Ligand Binding by the Tandem Glycine Riboswitch Depends on Aptamer Dimerization but Not Double Ligand Occupancy

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Ligand binding by the tandem glycine riboswitch depends on aptamer dimerization but not double ligand occupancy

KAREN M. RUFF and SCOTT A. STROBEL Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520-8114, USA

ABSTRACT 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. Keywords: riboswitch; glycine; tandem; aptamers; dimerization

INTRODUCTION populations to grow in high glycine (Tezuka and Ohnishi 2014). The riboswitch system allows organisms to respond Riboswitches are noncoding elements in mRNA that modu- quickly to changes in local environment, catabolizing glycine late expression of a gene in response to changes in the confor energy when environmental concentrations get too high, centration of a specific small-molecule ligand. The glycine and importing glycine from the environment when concen- riboswitch, which senses the concentration of the smallest trations fall too low. amino acid (Mandal et al. 2004), is a common riboswitch, The glycine riboswitch predominantly exists in a tandem with at least 350 known instances spread across the bacterial architecture, with two adjacent, homologous aptamers joined kingdom (Kazanov et al. 2007; Kladwang et al. 2012). Of by a short linker region, followed by a single expression plat- these, ∼60% are found regulating genes for the glycine cleav- form (Fig. 1A; Mandal et al. 2004). The two aptamers each age system, primarily gcvT and gcvP (Barrick and Breaker bind glycine with micromolar dissociation constants (1–30 2007; Kazanov et al. 2007). Another ∼20% of the known gly- µM K ), and, like many other riboswitches, the glycine ribo- cine riboswitches regulate a sodium/alanine- or glycine-sym- d switch undergoes conformational compaction upon the ad- porter (Mandal et al. 2004). At concentrations in excess of dition of glycine (Mandal et al. 2004; Lipfert et al. 2007; that necessary for protein synthesis, glycine binds to the ribo- Kwon and Strobel 2008; Baird and Ferré-D’Amaré 2013; switch and activates glycine cleavage genes, breaking down Zhang et al. 2014). It is the only known riboswitch where glycine into ammonia, a methylene unit in the form of meth- two aptamer domains control a single expression platform yl-THF, and NADH (Barrick et al. 2004). In addition to ex- (Mandal et al. 2004; Breaker 2011). Because the tandem ar- ploiting excess glycine as an energy source, bacteria must chitecture, rather than a simpler single-aptamer riboswitch, regulate concentrations to prevent glycine toxicity. High con- has been conserved against evolutionary drift, it is expected centrations of glycine interfere with cell wall biosynthesis, and glycine riboswitches are necessary for Streptomyces griseus © 2014 Ruff and Strobel This article is distributed exclusively by the RNA Society for the first 12 months after the full-issue publication date (see http:// Corresponding author: [email protected] rnajournal.cshlp.org/site/misc/terms.xhtml). After 12 months, it is available Article published online ahead of print. Article and publication date are at under a Creative Commons License (Attribution-NonCommercial 4.0 Inter- http://www.rnajournal.org/cgi/doi/10.1261/rna.047266.114. national), as described at http://creativecommons.org/licenses/by-nc/4.0/.

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Ruff and Strobel

2011) and as a single aptamer (Huang et al. 2010). The tandem riboswitch forms a semisymmetric dimer, with each aptamer domain binding a separate molecule of glycine in a bulge within helix P3 (Fig. 1B). An extensive network of interaptamer interactions, largely mediated by A-minor contacts between the P1 of one aptamer and the P3 of the other, form an interface between the two ligand-binding sites (Fig. 1C). The single- aptamer construct formed a homodimer in the crystals that closely matched the interaptamer interface in the tandem aptamer structure (Huang et al. 2010). While the discovery of the leader helix has reopened the question of cooperativity, modeling indicates that the kink- turn and P0 helix can be accommo- dated into the existing structural model (Kladwang et al. 2012). Therefore, the FIGURE 1. Secondary and tertiary structures of the tandem glycine riboswitch. (A) Secondary structure showing the tandem glycine aptamers, each binding one equivalent of glycine. Here, binding-site and interface interactions a transcriptional on-switch is depicted. The recently identified leader helix is boxed, and the predicted by the structure remain rele- P0 helix is shown. (B) Secondary and tertiary structures of the tandem glycine riboswitch from vant to understanding this intriguing reg- Fusobacterium nucleatum (Fnu) (Butler et al. 2011) (PDB ID 3P49). The two molecules of glycine ulatory system. are depicted in brown, and the α and β A-minor interactions are highlighted in green and red. (C) The interface between the aptamers, showing the α, β, γ, and δ interactions, which link the two Investigations of the glycine ribo- ligand-binding sites. switch have relied on assays that indi- rectly track riboswitch structural changes. However, because the aptamers dimer- to provide some benefit, possibly for ligand-binding affinity, ize, glycine binding in either aptamer is predicted to pro- kinetic response time, or complex genetic control. pagate structural changes throughout both aptamers. We The two aptamers in the tandem riboswitch each bind a have used an equilibrium dialysis-based assay that directly separate molecule of glycine, but, because of the tandem ar- monitors glycine binding in each aptamer with a set of gly- rangement, the binding sites are not necessarily independent cine-binding-site and interface mutants to understand the (Mandal et al. 2004). Tandem riboswitch architectures that relationship between dimerization and glycine binding. We act as “genetic logic gates” have been reported (Sudarsan et propose a revised model for tandem riboswitch function al. 2006). However, most of these tandem configurations that is consistent with these results, wherein ligand binding comprise two complete riboswitches, including an expres- in aptamer-1 is linked to aptamer dimerization and stabilizes sion platform for each aptamer element, so they function in- the P1 stem of aptamer-2, which controls the expression dependently (Welz and Breaker 2007). For many years, the platform. tandem glycine riboswitch was considered a unique cooperative RNA system (Mandal et al. 2004), behaving as a digital RESULTS sensor for the concentration of glycine. The recent identification of a leader helix (boxed in Fig. The tandem glycine riboswitch from Vibrio cholerae, includ- 1A), the inclusion of which eliminates cooperativity between ing the leader sequence (VC1-2) was the focus of this study the aptamer domains, has reopened the debate over the (see Supplemetal Material for full sequence). This particular purpose of the tandem structure of the glycine riboswitch glycine riboswitch controls the VC1422 gene, which encodes (Kladwang et al. 2012; Sherman et al. 2012). A recent calo- a sodium/alanine- or glycine-symporter. It was the initial gly- rimetry study, while showing that the leader helix promotes cine riboswitch characterized (Mandal et al. 2004) and has ligand binding and riboswitch compaction, has further ques- served as the prototype for subsequent biochemical study tioned whether the tandem riboswitch with the leader P0 (Lipfert et al. 2007; Kwon and Strobel 2008; Erion and helix binds one or two equivalents of glycine (Baird and Strobel 2011). For clarity, the positional nucleotide number- Ferré-D’Amaré 2013). ing remains consistent with previous reports, with the leader The glycine riboswitch lacking the P0 helix has been struc- nucleotides assigned positions -7 to -1 (Sherman et al. 2012; turally characterized in both the tandem form (Butler et al. Esquiaqui et al. 2014).

