite ligand [18,20]. As other ribozymes, such as the natural hairpin ribozyme [21,22] and the in vitro selected Diels–Alderase [23], also assemble rigid active sites, this disparity might reflect the different constraints under which ribozymes and RNAs that function by alternative folding evolved.
The two helical stacks of the thi-box riboswitch separately recognize the aminopyrimidine and pyrophosphate moieties of TPP. J3/2 of the ‘pyrimidine sensor helix’ (the P1P2-P3 stack) adopts a canonical T-loop fold [9,10,11]. Binding of the aminopyrimidine ring of TPP to G40 of this T-loop (Figure 2b) mimics a tertiary interaction between the D- and T-loops in the classic L-shaped fold of tRNA. The aminopyrimidine of TPP and G40 replace G18 and C55, respectively (purines and pyrimidines are reversed between the riboswitch and the tRNA). Mimicry of an allRNA structure by an exogenous small molecule is reminiscent of ATP binding by an in vitro selected aptamer RNA, whereby the ATP completes a GNRA tetraloop [27,28]. Rather than directly binding to the negatively charged pyrophosphate of TPP, the ‘pyrophosphate sensor helix’ (the P4-P5 stack) of the riboswitch coordinates the pyrophosphate mostly through two solvated divalent metals ions (the exception is G78) [9,10,11,29]. Thus, the riboswitch effectively binds a positively charged TPP– cation complex. Structures of the TPP riboswitch bound to three metabolite analogs suggest that the pyrimidine sensor helix is largely preformed, whereas the pyrophosphate sensor helix becomes organized concomitant with binding of the TPP–cation complex [11]. The SAM-I riboswitch sandwiches its ligand between two parallel helices [15]. P1 recognizes the ribose–sulfur backbone of SAM primarily through van der Waals contacts (Figure 2c). By contrast, the P3 helix binds the adenine ring and the amino acid by making several hydrogen bonds and stacking interactions. Interestingly, the RNA does not directly recognize the methionine e-methyl group. Rather, the positively charged sulfur atom makes a favorable electrostatic interaction with the partial negative charge on O2 of U7. This might explain the enhanced binding of SAM compared to S-adenosylhomocysteine and other non-positively charged analogs [12,30].
Ligand recognition by riboswitches The purine riboswitch recognizes its ligand almost exclusively through hydrogen-bonding interactions that satisfy nearly all possible acceptors and donors of the purine (Figure 2a). Purine riboswitch structures have been solved bound to 2,6-diaminopurine [24] and 2,4,6-triaminopyrimidine [25], in addition to the biological activators hypoxanthine [6], guanine [7] and adenine [7]. The purine ligand is primarily recognized by residue 74 of the riboswitch, a pyrimidine, through Watson–Crick pairing [6,7,26]. In addition, U51 and the 20 -OH of U22 hydrogen bond to the N3/N9 edge (corresponding to the sugar edge of nucleotides) and the N7, respectively, of the purine. Reliance on Watson–Crick (as opposed to Hoogsteen) pairing for recognition enables the same RNA scaffold to regulate either adenine or guanine metabolism by having U74 or C74, respectively. Gilbert et al. [24] noted that the purine ligand makes poor stacking interactions and proposed that this enhances the discriminatory role of Watson–Crick pairing with residue 74. www.sciencedirect.com
Recognition of GlcN6P, a simple phosphorylated hexosamine sugar, by the glmS ribozyme-riboswitch presents a challenge for RNA that is distinct from those posed by purines, TPP and SAM, all of which have nucleotide-like substructures. Crystal structures of the glmS ribozymeriboswitch were solved bound to glucose-6-phosphate (Glc6P), an isosteric competitive inhibitor (antagonist) of GlcN6P [18], and to the authentic activator GlcN6P ([19]; DJ Klein and AR Ferre´-D’Amare´, unpublished), revealing that GlcN6P and Glc6P are equivalently positioned in the glmS ribozyme-riboswitch active site. The sugar hydroxyl groups hydrogen bond to G1, C2, A50 and G65 (Figure 2d). The amine of GlcN6P hydrogen bonds to a water molecule, U51 and the 50 oxygen of G1; the last is the leaving group of the transesterification reaction catalyzed by the ribozyme. As in the TPP riboswitch, the phosphate of the metabolite interacts with the RNA through two solvated divalent metal ions ([19]; DJ Klein and AR Ferre´-D’Amare´, unpublished). TPP, SAM and Current Opinion in Structural Biology 2007, 17:273–279
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Figure 2
GlcN6P all present negatively charged moieties to the N1/N2 face of a guanine residue (G78, G11 and G1, respectively). This face of guanine has previously been shown to be an anion-binding site [31].
