Tuning a riboswitch response through structural

Tuning a riboswitch response through structural extension of a pseudoknot Marie F. Soulièrea,1, Roger B. Altmanb,1, Veronika Schwarza, Andrea Hallera, Scott C. Blanchardb,2, and Ronald Micuraa,2 a Institute of Organic Chemistry and Center for Molecular Biosciences, University of Innsbruck, A-6020 Innsbruck, Austria; and bDepartment of Physiology and Biophysics, Weill Medical College of Cornell University, New York, NY 10021

Edited by David A. Tirrell, California Institute of Technology, Pasadena, CA, and approved July 23, 2013 (received for review March 8, 2013)

Structural and dynamic features of RNA folding landscapes represent critical aspects of RNA function in the cell and are particularly central to riboswitch-mediated control of gene expression. Here, using single-molecule fluorescence energy transfer imaging, we explore the folding dynamics of the preQ1 class II riboswitch, an upstream mRNA element that regulates downstream encoded modification enzymes of queuosine biosynthesis. For reasons that are not presently understood, the classical pseudoknot fold of this system harbors an extra stem–loop structure within its 3′-terminal region immediately upstream of the Shine–Dalgarno sequence that contributes to formation of the ligand-bound state. By imaging ligand-dependent preQ1 riboswitch folding from multiple structural perspectives, we reveal that the extra stem–loop strongly influences pseudoknot dynamics in a manner that decreases its propensity to spontaneously fold and increases its responsiveness to ligand binding. We conclude that the extra stem–loop sensitizes this RNA to broaden the dynamic range of the ON/OFF regulatory switch.

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SHAPE single-molecule FRET RNAs ligand recognition

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variety of small metabolites have been found to regulate gene expression in bacteria, fungi, and plants via direct interactions with distinct mRNA folds (1–4). In this form of regulation, the target mRNA typically undergoes a structural change in response to metabolite binding (5–9). These mRNA elements have thus been termed “riboswitches” and generally include both a metabolite-sensitive aptamer subdomain and an expression platform. For riboswitches that regulate the process of translation, the expression platform minimally consists of a ribosomal recognition site [Shine–Dalgarno (SD)]. In the simplest form, the SD sequence overlaps with the metabolite-sensitive aptamer domain at its downstream end. Representative examples include the S-adenosylmethionine class II (SAM-II) (10) and the S-adenosylhomocysteine (SAH) riboswitches (11, 12), as well as prequeuosine class I (preQ1-I) and II (preQ1-II) riboswitches (13, 14). The secondary structures of these four short RNA families contain a pseudoknot fold that is central to their gene regulation capacity. Although the SAM-II and preQ1-I riboswitches fold into classical pseudoknots (15, 16), the conformations of the SAH (17) and preQ1-II counterparts are more complex and include a structural extension that contributes to the pseudoknot architecture (14). Importantly, the impact and evolutionary significance of these “extra” stem–loop elements on the function of the SAH and preQ1-II riboswitches remain unclear. PreQ1 riboswitches interact with the bacterial metabolite 7-aminomethyl-7-deazaguanine (preQ1), a precursor molecule in the biosynthetic pathway of queuosine, a modified base encountered at the wobble position of some transfer RNAs (14). The general biological significance of studying the preQ1-II system stems from the fact that this gene-regulatory element is found almost exclusively in the Streptococcaceae bacterial family. Moreover, the preQ1 metabolite is not generated in humans and has to be acquired from the environment (14). Correspondingly, the preQ1-II riboswitch represents a putative target for antibiotic intervention. Although preQ1 class I (preQ1-I) riboswitches have E3256–E3264 | PNAS | Published online August 12, 2013