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Glycine riboswitch depends on aptamer dimerization

Glycine binding by the wild-type full-length RNA To investigate ligand binding by the tandem glycine riboswitch independent from any conformational changes, we used a binding assay that directly measures glycine binding using equilibrium dialysis. This analysis was enabled by the inclusion of the leader, which allows the riboswitch to behave well at higher RNA concentrations. We confirmed that wild- type (WT) VC1-2 containing the leader sequence binds glycine noncooperatively with low micromolar affinity, in agreement with other recent reports (Kladwang et al. 2012; Sherman et al. 2012; Baird and Ferré-D’Amaré 2013). Because the inclusion of the leader sequence dramatically changes the ligand-binding activity of the riboswitch, we wished to verify if each ligand-binding site in the tandem FIGURE 2. Binding-site mutants of the tandem riboswitch bind a sin- riboswitch with leader binds to a separate molecule of glycine. gle molecule of glycine in the unmutated aptamer. (A) Detailed struc- Glycine binding to in vitro transcribed WT VC1-2 ture of the glycine-binding site from the Fnu glycine riboswitch was monitored by equilibrium dialysis (see Materials and (Butler et al. 2011) (PDB ID 3P49), showing the uracil that contacts Methods), and the fraction of bound glycine increases with the ligand. (B) Secondary structures of the tandem riboswitch showing the U to A mutations that disrupt ligand binding. (C) Glycine binding RNA concentration. The data were fit well by a standard bind- by WT VC1-2 and its binding-site mutants, showing that the singly ing curve (Hill coefficient of 1) with an equilibrium dissocia- binding U78A (Lig2, fuchsia) and U207A (Lig1, blue) bind with near wild-type affinity while the doubly mutated U78A/U207A (brown) tion constant (Kd) of 2.0 µM (Table 1 and Fig. 2C). The shows no binding activity. Here, ligand binding by WT VC1-2 is fit us- measured ligand-binding affinity is in agreement with pre- ing a single-binding-site model. (D) WT VC1-2 binds to glycine approx- vious reports, and the Hill coefficient corroborates the recent imately twofold more tightly than predicted for two sites with the Lig1 findings that glycine binding is not cooperative in ribo- and Lig2 affinities of 8.5 and 3.7 μM (dashed line). The predicted bind- switches that include the leader sequence. The data fit equally ing curve for two sites with equivalent affinities of 4.0 μM is shown for comparison (dotted line). well to a one- or two-site binding model, indicating that either the two sites have very similar affinities or binding to the second site is weak or nonexistent (Fig. 2D). If the two sites are tained the same results when glycine was added to prefolded assumed to have equivalent affinities, these data are consistent WT VC1-2. When binding was monitored at even higher with two binding events with Kds of 4 µM. RNA and ligand concentrations (∼100 times the Kd), the tan- To test whether binding occurs in both sites, we repeated dem riboswitch binds a full two equivalents of glycine. Our the binding assay with a known excess of glycine. At RNA con- results demonstrate that both binding sites are able to bind li- ∼ centrations 30 times the Kd, the WT tandem riboswitch con- gand in the tandem riboswitch with leader. taining the leader sequence binds 1.8 equivalents of glycine (Table 1), in agreement with the original studies on the glycine riboswitch, but in contrast to the recent report of a single Mutations to create tandem riboswitches equivalent bound as reported using isothermal titration calo- with a single glycine-binding site rimetry (ITC) (Baird and Ferré-D’Amaré 2013). The disparity Because of the evolutionary conservation of the tandem struc- might arise from a variety of differences between the tech- ture of glycine riboswitches, the presence of two aptamers niques, including longer equilibration times or refolding the is expected to contribute to ligand binding or gene control. riboswitch in the presence of the ligand. However, we ob- However, it was not known if glycine binding by both aptamers is necessary to achieve high-affinity binding to either aptamer. Mutation of a conserved guanosine (G) in the TABLE 1. Glycine-binding affinity and equivalents bound of WT three-helix junction of one aptamer has been shown to have VC1-2 and its binding-site mutants no effect on the glycine-binding affinity of the other aptamer, K μ VC1-2 d for glycine ( M) Equivalents bound as monitored by in-line probing (Sherman et al. 2012). WT 2.0 ± 0.1a/4.0 ± 0.3b 1.8 ± 0.1 However, based on the current structural model, these Gs U78A (Lig2) 3.7 ± 0.3 0.95 ± 0.06 do not directly contact the ligand (Butler et al. 2011), and U207A (Lig1) 8.5 ± 0.6 0.92 ± 0.06 the effects of these mutations on each aptamer’s structure U78A/U207A Not detectable 0.06 ± 0.02 and ligand-binding capacity are unclear. In order to investi- aFitting the data with a single-site equation gives an affinity of gate the interdependence of the two aptamers in the tandem 2 μM. structure, we directly mutated the ligand-binding site and de- b Fitting the data with a two-site equation where both binding sites termined if disruption of ligand binding in one aptamer af- have the same affinity gives K s equal to 4 μM. d fects the affinity of the second.

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Ruff and Strobel

The crystal structure of the F. nucleatum (Fnu) tandem gly- rupted independently (Lig1 and Lig2), there is at most a two- cine riboswitch includes a conserved uracil (U) in each ligand- fold effect on ligand binding to the remaining. This is reflected binding pocket that directly contacts glycine (U78 and U207) in the predicted binding curve for two sites with affinities (Fig. 2A; Butler et al. 2011). We hypothesized that substitu- matching those of Lig1 and Lig2 (Fig. 2D, dashed line), which tion with an adenosine (A) would disrupt this interaction deviates only slightly from the data for WT VC1-2 (squares). and obstruct the pocket, therefore displacing glycine from When both binding sites are disrupted, the tandem riboswitch the binding site. We incorporated this binding-site muta- no longer binds glycine acrossthe range of concentrationstest- tion into VC1-2 aptamer-1 (U78A), aptamer-2 (U207A), ed. Therefore, the ability to bind glycine in one aptamer has or both aptamers (U78A/U207A) (Fig. 2B). These mutants only a small effect on the other aptamer’s ligand-binding site. have not been studied previously because, prior to the struc- Because the double-binding-site mutant does not bind gly- ture, ligand binding was thought to occur in the three-helix cine at any of the tested RNA concentrations, and because junction rather than the P3 bulge. each single mutant binds only one equivalent of glycine, we The number of glycine-equivalents bound was determined conclude that the Lig1 and Lig2 constructs only bind ligand for the binding-site mutants (Table 1). As predicted, when ei- in the unmutated ligand-binding site. Therefore, with these ther ligand-binding site is disrupted independently (U78A or constructs it is possible to monitor ligand binding in a specif- U207A), the tandem riboswitch only binds one glycine equiv- ic ligand-binding site, in the context of the full-length tan- alent. Furthermore, the disruption of both aptamer binding dem riboswitch. sites (U78A/U207A) abolished glycine binding by the riboswitch. Therefore, these single-site mutations can be used to Mutations that disrupt the aptamer–aptamer interface selectively eliminate a ligand-binding site. They also corrobo- rate the conclusion that the WT tandem riboswitch binds two Dual ligand binding by the tandem riboswitch is not nec- equivalents of glycine. To draw attention to the occupied li- essary for high-affinity ligand binding and cannot explain gand-binding site in these singly glycine-binding mutants, the evolutionary conservation of the tandem riboswitch. An we named them according to the unmutated site. Therefore, alternative explanation for the tandem riboswitch invokes U78A is referred to as Lig2, while U207A is Lig1. aptamer dimerization as a requirement for ligand binding. Having identified mutants that selectively eliminate glycine Therefore, we tested if the aptamers need to dimerize in order binding to either of the tandem aptamers, we determined if to bind ligand. disruption of ligand binding by one affects the affinity of the The crystal structure of the tandem riboswitch identified a second. The single-glycine-binding mutants of VC1-2 were series of tertiary interactions between the two aptamers (Figs. folded in the presence of trace glycine, and the affinity for 1C, 3A; Butler et al. 2011). These interactions create an in- the unmutated site was determined by equilibrium dialysis terface between the two ligand-binding sites and include (Table 1 and Fig. 2C). When each ligand-binding site is dis- two pseudo-symmetric series of A-minor interactions (the