Aptamers and riboswitches The discovery of RNA ‘aptamers’ that bind small molecules through in vitro selection (reviewed in [32]) preceded the discovery of their naturally occurring counterparts, riboswitches. We compared RNA and ligand solvent accessibilities for several aptamer–ligand (reviewed in [33]) and riboswitch–effector structures. Riboswitches bury 90 6% (mean standard deviation) of the accessible surface of their effectors. Aptamers, by contrast, bury 71 14% of the solvent-accessible surface of their cognate ligand, consistent with an in vitro selection methodology in which the ligand is tethered to beads. Furthermore, riboswitches tend to have more compact structures than aptamers, as indicated by solvent-accessible surface areas of 156 5 A˚2/nt for riboswitches and 171 8 A˚2/nt for aptamers (end correction as in [34]). Despite these differences and the different evolutionary origins of aptamers and riboswitches, the binding energy correlates linearly with the buried surface area of the ligand (Figure 3). Such a relationship might be expected on theoretical grounds [35]. This relationship implies that the vitamin B12 riboswitch (Kd 0.3 mM [36]) and the tetracycline aptamer (Kd 0.8 nM [37]) can achieve their known affinity by burying 40% (of 1200 A˚2) and nearly all (600 A˚2), respectively, of the surface area of their ligands. Exceptions to the trend are RNAs that bind the smallest ligands, such as the purine riboswitches and the theophylline aptamer, which completely envelop the small molecule. For these ligands, higher affinity must result from other factors, such as RNA–RNA interactions. Our analysis implies that the glycine riboswitch will also be an outlier, consistent with the cooperative binding exhibited by this RNA [38]. Structural and biochemical analyses of several GTP aptamers of varying degrees of complexity show that increased RNA complexity correlates with higher affinity [39]. However, higher affinity does not result in higher specificity [40]. These studies suggest that higher affinity is derived from additional RNA–RNA interactions that stabilize the global RNA fold and that explicit selection is needed to achieve higher specificity. Compared to aptamers, riboswitch structures are stabilized by a variety of peripheral RNA contacts (Figure 1) and these cellular regulators presumably have undergone stringent natural selection, thus accounting for their higher affinities and selectivity. RNA–ligand interaction in the (a) purine, (b) thi-box, (c) SAM-I and (d) glmS ribozyme riboswitches. Color scheme as in Figure 1, with the addition of metal ions (green spheres) and water molecules (red spheres). Dotted lines denote hydrogen bonds; solid lines denote inner-sphere metal ion coordination. In (a), C74 is colored with nitrogens in blue and oxygens in red to illustrate hydrogen bonding with hypoxanthine (HX). Current Opinion in Structural Biology 2007, 17:273–279
Riboswitch folding and regulation of gene expression Although chemical probing has been used to investigate the small-molecule-dependent alternative folding of some full-length riboswitches (reviewed in [1]), most biochemical and biophysical studies of riboswitches have www.sciencedirect.com
Metabolite recognition by riboswitches Edwards, Klein and Ferre´-D’Amare´ 277
Figure 3
thus far focused on isolated metabolite-binding domains. Published studies include ensemble and single-molecule fluorescence resonance energy transfer (FRET) characterization of the adenine riboswitch (demonstrating the presence of Mg2+-dependent folding intermediates [41]), solution NMR experiments on the Mg2+ and effector dependence of the terminal loop–loop interaction in the purine riboswitches [42], and small-angle X-ray scattering analysis of the shape of the glycine riboswitch in the unliganded, Mg2+-induced folded and effector-bound states [43]. With the exception of the glmS ribozyme, riboswitchmodulated gene regulation results from the sequestration of a switch segment (red in Figure 1) into either a metabolite-bound or a metabolite-free conformational fold. For riboswitches that exert genetic control at the transcriptional level, co-transcriptional mRNA folding is an essential component of the mechanism of action [44] and