been extensively investigated (18–28), preQ1 class II (preQ1-II) riboswitches have been largely overlooked despite the fact that a different mode of ligand binding has been postulated (14). The consensus sequence and the secondary structure model for the preQ1-II motif (COG4708 RNA) (Fig. 1A) comprise ∼80–100 nt (14). The minimal Streptococcus pneumoniae R6 aptamer domain sequence binds preQ1 with submicromolar affinity and consists of an RNA segment forming two stem–loops, P2 and P4, and a pseudoknot P3 (Fig. 1B). In-line probing studies suggest that the putative SD box (AGGAGA; Fig. 1) is sequestered by pseudoknot formation, which results in translational-dependent gene regulation of the downstream gene (14). Here, we investigated folding and ligand recognition of the S. pneumoniae R6 preQ1-II riboswitch, using complementary chemical, biochemical, and biophysical methods including selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE), mutational analysis experiments, 2-aminopurine fluorescence, and single-molecule fluorescence resonance energy transfer (smFRET) imaging. In so doing, we explored the structural and functional impact of the additional stem–loop element in the context of its otherwise “classical” H-type pseudoknot fold (29– 32) (Fig. 1C). Our results reveal that the unique 3′-stem–loop element in the preQ1-II riboswitch contributes to the process of SD sequestration, and thus the regulation of gene expression, by modulating both its intrinsic dynamics and its responsiveness to ligand binding. Results and Discussion Temperature-Dependent SHAPE Indicates preQ1-II Pseudoknot Preorganization. Previous bioinformatics and in-line probing

studies on the preQ1-II riboswitch sequence from S. pneumonia Significance The three-dimensional fold and inherent structural dynamics of RNA are critical determinants of function. Although the hierarchical folding of RNA secondary structure followed by tertiary structure formation is well established, insights into how secondary structure units impact tertiary structure formation and dynamics remain elusive. Using single-molecule FRET imaging of the preQ1 class II bacterial riboswitch, we reveal that RNA employs stem–loop insertions into classical pseudoknots as a tool to tune ligand response and hence the outcome of natural gene-regulatory elements. Author contributions: M.F.S., S.C.B., and R.M. designed research; M.F.S., R.B.A., and V.S. performed research; A.H. contributed new reagents/analytic tools; M.F.S., R.B.A., V.S., A.H., S.C.B., and R.M. analyzed data; and M.F.S., S.C.B., and R.M. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. Freely available online through the PNAS open access option. 1

M.F.S. and R.B.A. contributed equally to this work.

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To whom correspondence may be addressed. E-mail: [email protected] or [email protected].

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1304585110/-/DCSupplemental.

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Fig. 1. PreQ1 class II riboswitch. (A) Chemical structure of 7-aminomethyl7-deazaguanosine (preQ1); consensus sequence and secondary structure model for the COG4708 RNA motif (adapted from reference 14). Nucleoside presence and identity as indicated. (B) S. pneumoniae R6 preQ1-II RNA aptamer investigated in this study. (C) Schematics of an H-type pseudoknot with generally used nomenclature for comparison.

R6 have yielded the putative secondary structure depicted in Fig. 1B (14). The characteristic pseudoknot (P3) interaction is defined by the pairing of the 3′-terminal sequence with junction J2–3, where the stem–loop P4 element is inserted immediately upstream of this interaction. To explore the structural and dynamic properties of this model, we used the chemical probing technique SHAPE (33) to probe the conformation of the preQ1-II riboswitch as a function of temperature. To do so, a transcribed riboswitch aptamer domain (85 nt in length; Materials and Methods and Fig. 2) was subjected to reaction with benzoyl cyanide (BzCN) at temperatures ranging from 25 to 70 °C in 5 °C increments. After reverse transcription, the discrete bands observed by gel electrophoresis were quantified using SAFA software (Fig. S1) (34). The relative 2′-OH reactivities were plotted as a function of temperature to evaluate the denaturation temperature of the RNA at the single-nucleotide level (Fig. 2 A and B, and Fig. S2). Here, band intensities represent the degree of 2′hydroxyl acylation of the base identified by reverse transcription. This analysis was repeated for the preQ1-II RNA in the absence or presence of magnesium ions and the preQ1 ligand. The results from these experiments are presented on the proposed secondary structure and color-coded according to their denaturation temperature (Fig. 2C). The data obtained demonstrate that P2 and P4 are preformed in the absence of both Mg2+ and preQ1 ligands, whereas the pseudoknot nucleotides (U15–U19, A50– G54) are disordered and highly sensitive to thermal denaturation. The overall reactivity of the entire riboswitch decreases in the presence of magnesium over the full range of temperatures examined, especially the nucleotides of the pseudoknot and J2–3 regions (Fig. 2C). These residues were shown to possess an even higher denaturation temperature than neighboring pseudoknot nucleotides. With the addition of preQ1 ligand, a further increase in the denaturation temperature of the pseudoknot nucleotides was observed, with the exception of A50, which showed a 5 °C-lower denaturation temperature. This base