FIGURE 3. Structure-guided perturbation of the aptamer interface. (A) Secondary structure of VC1-2, showing the ligand-binding site and interface residues. Glycine is depicted in brown. The α, β, γ, and δ interactions are based on the crystal structure, shown in Figure 1. Interface positions mutated in this study are shown in bold colors. The L3TL mutation replaces the L3 loop of aptamer-1 with a UUCG tetraloop (black box). (B) Split aptamer constructs used to probe aptamer dimerization in trans by gel-shift electrophoresis. (C) Aptamer–aptamer dimerization curves of interface mutants. WT is shown in black, α mutants in green, β in red, γ in orange, and δ in purple. (D) Sample gel shift showing a tightly associated dimer (A73C) and a weakly associated dimer (U74C).

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Glycine riboswitch depends on aptamer dimerization

α and β interactions), a cis Hoogsteen base pair (the γ inter- where L3 of aptamer-1 was mutated to a UUCG tetraloop, action), and a pair of adenosines that flip out of the ligand- which is predicted to disrupt both the β and γ interactions. binding sites and stack against the three-helix junction and This mutation dramatically weakens dimerization, causing the P1 helix of the cis aptamer (here labeled δ interactions). a 180-fold loss in dimer affinity. Mutations were incorporated into the riboswitch to disrupt The γ interaction is an A–U Hoogsteen base pair at the cen- these interactions (Fig. 3A), as discussed below. The effects of ter of the dimer interface. Mutation of either the A to a G or the mutations on dimerization were determined by measur- the U to a C should disrupt the interaction. While these mu- ing the affinity between the two aptamers in a trans gel-shift tant constructs could rearrange to allow wobble Hoogsteen assay (Erion and Strobel 2011; Sherman et al. 2012), with pairs or wobble Watson–Crick pairs, either possibility would the aptamer-1 portion retaining the 5′-half of the P0 helix require remodeling of the interface to accommodate the dif- and the aptamer-2 construct containing the 3′-half (Table 2 ferent pairing distances, which would disturb the arrange- and Fig. 3B,C). Dimer formation was monitored in the pres- ment of the α and β interactions. U74C causes a 19-fold loss ence of saturating glycine by the appearance of a higher mo- in dimer affinity, while A203G reduces the affinity by 89-fold. lecular-weight complex during gel electrophoresis (Fig. 3D). These mutations provide the greatest disruption of dimeriza- Disruption of the predicted tertiary interactions generally tion of any of the point mutants tested. weakens dimerization of the aptamers. The α and β interac- The adenines that form each δ interaction are flipped out tions consist of four and three A-minor interactions, respec- of the ligand-binding pockets to stack against the three-helix tively, between the P1 stem of one aptamer and a loop or junction and the P1 stem of the cis aptamer. Replacing the bulge in P3 of the other aptamer. A-minor interactions are adenosines with pyrimidines may weaken the stacking inter- disrupted by mutation of the adenosines to cytosines or by actions, which will affect P1 stability and could have long- the formation of a wobble pair in the P1 stem (Doherty range consequences, given the participation of the P1 stems et al. 2001; Kwon and Strobel 2008; Erion and Strobel in the α and β interactions. The A64U and A171U mutations 2011). We concentrated on the Type I A-minor interactions cause 15-fold and twofold weaker aptamer dimerization, formed at the top of each P1 stem. At this position in the α respectively. As with the A73C mutation above, the A171U interaction, A202 contacts G14:C125 (in the corresponding mutation, also located at the top of P1 of aptamer-2, is β interaction, A73 contacts G145:C220). The A202C muta- less detrimental than the equivalent mutation in aptamer-1. tion weakens dimerization of the two aptamers eightfold. While the differences in the two δ mutations’ effects were un- The corresponding A73C mutation in the β interaction did expected, both can be used to perturb dimerization to varying not cause similar weakening of dimerization (see Discus- degrees. sion). However, C220U, which forms a wobble pair at the po- The observed effects of the interface mutations on aptamer sition where A73 contacts the P1 stem, weakens dimerization dimerization in trans confirm the interactions proposed in in trans eightfold. C220U’s effects on dimerization cannot be the structure of the tandem glycine riboswitch. These mu- isolated to just the β interaction because G145:C220 is the tants can be used to perturb interface interactions and disrupt base pair against which A171 and A219 stack in the δ inter- dimerization. action. However, C220U causes a fourfold larger disruption of dimerization than the A171U mutation, which lends sup- Mutations that disrupt dimerization weaken glycine port to the importance of the β interaction for aptamer dime- binding in aptamer-1 rization. We also created a more extreme mutation L3TL, We used the interface mutations to determine the importance of aptamer dimerization for glycine binding by the ribo- TABLE 2. Aptamer-1/aptamer-2 dimerization affinities in trans of switch. Because the Lig1 and Lig2 constructs cannot bind li- interface mutants of VC1-2 gand in the mutated ligand-binding site, these constructs can be used to monitor glycine binding specifically in the re- Interaction VC1-2 Fold-change K μ a maining functional aptamer in the context of the full-length mutated mutant d ( M) versus WT tandem riboswitch. By combining the binding-site muta- – – WT 0.17 ± 0.03 tions with interface mutations, we tested the importance of α A202C 1.3 ± 0.5 7.7 β A73C 0.18 ± 0.03 ∼1 aptamer dimerization for the ligand-binding affinity of each C220U 1.3 ± 0.1 7.6 binding site. γ U74C 3.3 ± 0.3 19 We determined the glycine-binding affinity of a series of A203G 15 ± 3 89 double mutants of VC1-2, each combining an interface mu- δ A64U 2.6 ± 0.3 15 tation with a binding-site mutation (Table 3 and Fig. 4A,B). A171U 0.39 ± 0.09 2.3 β and γ L3TL 30. ± 5b 180 The affinity of each double-mutant was compared with the affinity of the parent RNA containing only the glycine-bind- a B Curve fits were calculated by fixing max at 0.92. ing-site mutation. In each case tested, the interface point mu- bCalculated by fixing nonspecific binding at zero. tations to α, β, and γ interactions disrupt the glycine-binding

