Edwards et al., Curr Opin Struct Biol 2007

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276 Nucleic acids 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 5Å 2 /nt for riboswitches and 171 8Å 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 B 12 riboswitch (K d 0.3 mM [36]) and the tetracycline aptamer (K d 0.8 nM [37]) can achieve their known affinity by burying 40% (of 1200 Å 2 ) and nearly all (600 Å 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 Ferré-D’Amaré 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 Mg 2+ -dependent folding intermediates [41 ]), solution NMR experiments on the Mg 2+ 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, Mg 2+ -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 Mg 2+ [48] and temperature [49]. A series of ‘RNA thermometers’ in the 5 0 -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 Å 2 of buried area. Red and blue represent riboswitches and aptamers, respectively; squares and circles denote structures solved by X-ray crystallography or NMR, 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 Å 2 , light blue 1–10 Å 2 , green 10–20 Å 2 , yellow 20–30 Å 2 , orange 30–40 Å 2 , red 40–60 Å 2 ) mapped onto the molecular surface overlaying it. Buried surface areas were calculated using a 1.4 Å radius probe [50]. Aptamer structures are reviewed in [32]. www.sciencedirect.com Current Opinion in Structural Biology 2007, 17:273–279
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Edwards et al., Curr Opin Struct Biol 2007

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