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exhibited enhanced SHAPE reactivity in the presence of preQ1 ligand (see ref. 35 and Fig. S3), suggesting that its solvent exposure increases as a consequence of preQ1 binding. Changes of this kind likely stem from disruption of the U19–A50 base pair in the ligand-bound complex. This conclusion is corroborated by the observation that a 2-aminopurine (2-Ap) nucleoside at position 50 exhibits an increase in fluorescence in the presence of Mg2+ and preQ1, trends that are in line with its movement from a stacked, or intrahelical, position to one that is extrahelical (35). Variations in denaturation temperature were also ob-

Fig. 2. Temperature-dependent SHAPE analysis of the preQ1-II RNA. (A) Representative gel for the SHAPE probing of the preQ1-II RNA structure with BzCN. Lanes from Left to Right: G and C base ladders, control in the absence of probing reagent, probing in the presence of 5 mM Mg2+ ions and 10 μM preQ1 across a temperature gradient from 25 to 70 °C in 5 °C increments. (B) Representative gel analysis for the estimation of denaturation temperatures Td of individual nucleotides (here A50) without ligands, with Mg2+ and with Mg2+ and preQ1. Td values were determined by the inflection point of the plot of relative reactivities over increasing temperature. (C) The apparent denaturation temperatures Td are colored as indicated onto the secondary structure for the three different conditions tested (free RNA, RNA with Mg2+, RNA with Mg2+ and preQ1).