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fect on glycine binding in aptamer-2. TABLE 3. Glycine-binding affinities of interface/binding-site double mutants Mutation of the δ adenosine in aptamer- Binding site Interaction VC1-2 Fold-change 2 (A171U) has a moderate effect on gly- K μ background mutated mutant d ( M) versus parent cine binding in both aptamers (nine- Lig1 (U207A) α Lig1 A202C 87 ± 4a 10. and fourfold). β Lig1 A73C 310 ± 20a 37 We plotted the effect of each interface a Lig1 C220U 310 ± 40 36 mutation on aptamer dimerization in γ Lig1 U74C 230 ± 20a 27 a trans against its effect on glycine binding Lig1 A203G 610 ± 120 72 δ Lig1 A64U 520 ± 40a 62 in aptamer-1 (Fig. 4D). Not only do mu- Lig1 A171U 35 ± 2a 4.1 tations that disrupt dimerization show β and γ Lig1 L3TL Not detectable at 500 μM >75 significantly weaker ligand binding in Lig2 (U78A) α Lig2 A202C 6.2 ± 0.3 1.7 aptamer-1, these effects are directly pro- β Lig2 A73C 5.1 ± 0.2 1.4 portional, indicating a linkage between Lig2 C220U 9.2 ± 1.1 2.5 γ Lig2 U74C 4.8 ± 0.4 1.3 the two equilibria. The mutants that fall Lig2 A203G 8.2 ± 0.6 2.3 below the line are A64U and the β mu- δ Lig2 A64U 5.7 ± 0.2 1.6 tants. A64 is located in the ligand-binding a Lig2 A171U 32 ± 2 8.7 site of aptamer-1, and so it is reasonable β γ a and Lig2 L3TL 39 ± 1 11 that A64U would affect ligand binding a B Curve fits were calculated by fixing max at 0.98. by aptamer-1 in excess of the amount predicted by its effect on dimerization. Indeed, the equivalent mutation in activity of the Lig1 constructs, but have little to no effect on aptamer-2, A171U, disrupts ligand binding by aptamer-2 binding by the Lig2 constructs (Fig. 4B). Since the Lig1 con- ninefold, the only point mutant to have such an effect. structs only harbor an active aptamer-1 ligand-binding site, Therefore, once the effects on the cis binding site are these results indicate that disruption of the dimer interface disproportionately impacts ligand binding in aptamer-1. For example, when either side of the γ interaction is mutated (U74C or A203G), ligand binding is only slightly affected in the Lig2 constructs (about twofold). In contrast, the identical γ mutations reduce ligand binding by 27- and 72-fold, respectively, for the Lig1 constructs. Similarly, mutation of the α (A202C) or β (A73C or C220U) interaction has little or no effect on ligand binding in aptamer-2 but significantly reduces ligand binding in aptamer-1 (10-, 37-, and 36-fold, respectively). The L3 tetraloop mutation (L3TL), designed to disrupt both the β and γ interactions, has the greatest effect on glycine binding by aptamer-1 (unde- tectable binding, which is a >75-fold FIGURE 4. Mutations that disrupt the dimer interface have little effect on glycine binding by change). This extreme mutation weakens aptamer-2, but disrupt glycine binding by aptamer-1 to a degree proportional to their effect on dimerization. (A) Sample binding curves for U74C mutants. Parent binding-site mutants glycine binding by aptamer-2 by a more are shown in black, U74C mutants in orange. (B) Fold change in glycine-binding affinity of an modest 11-fold. interface mutant relative to the single-glycine-binding parent, for Lig1 (dark gray) and Lig2 (light Disruption of the δ interactions does gray). (C) Comparison of various ligand-binding models to the U74C glycine-binding data. The μ μ not fully follow the pattern identified one-site model (dotted line, 9.2 M Kd) and two-site model (solid line, 9.2 and 300 M Kds) are largely superimposed and fit the data well (R2 > 0.995). A predicted two-site binding curve based above, perhaps because the adenosine in- on the affinities measured in the Lig1 and Lig2 backgrounds (dashed line, 230 and 4.8 μM Kds) volved in the δ interaction is located in would have approximately threefold tighter affinity than the measured binding data. (D) the glycine-binding pocket. Mutation of Comparison of interface mutations’ effects on dimerization and glycine binding by aptamer-1. α β γ δ the δ adenosine in aptamer-1 (A64U) has Mutations that disrupt the interaction are shown in green, in red, in orange, and in purple. The L3TL mutation disrupts both the β and γ interactions. The dashed line illustrates a trend and a significant effect (62-fold) on glycine is not a fit. (#) Lig1 L3TL shows no detectable binding at 500 μM glycine, which is at least 75-fold binding in aptamer-1 with little to no ef- weaker than the parent construct.

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Glycine riboswitch depends on aptamer dimerization