must be considered alongside the thermodynamics and kinetics of ligand binding [24,45], as well as the relative speed of transcription [46]. In a study of the flavin mononucleotide (FMN) riboswitch, it was shown that the speed of transcription relative to the kinetics of FMN binding precludes this RNA from reaching thermodynamic equilibrium before the point at which a genetic decision must be made [46]. Consequently, metabolite concentrations needed to trigger this riboswitch in vivo must be considerably higher than the in vitro dissociation constant measured with previously transcribed RNA. These studies also implicate transcriptional pause sites as critical components of riboswitch-mediated regulation. Several ribosensors have been described that function by means other than direct metabolite binding. These include non-coding mRNA elements that respond to the presence of uncharged tRNAs [47], the concentration of Mg2+ [48] and temperature [49]. A series of ‘RNA thermometers’ in the 50 -UTRs of heat shock response genes unfold at elevated temperatures, releasing a Shine–Dalgarno sequence sequestered at lower temperatures (Figure 4a). A solution NMR structure of the lower temperature state has been determined, in which several residues form noncanonical base pairs (Figure 4b) [49]. The imino proton resonances of these residues disappear at elevated temperatures, indicating that these pairs melt. The coupling of folding with the control of gene expression is a feature shared by these ribosensors and riboswitches.
Small-molecule binding by aptamers and riboswitches. (a) The solvent-accessible surface area of the ligand that is buried when in complex with aptamers or riboswitches correlates linearly with affinity, with a binding energy of 19 cal/mol per A˚2 of buried area. Red and blue represent riboswitches and aptamers, respectively; squares and circles denote structures solved by X-ray crystallography or NMR, www.sciencedirect.com
respectively; 95% prediction intervals are shown as dashed lines. The purine and theophylline complexes were excluded from the analysis. (b) Solvent-accessible surfaces of riboswitch- or aptamerbound ligands. Panels show the absolute solvent-accessible area of each ligand atom (dark blue 0–1 A˚2, light blue 1–10 A˚2, green 10–20 A˚2, yellow 20–30 A˚2, orange 30–40 A˚2, red 40–60 A˚2) mapped onto the molecular surface overlaying it. Buried surface areas were calculated using a 1.4 A˚ radius probe [50]. Aptamer structures are reviewed in [32]. Current Opinion in Structural Biology 2007, 17:273–279
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Figure 4
Foundation. TEE and DJK contributed equally to the writing of this review.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
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Secondary structure (a) and NMR structure (PDB code 2GIO) (b) of the ROSE (repressor of heat-shock gene expression) ribosensor, which functions as an RNA thermometer. Upon increase in temperature (D), a helix primarily consisting of non-canonical Watson–Crick pairings melts to liberate a Shine–Dalgarno sequence (depicted in gray boxes). Except for line drawings, figures were prepared with PyMol [51].
Conclusions The recent determination of the structures of the metabolite-binding domains of riboswitches opens the way to multiple research avenues that take advantage of highresolution information. These include analysis of the molecular recognition mechanisms employed by riboswitches and design of artificial effectors, elucidation of the mechanism of action of the coenzyme GlcN6P in the glmS ribozyme and, most generally, coupling of ligandinduced folding of RNA to the cellular processes of transcription, post-transcriptional RNA processing and translation.
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Acknowledgements
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TEE and DJK are Damon Runyon Fellows, and ARF is a Distinguished Young Scholar in Medical Research of the WM Keck Foundation. Supported by grants from the Damon Runyon Cancer Research Foundation (DRG-1844-04 to TEE and DRG-1863-05 to DJK), the National Institutes of Health (GM63576 to ARF) and the WM Keck
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