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served in junction J2–3 with certain nucleosides exhibiting greater temperature sensitivity (U9, G12, U14) than others (C13). These observations are consistent with conformational rearrangements in J2–3 upon formation of the preQ1-bound complex. Junction J2–4 nucleosides, close to P4 (C34 to C36), also become rigidified in the preQ1-bound complex, whereas those closer to P2 (A28, U29) become more flexible. Strikingly, only minor changes in denaturation temperatures were observed for the majority of nucleosides in the P4 stem–loop under the conditions tested, suggesting that this extra arm is highly stable relative to the rest of the structure. Mutational Analysis of the preQ1-II Riboswitch. To assess the contribution of specific residues to pseudoknot formation and preQ1 binding, SHAPE analysis was performed on riboswitch constructs containing single point mutations. To test the role of U19 in preQ1 ligand recognition (discussed above), we first replaced this nucleotide with cytidine. Consistent with U19 being strictly required for pseudoknot preorganization and preQ1 binding, this mutant construct failed to exhibit any detectable changes in reactivity in the presence of magnesium and preQ1 (Fig. 3A). A similar analysis on constructs containing a C8-to-U mutation, a perturbation that causes an approximately two order of magnitude reduction in preQ1 affinity (14), also showed severely reduced binding (Fig. S3). We conclude that residues C8 and U19 either contribute to formation of the ligand binding pocket or directly interact with the preQ1 ligand in the bound complex. To evaluate whether nucleosides in the upper portion of junction J2–3 are also required for ligand recognition, residues A11 and G12 were mutated to uridines. These mutations were apparently tolerated, as SHAPE probing revealed a pattern of modification that was similar to the WT RNA (Fig. 3B and Fig. S3). Consistent with the “extra” P4 stem–loop being critical for the preQ1-II riboswitch function, a double mutation (G39C/G40C) within this region was shown to abrogate interactions with preQ1 (14). Reasoning that the lack of binding may arise from the disruption of pseudoknot formation due to pairing of the G-rich sequence at the 3′ terminus (G53–G56) with the newly generated C track (C36–C40), a truncated aptamer was synthesized that entirely lacks the P4 stem–loop element (ΔP4; Fig. 3C). Here, a single guanosine (G37) was kept to directly bridge C36 of junction J2–4 and C49 of the aptamer 3′ terminal sequence. Strikingly, this construct retained the capacity to bind the preQ1 ligand (Fig. 3C). PreQ1-II RNAs with triple-uridine (ΔP4/U3) or triple-adenosine (ΔP4/A3) mutations of the bases surrounding the truncation (C36, G37, C49) also retained preQ1 binding, suggesting that the contribution of the J2-4 junction to preQ1 binding is largely sequence independent (Fig. S3 and Table S1). To evaluate the relative ligand binding affinities of the WT and truncated aptamers in parallel, binding experiments were performed using riboswitch constructs containing a 2-Ap substitution at position A11 (Fig. S4). These experiments revealed that the truncated ΔP4 construct exhibited a Kd for preQ1 binding of 4.2 ± 0.1 μM, a roughly 10-fold decrease in affinity compared with the WT (A11Ap) aptamer construct (Kd = 0.43 ± 0.01 μM). Additional deletions in J2-4, which reduced the preQ1-II riboswitch motif to 41 nt, were also tolerated without further losses in preQ1 binding affinity (Table S1). We conclude that the P4 stem–loop is not essential but serves an important role of increasing the affinity of preQ1 binding. To reveal the kinetics of the preQ1–aptamer interaction, pre– steady-state measurements were performed, where preQ1 binding was tracked by the increase in 2-Ap fluorescence over time. Here, the WT A11Ap system exhibited an apparent on rate, kon, of 2.9 ± 0.1 × 104 M−1·s−1 (Fig. S5). This rate, together with the estimated binding affinity, Kd, enabled us to estimate the dissociation rate of the preQ1 ligand, koff, as 1.2 × 10−2 s−1 (koff = Kd × kon). These estimates, which are based on local structural E3258 | www.pnas.org/cgi/doi/10.1073/pnas.1304585110

Fig. 3. SHAPE analysis of preQ1-II RNA mutants. (A) Secondary structure and representative gel for the SHAPE probing of the preQ1-II C19U mutant with BzCN. Lanes from Left to Right: control in the absence of probing reagent (DMSO lane), probing in the absence of MgCl2 or ligand (BzCN) and presence of 5 mM Mg2+ ions, and 5 mM Mg2+ ions plus 10 μM preQ1. (B) Same as A for preQ1-II A11U/G12U mutant. (C) Same as A for a deletion mutant of preQ1-II RNA with ΔP4. The region that is highly indicative of preQ1 binding is highlighted by a red square. Secondary structures are color-coded to facilitate correlation of nucleoside positions with gel electrophoresis band shifts. Mutations and deletions highlighted by red lettering.

rearrangements of the RNA that report indirectly on the ligand binding process, are in line with the parameters reported previously for a number of other riboswitch aptamer domains (for comparison, see table S1 in ref. 22). Strikingly, the estimated kon for the truncated, ΔP4/A3 construct was ∼1.6 ± 0.1 × 106 M−1·s−1 (Fig. S5), more than an order of magnitude faster than the WT construct. Consequently, koff must also increase by an even greater extent (ca. 5 s−1). These findings show that the P4 helical element serves to modulate ligand binding affinity by altering both the rate of productive preQ1-riboswitch aptamer domain interactions as well as the stability of the ligand-bound state. Dynamics of the preQ1-II Riboswitch Revealed by smFRET Imaging. To elucidate dynamic features of the preQ1-II riboswitch RNA underpinning the folding and ligand recognition process, three distinct fluorescently labeled RNA constructs were created for Soulière et al.