be observed. Using the experimentally determined K values TABLE 4. Glycine-binding affinities of interface mutants in WT- d binding-site background for the two ligand-binding sites in the Lig1 and Lig2 backgrounds, we predicted the binding site occupancy at RNA K μ a Interaction mutated VC1-2 Mutant d ( M) fit as single site and glycine concentrations in the dynamic region of the α A202C 42 ± 4 binding curve (Supplemental Material). We then experimen- β A73C 3.1 ± 0.2 tally determined the equivalents of glycine that were bound C220U 58 ± 9 by the tandem riboswitch at that same concentration γ U74C 9.2 ± 0.5 A203G 60. ± 6 (Table 5). δ A64U 11 ± 1 For example, the mutant Lig2 U74C has a glycine affinity A171U 56 ± 6 of 4.8 μM, and consequently, aptamer-2 is predicted to be β and γ L3TL 46 ± 9 >99% occupied at 140 μM RNA and 380 μM glycine. In con- μ aThe one-site binding model fits the data very well, with all R2 > trast, Lig1 U74C has an affinity of 230 M; thus, aptamer-1 is 0.994. predicted to be 20% occupied under those same conditions. When these values are adjusted to reflect the approximately twofold tighter ligand binding in the wild-type-binding-site considered, the A64U mutation exhibits proportional effects background (see Supplemental Material for full calculations), on glycine affinity and dimerization. It should be noted that aptamer-1 is predicted to be 40% occupied when the U74C these two affinities, ligand binding and dimerization, are not mutation is introduced into a WT-binding-site background. independent (Sherman et al. 2012 and results below). This VC1-2 μM binds 1.2 equivalents of glycine at these concen- analysis illustrates a trend, rather than quantitatively charac- trations of RNA and ligand, which is consistent with the ex- terizing a dependence. In general, disruption of the dimer in- pected range of 1.2–1.4 equivalents. terface weakens ligand-binding affinity, particularly for As shown in Table 4, glycine binding in the WT-binding- aptamer-1, which is weakened proportionally. site background is consistent with the affinities measured in the Lig1 and Lig2 backgrounds for most of the interface mutants. The outliers are A73C and C220U, which disrupt the β Ligand-binding affinities in WT-binding- interaction, and A203G, which disrupts the γ, all of which site background bind ∼20% more glycine in the WT background than we We next determined if the effects of interface mutations on li- would predict based on the affinities measured in the single- gand binding in the Lig1 and Lig2 backgrounds were indi- site backgrounds (see Discussion). In general, the behavior cative of the effects of those same mutations on ligand of interface mutations in the Lig1 and Lig2 backgrounds is a binding in the wild-type-binding-site background. We deter- good predictor for their behavior in the WT-binding-site mined the glycine-binding activity of the interface mutants in background. In the tandem glycine riboswitch, disruption RNAs with WT glycine-binding sites across a range of RNA of the dimer interface weakens ligand-binding affinity, partic- concentrations (Table 4). As with the fully WT VC-12, data ularly for aptamer-1. for interface mutants in the WT-binding-site background fit equally well to a one- or two-site model (Fig. 4C). In some cases, the affinity measured in the TABLE 5. Glycine-equivalents bound by VC1-2 interface mutants at the indicated RNA WT background was as strong as the and glycine concentrations tightest single-binding site measurement Experimental Predicted Predicted with (A73C, U74C, A64U, A171U, L3TL) Interaction VC1-2 equivalents equivalents adjustment to and was presumably dominated by bind- mutated mutant bound bound WT ing in aptamer-2. In other cases, the affin- α A202C 1.8 ± 0.1a 1.7 1.8 ity measured in the WT background β A73C 1.8 ± 0.1a 1.5 1.6 approached the average of the two affini- C220U 1.6 ± 0.1b 1.2 1.3 ties measured in the single-binding site γ U74C 1.2 ± 0.1b 1.2 1.4 backgrounds (A202C, C220U, A203G). A203G 1.6 ± 0.1a 1.3 1.4 δ a Given the difficulties of deconvoluting A64U 1.4 ± 0.1 1.3 1.4 A171U 1.7 ± 0.1b 1.5 1.6 the two binding sites in experiments per- β and γ L3TL 1.0 ± 0.1a 1.0–1.2c 1.1–1.4c formed with trace glycine, we next ana- K lyzed the interface mutants in the WT- Predicted equivalents bound are based on the measured ds in the Lig1 and Lig2 backgrounds. The adjusted predictions account for the affinity difference between the Lig1 and binding-site background using equilibri- WT backgrounds. Full calculations are in the Supplemental Material. um dialysis experiments with excess gly- a350 μM RNA, 1000 μM glycine. At these concentrations, WT binds 2.0 ± 0.1 equivalents. b cine. Under these conditions, ligand 140 μM RNA, 380 μM glycine. At these concentrations, WT binds 1.8 ± 0.1 equivalents. cAssumes Lig1 L3TL affinity of 500 and 2000 μM for purposes of calculation. binding in the weaker binding site can

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Aptamer-2 constructs only bind glycine when able plex during gel electrophoresis. The glycine-binding affinity to form a homodimer of the aptamer-2 constructs was determined by equilibrium dialysis of a mixture of RNA and trace, radiolabeled glycine. As described above, interface mutations have little to no effect The aptamer-2 construct that includes the linker region on ligand binding by aptamer-2 in the context of the tandem and the last several nucleotides of aptamer-1, VC2, binds gly- riboswitch. One potential explanation for this observation is cine with 35 µM affinity (Fig. 5C), consistent with previous re- that aptamer-2 is functional as a monomer. Consistent with ports. This construct forms a homodimer with 200 nM this hypothesis, an aptamer-2 construct, VC2 (Fig. 5A), has affinity in saturating glycine. Attempts to render VC2 mono- been shown to bind glycine with reasonably high affinity meric were unsuccessful (Fig. 5B), and VC2 homodimerizes (Mandal et al. 2004; Erion and Strobel 2011). However, struc- at approximately the same affinity in saturating glycine and tural (Huang et al. 2010) and biochemical analysis of this and in alanine (Fig. 5D). This ligand-independent dimerization similar constructs indicate that they form a homodimer with a is likely mediated by a 10 bp helix, which can form between Kd value for dimerization close to the concentration at which the “tails” of two VC2 constructs (Fig. 5A). This 10 bp helix those ligand-binding experiments were performed. Another is consistent with the recent report of construct inhibition aptamer-2-only construct, VC2s (Fig. 5A), which lacks the when aptamer-2-only constructs are lengthened to include linker region, is active when aptamer-1 is added in trans but pairing regions that would interfere with its formation has never shown glycine-binding activity in isolation (TV (Sherman et al. 2014). Therefore, VC2 forms an obligate Erion, pers. comm.). A third aptamer-2-only construct, which homodimer and cannot be used to determine if monomeric included the linker region, showed barely detectable glycine- aptamer-2 can bind glycine. binding affinity in isolation, but addition of aptamer-1 in trans VC2s, the aptamer-2 construct lacking the linker region, rescued near WT affinity (Sherman et al. 2012). binds glycine much more weakly than VC2 (Fig. 5C). The In order to determine if homodimerization is important for glycine-binding curve for VC2s is best fit by a model for co- glycine binding by the aptamer-2-only constructs, VC2 and operative ligand binding with a Hill coefficient of 1.8 and an VC2s, we mutated all three adenosines involved in type-I A- equilibrium constant equal to 300 μM, consistent with the li- minor interactions in the α interaction and then determined gand-binding equilibrium depending on two molecules of if this affected the homodimerization and glycine-binding aptamer-2, likely because of homodimerization. VC2s WT affinities (Table 6 and Fig. 5). In order to monitor homodime- forms a homodimer in saturating glycine with an affinity of rization, a small amount of radiolabeled aptamer-2 construct 10 μM (Fig. 5B), and VC2s dimerization is ligand dependent wasmixedwithanexcessofthesameRNAspeciesandrefolded (Fig. 5D). The glycine-binding curve for VC2s probably cor- in the presence of saturating glycine. Dimer formation was ob- responds to homodimerization, even though the affinity is served by the appearance of a higher molecular-weight com- >35-fold weaker than that measured by gel shift, because

FIGURE 5. Aptamer-2 constructs that bind glycine function as homodimers, and mutations that disrupt homodimerization also disrupt glycine binding (green). (A) Aptamer-2 constructs tested for homodimerization and glycine binding. VC2 homodimerization is likely mediated by a helix formed between two VC2 “tails.” (B) Homodimerization affinity of aptamer-2 constructs. (C) Glycine-binding affinity of aptamer-2 constructs. (D) VC2 homodimerizes in a glycine-independent manner, while VC2s requires glycine for homodimerization, as seen by gel shift in saturating glycine (closed symbols) or an equivalent concentration of alanine (open symbols).

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weakened but not abrogated. The Lig1 TABLE 6. Homodimerization and glycine-binding affinities of aptamer-2-only constructs and Lig2 constructs dimerize in saturating Homodimerization Glycine binding glycine with nine- and fivefold weaker af- Fold versus Fold versus finities than WT, but still >10-fold stron- α K μ K μ Construct interaction? d ( M) parent d ( M) parent ger than the no-glycine cases. Therefore, ligand binding in each binding site con- VC2 WT 0.20 ± 0.04 – 35 ± 5a – mutα 0.39 ± 0.02 2.0 >600b >17b tributes to promoting dimerization of P3b truncation 0.36 ± 0.03 1.8 >600b >17b the two aptamer domains into awell-fold- WT in alanine 0.40 ± 0.04 2.0 ––ed tertiary structure, and ligand bind- VC2s WT 10. ± 1 – 310 ± 80c – ing and aptamer dimerization are linked α b b b b mut >450 >45 >600 >2 equilibria. WT in alanine >450b –– a B Curve fit was calculated by fixing max at 0.98. bOutside limit of detection. DISCUSSION cCurve fit represents a cooperative binding curve with a Hill coefficient of 1.8 ± 0.3 and B In this study, we examined two aspects of max constrained at 1. the tandem riboswitch that might provide selective advantage over a single- aptamer system: double binding-site oc- the gel shift occurs in saturating glycine while ligand binding cupancy and aptamer dimerization. We show that double was studied in trace glycine. Mutating all three adenosines in- binding-site occupancy is not necessary for high-affinity volved in type-I A-minor interactions in the α interaction dis- ligand binding. In contrast, aptamer dimerization is energet- rupts dimerization entirely at the concentrations tested, a ically linked to ligand binding, particularly in aptamer 1. Based >45-fold loss in dimer affinity (Fig. 5B). This VC2s α mutant on our results, we propose a model for riboswitch function shows barely detectable glycine binding at the highest RNA (Fig. 7), wherein ligand binding in aptamer-1 is linked to concentration tested (Fig. 5C). Therefore, aptamer-2 is not aptamer dimerization and stabilizes the P1 stem of aptamer- able to function in isolation as a monomer. 2, which controls the expression platform. Our analysis confirms that the tandem glycine riboswitch from V. cholerae containing the leader sequence non- Aptamer dimerization depends on cooperatively binds glycine with low micromolar affinity, in ligand binding agreement with other recent reports (Kladwang et al. 2012; The decrease in ligand-binding affinity of VC1-2 and VC2 Sherman et al. 2012; Baird and Ferré-D’Amaré 2013). This upon disruption of the dimer interface indicates that dimeri- lack of cooperativity has reopened the debate over the purpose zation is important for glycine binding. Therefore, the re- of the tandem structure of the glycine riboswitch. Because the ciprocal dependence should also be present, where ligand tandem architecture has been conserved against evolution, binding promotes dimerization of the two aptamer domains rather than being reduced to a simpler single-aptamer ribo- into a well-folded tertiary structure. To test this interdepen- switch, it is expected to provide some benefit in ligand-bind- dence, we analyzed the dimerization affinity of the two ing affinity, kinetic response time, or complex genetic control. aptamer domains in trans in the absence of glycine binding, In contrast to a recent binding study using isothermal either by disrupting the ligand-binding sites or performing titration calorimetry (Baird and Ferré-D’Amaré 2013), gel shifts in the presence of alanine in place of glycine we show that the tandem glycine riboswitch binds two (Table 7 and Fig. 6). In both cases, the dimer affinity weakens by >80-fold, indicating that the tandem riboswitch re- TABLE 7. Aptamer-1/aptamer-2 dimerization affinities in trans when glycine binding is quires glycine binding to form a well- disrupted folded dimer. Interestingly, the dimeriza- VC1-2 Glycine Fold-change K μ a tion affinity of the L3TL mutant in both construct Description present? d ( M) versus WT glycine and alanine is similar to that of WT Doubly glycine binding Yes 0.17 ± 0.03 – the WT construct in alanine, indicating No 34 ± 7 200 that the residual affinity is not dependent Lig1 Singly binding Yes 1.5 ± 0.3 8.8 on the aptamer–aptamer interface. We Lig2 Singly binding Yes 0.82 ± 0.16 4.8 attribute the residual ∼20 μM affinity to U78A/U207A Cannot bind glycine Yes 16 ± 2 94 No 17 ± 4 100 base-pairing within the P0 helix, which L3TL Interface severely Yes 30.0 ± 5 180 is split between the two constructs. disrupted No 20 ± 20 120 When a single ligand-binding site is a B disrupted, dimer formation in trans is Calculated by fixing max = 0.92 and nonspecific binding = zero.

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ing. Mutations to the predicted interface interactions disrupt aptamer dimerization in trans in a manner largely consistent with the structural model (Fig. 3). The exceptions cluster around the top of the P1 helix in aptamer-2. Given the con- siderable consequences of the β mutants for glycine binding by aptamer-1, we propose that remodeling of the interaction conceals the effects of these mutations on aptamer dimerization, as discussed further below. We also studied a more extreme mutation, L3TL, which should disrupt both the β and γ interactions. Because this mutant’s dimerization affinity was equally poor in the presence and absence of glycine (Fig. 6), we consider L3TL to completely disrupt the interface. L3TL and the point mutants provide a range of interface mutations that can be used to disrupt aptamer dimerization. We used these interface mutations to determine the importance of aptamer dimerization for glycine binding by the riboswitch. Intriguingly, glycine binding by aptamer-1 is much more sensitive to dimerization than glycine bind- FIGURE 6. Aptamer dimerization depends on glycine binding. (A) ing by aptamer-2. In each case tested, the interface point mu- When both ligand-binding sites are disrupted (U78A/U207A, in tations to α, β, and γ interactions all disrupted the glycine- brown) or no ligand is present (alanine, open symbols), dimerization is substantially weakened. The L3TL interface mutation (red) does not binding activity of the Lig1 constructs, while having little- further weaken the affinity. (B) Disrupting the ligand-binding sites to-no effect on binding by the Lig2 constructs (Fig. 4C). singly (Lig1, blue and Lig2, fuschia) has an intermediate effect on The L3TL mutant, which disrupts both the β and γ interac- dimerization. tions, has the largest effect on ligand binding in aptamer-1. In addition, this extreme interface mutation also weakens li- equivalents of glycine. The disparity does not appear to result gand binding in aptamer-2. Therefore, both aptamers require from technical differences between the techniques. Given the a well-folded dimer interface for ligand binding, but aptamer- relationship that we have demonstrated between ligand bind- 1 is much more sensitive to perturbations of that interface. ing and dimerization (Fig. 6B), we speculate that the heat Furthermore, each interface mutation’s effect on ligand evolved upon ligand binding in the ITC experiments results binding in aptamer-1 and dimerization are directly pro- from dimerization, which is significantly promoted by bind- portional (Fig. 4D), indicating a linkage between the two equi- ing of the first equivalent of ligand. It is worth noting that libria. The mutants that fall below the line are A64U and the β many transcriptionally controlled riboswitches are not under mutants. A64 is located in the ligand-binding site of aptamer- thermodynamic control (Serganov and Patel 2012) and refer- 1,andoncetheeffectsonthecisbindingsiteareconsidered,the ences therein), so neither technique entirely describes the A64U mutation exhibits proportional effects on glycine affin- riboswitch–ligand interaction. However, our results demon- ity and dimerization. The β mutants, A73C and C220U, both strate that both binding sites are able to bind ligand in the tandem riboswitch with leader. Because Lig1 and Lig2 bind almost as well as WT, the two ligand-binding sites are independent. This analysis assumes that the U-to-A binding-site mutation displaces the glycine without stabilizing the binding site in a “bound-like” conformation. As a counter-example, the C64U mutant of the adenine riboswitch fails to respond to ligand, instead causing constitutive activation of the downstream gene (Tremblay et al. 2011). Given the significant disruption of dimerization when both binding sites are mutated, and the similarity of U78A/U207A’s dimer affinity to that of the WT constructs in the absence of ligand (Fig. 6), the U-to-A mutations FIGURE 7. Proposed model for glycine binding by the tandem glycine riboswitch. In the absence of glycine (top), dimerization of the two do- behave like empty sites. Therefore, dual ligand binding by mains is disfavored. In this case, the P1 stem of aptamer-2 is largely not the tandem riboswitch is not necessary for high-affinity bind- formed, instead interacting with the downstream expression platform ing and cannot explain the evolutionary conservation of the (red domains form an alternative helix). Upon addition of glycine (bot- tandem riboswitch. tom), the equilibrium is shifted toward aptamer dimerization, with the P1 stem of aptamer-1 providing a scaffold for the dimer interface. An alternative explanation for the tandem riboswitch in- Dimerization stabilizes the P1 stem of aptamer-2, which modulates vokes aptamer dimerization as a requirement for ligand bind- the structure of the downstream expression platform.