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SHAPE probing data and mutational analysis, together with the 2-Ap data obtained here and elsewhere (35). We refer to these constructs as WT/11–57, WT/11–42, and ΔP4/11–57, where the acceptor fluorophore (Cy5) was linked at position 11 and the donor fluorophore (Cy3) at position 57 or 42. In each case, the dye linkage to the RNA was engineered via an extended linker to a 5aminoallyluridine residue (Materials and Methods and Fig. S6). To enable surface immobilization of each construct for smFRET imaging, a 5′-biotin moiety was linked to the 5′-terminus. The final products were confirmed by liquid chromatography–electrospray ionization (LC-ESI) mass spectrometry (Fig. S6). Single-molecule imaging was performed using a wide-field total internal reflection fluorescence microscope as previously

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smFRET imaging. These investigations focused on evaluating features of the regulatory interaction, namely the sequestration of the SD sequence through pairing with junction J2–3, as well as the role of the “extra” P4 stem–loop element. Two WT preQ1-II RNA constructs were synthesized carrying donor and acceptor fluorophores within junction J2–3 and very close to the SD sequence (Fig. 4A and Table S2), and within junction J2–3 and at the tip of P4, respectively (Fig. 4B and Table S2). To further examine the role of the P4 stem–loop element, a ΔP4 preQ1-II RNA construct was prepared, where donor and acceptor fluorophores were linked within the J2–3 and SD regions, respectively (Fig. 4C and Table S2). In each construct, the specific positions for fluorophore attachment were chosen based on our

Fig. 4. Dynamics of the preQ1 riboswitch aptamer analyzed by smFRET imaging and cartoon representations of hypothetic folding states predicted from secondary structure analysis and experimental FRET data. (A) Schematics of labeling pattern to sense pseudoknot formation. (B) Same as A but with labeling pattern to sense dynamics of the extra stem–loop P4. (C) Same as A but with labeling pattern to sense dynamics of pseudoknot formation of the ΔP4 deletion mutant. (D) Population FRET histograms showing the mean FRET values and percentage (%) occupancies of each state observed for the preQ1-II riboswitch (WT/11–57) in the absence of Mg2+ and preQ1 ligand, in the presence of 2 mM Mg2+ ions, and in the presence of 2 mM Mg2+ ions and 100 μM preQ1; corresponding fluorescence (green, Cy3; red, Cy5) and FRET (blue) trajectories of individual preQ1 aptamer molecules under the same conditions, where idealization of the data to a two-state Markov chain is shown in red. (E) Same as D but with WT/11–42 labeling scheme. (F) Same as D but with ΔP4 deletion.

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described (36). The dynamics of hundreds of individual surfaceimmobilized preQ1-II riboswitch molecules were tracked simultaneously over extended periods using an oxygen scavenging system in the presence of solution additives (37). Fluorescence resonance energy transfer efficiency (FRET) was calculated ratiometrically [FRET = ICy5/(ICy5 + ICy3)] to provide estimates of time-dependent changes in distance between donor and acceptor fluorophores (38, 39). Here, τFRET was ∼4.0 s, predominantly limited by photobleaching of the Cy5 fluorophore. The dynamic behaviors of individual molecules were assessed using hidden Markov modeling procedures (Materials and Methods), and ensemble information was obtained by combining FRET trajectories from individual molecules into population FRET histograms. Measurements of each construct were performed under three distinct conditions: (i) in the absence of Mg2+ and preQ1; (ii) in presence of 2 mM Mg2+ ions and absence of preQ1; and (iii) in presence of 2 mM Mg2+ ions and saturating concentrations of the preQ1 ligand (100 μM) (Fig. 4). In the absence of both ligands, the WT/11–57 preQ1-II construct exhibited a dominant low-FRET (0.3) configuration (Fig. 4D, Top Left). This FRET value is consistent with a relatively open conformation in which the pseudoknot is not formed and the dyes are separated by ∼60–70 Å [assuming an R0 of ∼56 Å (40, 41)]. Under these conditions, this construct also exhibited roughly 20% high-FRET state occupancy (∼0.84 FRET) on a time-averaged basis, consistent with an interdye distance of