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Glycine riboswitch depends on aptamer dimerization significantly disrupt ligand binding in aptamer-1 while having (Mandal et al. 2004; Sherman et al. 2012). Similar differences little or no effect on aptamer dimerization affinity in trans.In in P1 helix length and stability have been shown to cause pro- addition, A73C and C220U are two of the three mutants that nounced differences in structure and ligand responsiveness bind glycine better in the WT-binding-site background than for two adenine riboswitches (Nozinovic et al. 2014). would be predicted based on the measured values in the Lig1 We propose that this additional stability causes the P1 of and Lig2 backgrounds. These discrepancies suggest there is re- aptamer-1 to form independently of ligand binding. In-line modeling around the top of the P1 stem of aptamer-2 when probing experiments have shown that the P1 and P0 helices mutations disrupt the β interaction. As this stem is predicted of aptamer-1 are protected in the presence and absence of to change conformations between the on- and off-states glycine (Sherman et al. 2012), and in recent spin labeling ex- (Mandal et al. 2004), interactions that stabilize the appropriate periments, P0 is largely formed upon addition of monovalent conformation could have important consequences for gene cations (Esquiaqui et al. 2014). In contrast, aptamer-2’sP1 control beyond their effects on ligand-binding affinity. is predicted to undergo a conformational change on ligand At least two possible models of riboswitch function could binding, favoring aptamer formation over an alternative account for the asymmetric effect that disrupting dimeriza- helix with the expression platform, although, in experiments tion has on ligand binding by the two aptamers. The simplest performed without the competing expression platform, explanation would be that aptamer-2 is able to function as a aptamer-2’s P1 does not show differential reactivity in the monomer, while aptamer-1 requires dimerization for ligand presence and absence of ligand (Mandal et al. 2004). binding to occur. We tested the homodimerization and gly- Because aptamer-1’s P1 stem is preformed, aptamer-2 is cine-binding affinities of two aptamer-2-only constructs able to take advantage of aptamer-1’s structure and more (Fig. 5), and showed that aptamer-2 is not able to function readily form dimer, even when the tertiary interface is par- in isolation as a monomer. tially disrupted. In contrast, aptamer-1 requires an intact ter- Our proposed model for riboswitch function, outlined in tiary interface in order to constrain aptamer-2 in a bound- Figure 7, invokes aptamer dimerization as a key modulator like dimeric structure, particularly when ligand binding in of P1 formation in aptamer-2. Therefore, the tandem glycine aptamer-2 is disrupted. Functionally, this asymmetry in P1 riboswitch can be considered an extreme example of “inverse stability could allow aptamer-1 to scaffold the dimer inter- junctional architecture” (Serganov and Patel 2012; Serganov face, which forms along the P1 stems of the two aptamers. and Nudler 2013), wherein ligand binding affects P1 stability Other riboswitches have been shown to use scaffolding through stabilization of global conformation, including the to preform significant portions of the aptameric secondary formation of long-range tertiary interactions between the structure prior to ligand binding, including prequeuo- two domains. This model presumes that dimerization and li- sine class-II (Soulière et al. 2013), S-adenosylmethionine gand binding are linked equilibria and predicts that dimeriza- (SAM)-I (Heppell et al. 2011), SAM-II (Haller et al. 2011), cy- tion of the two aptamers in trans is weakened when ligand clic-di-guanosine monophosphate (c-di-GMP) (Wood et al. binding is impaired. We analyzed the dimerization affinity 2012), and the purine riboswitches (Lemay et al. 2006; of the two aptamer domains in trans in the absence of glycine Brenner et al. 2010; Nozinovic et al. 2014). Scaffolding can binding (Fig. 6A), and the dimer affinity weakens signifi- have important consequences for kinetically controlled ribo- cantly, indicating that the tandem riboswitch requires glycine switches (Wickiser et al. 2005a,b; Trausch and Batey 2014), al- binding to form a well-folded dimer. When a single ligand- lowing ligand binding to occur on transcriptionally relevant binding site is disrupted, dimer formation in trans is weak- time scales. As some glycine riboswitches control transcrip- ened but not abrogated (Fig. 6B). Therefore, ligand binding tion termination, they are likely to be kinetically controlled in each binding site contributes to promoting dimerization (Serganov and Patel 2012). of the two aptamer domains into a well-folded tertiary struc- In contrast, many glycine riboswitches in γ proteobacteria ture, with the interdependence between ligand binding and control translation (Mandal et al. 2004), and these ribo- dimerization particularly strong for aptamer-1. switches are likely to be thermodynamically controlled This model proposes that, in order for ligand binding to oc- (Rieder et al. 2007; Lemay et al. 2011; Serganov and Patel cur in either aptamer, both aptamers must form the dimer in- 2012). In these cases, ligand binding in aptamer-1 could pro- terface. The asymmetric effects on ligand binding by the two mote dimerization. Because many of the tertiary interactions aptamers could be explained by differences in the relative involve the P1 stem of aptamer-2, dimerization could stabi- stabilities of the P1 stems, as the P1/P0 helix of aptamer-1 is lize the P1 switch, providing extra energy not provided by twice as long as that of aptamer-2. The predicted folding en- the binding of the small ligand (Zhang et al. 2014). In addi- ergies for helices with these lengths and sequences show the tion, based on many other riboswitch systems, it is likely P1/P0 helix of aptamer-1 is 8 kcal/mol more stable than that ligand binding in aptamer-2 directly stabilizes the cis the P1 of aptamer-2 (Zuker 2003). This difference in stem- P1 stem. In this way, binding events in each aptamer could length is conserved across tandem glycine riboswitches, independently stabilize the P1 switch, with dimerization with aptamer-1 always containing a 3–7 base P0 with an 8– and the interface relaying energy from the binding site in 9 base P1, while aptamer-2 has only a 5–6 base P1 helix aptamer-1.

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While the tandem glycine riboswitch remains the only plasmid. Mutant riboswitch constructs were made by PCR reaction riboswitch system with two homologous aptamer domains using corresponding primers. that regulate a single expression platform, another riboswitch has recently been shown to bind two ligand molecules within a single-aptamer domain, tetrahydrofolate (THF) (Trausch In vitro transcription et al. 2011; Trausch and Batey 2014). In the case of the THF Plasmid DNA encoding the glycine riboswitch was linearized by re- riboswitch, both kinetic scaffolding and thermodynamic striction digest and used as template for transcription by T7 RNA cooperativity have been suggested as rationale for dual polymerase. RNAs were transcribed in 40 mM Tris–HCL (pH ligand binding. Several natural variants of the THF riboswitch 7.5), 4 mM spermidine, 10 mM DTT, 55 mM MgCl2, 0.05% ′ were analyzed using ITC and structure probing, and, while the Triton X-100, and 4 mM of each 5 -nucleotide triphosphate (7 Hill coefficients varied, dual ligand binding was conserved. mM for GTP) for 2 h at 37°C. The HDV ribozyme was allowed to Mutants that disrupt each site were identified and analyzed self-cleave by heat denaturing at 70°C and slow refolding in an additional 100 mM NaCl and 20 mM MgCl . All RNAs were purified in one of the cooperative parent constructs. In transcriptional 2 by 6% PAGE, eluted into 0.3 M NaOAc (pH 5.2), precipitated with termination assays, the two singly binding mutants of the ethanol, and resuspended in the appropriate buffer. RNA transcripts THF riboswitch diverge significantly, with one continuing were then buffer-exchanged four times and concentrated using to control gene expression, albeit with a moderately reduced Amicon Ultra centrifugal filters. RNA concentrations were deter- effective concentration, while the other mutant fails to re- mined by UV absorbance at 260 nm. Absorption coefficients were spond to ligand. The authors propose that binding at the determined by digestion with Nuclease P1, according to established pseudoknot site is critical for switching, while binding at the protocols (Cavaluzzi and Borer 2004; Wilson et al. 2014). Briefly, ∼1 distal site could play a scaffolding role and/or allow a cooper- nmol of RNA was incubated at 50°C for 1 h with 1 unit of Nuclease ative response to changes in THF concentration. P1 in 200 mM NaOAc, pH 5.3 with 5 mM EDTA and 10 mM Zn In this study, we demonstrate that aptamer dimerization is (OAc)2. Based on extinction coefficients for the individual nucleotides, the extinction coefficient of fully digested VC1-2 WT is 2.7 energetically linked to ligand binding in aptamer-1. Based on − − − − M 1 cm 1 and that of the intact, folded RNA is 2.0 M 1 cm 1. our results, we propose a model for riboswitch function (Fig. The extinction coefficients for fully digested and intact, folded − − 7), wherein (1) both aptamers must adopt a dimeric tertiary aptamer-1 are 1.6 and 1.1 M 1 cm 1, respectively. structure for ligand binding to occur, (2) aptamer-1 has a sta- bilized P1 that acts as a scaffold for dimerization, and (3) dimerization and ligand binding stabilize the P1 helix of Equilibrium dialysis assay aptamer-2, which serves as the switch for gene control. 14 Such a model for riboswitch function could explain the prev- VC1-2 RNA transcripts were combined with trace C-labeled glycine in TB buffer (90 mM Tris-borate at pH 8.3) containing 10 alence of the tandem riboswitch in two different ways. mM MgCl2 and 100 mM KCl. Samples were heated to 60°C then al- Dimerization of the two domains could act thermodynami- lowed to slow cool to ∼30°C over an hour. The RNA/glycine mixture cally, providing extra energy to counter-balance the alterna- was equilibrated overnight at 23°C across from an equal volume of tive conformation of the expression platform. In contrast or buffer in a 5000 MW cut-off Dispo Equilibrium Dialyzer from in addition, dimerization could be important kinetically, Harvard Apparatus. For the highest RNA concentrations tested, with scaffolding by aptamer-1 playing an important role in osmosis resulted in increased volume on the RNA side of the dia- the speed at which aptamer-2 folds and binds ligand. lyzer, and the estimated RNA concentration was adjusted accord- ingly. The amount of 14C-labeled glycine on each side of the dialyzer was determined by scintillation counting in Ultima Gold MATERIALS AND METHODS on a PerkinElmer Tri-Carb 2910TR scintillation counter. The fraction bound was determined for each sample ([counts on RNA side − DNA oligonucleotides and chemicals counts on buffer side]/counts on RNA side). The Kd value for glycine binding was determined by plotting the fraction bound value DNA oligonucleotides were synthesized by the W.M. Keck versus the concentration of RNA and fitting to a standard equation Foundation Biotechnology Resource Laboratory at Yale University for one-site binding, using Prism to perform a least squares regres- and used without further purification. Glycine and other chemicals sion: were obtained from Sigma. Y = Bmax × X/(Kd + X)+NS × X + background, where Y is the fraction bound, X is the concentration of RNA, and NS is a constant term for nonspecific binding. DNA constructs For mutants that failed to saturate at the RNA concentrations test- The V. cholerae VC1422 glycine riboswitch (VC1-2 WT) and single- ed, Bmax values were fixed at 0.98, as indicated in the table legends. aptamer constructs were made by adding the seven base leader se- For doubly glycine-binding mutants, the data were also fit to an quence to previously reported plasmids using a PCR reaction with equation for binding to a two-site model: corresponding primers (Erion and Strobel 2011). The plasmids con- Y = background + NS × X + Bmax ×[Kd1 × X + Kd2 × X]/[Kd1 sisted of the T7 promoter sequence, riboswitch DNA sequence, and × + × + × ], the anti-genomic HDV ribozyme sequence in the pUC19 (NEB) Kd2 Kd1 X Kd2 X

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Glycine riboswitch depends on aptamer dimerization

where Y is the fraction bound, X is the concentration of RNA, and ACKNOWLEDGMENTS NS is a constant term for nonspecific binding. In all cases, the We thank Meghan Griffin and Dave Hiller for critical comments on data fit equally well to a one-site or two-site model, and the error 10 the manuscript, Cambria Alpha, Brian Dunican, Thanh Erion, and on fitting the weaker site was >10 . Dave Hiller for helpful discussion, and Patricia Gordon for tran- For the aptamer-2-only constructs, for which homodimerization scription reagents and general laboratory support. This work was was in question, the data were also fit to an equation for cooperative supported by National Institutes of Health Grant GM022778. binding: = × n/( n + n)+ , Y Bmax X Kd X background Received July 10, 2014; accepted August 22, 2014. where Y is the fraction bound, X is the concentration of RNA, and n is the Hill coefficient, which describes cooperativity. REFERENCES Baird NJ, Ferré-D’Amaré AR. 2013. 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Ligand binding by the tandem glycine riboswitch depends on aptamer dimerization but not double ligand occupancy

Karen M. Ruff and Scott A. Strobel

RNA published online September 22, 2014

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