Tuning a Riboswitch Response Through Structural Extension of a Pseudoknot

Tuning a riboswitch response through structural PNAS PLUS extension of a pseudoknot

Marie F. Soulièrea,1, Roger B. Altmanb,1, Veronika Schwarza, Andrea Hallera, Scott C. Blanchardb,2, and Ronald Micuraa,2

aInstitute 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 rep- been extensively investigated (18–28), preQ1 class II (preQ1-II) resent critical aspects of RNA function in the cell and are par- riboswitches have been largely overlooked despite the fact that ticularly central to riboswitch-mediated control of gene expression. a different mode of ligand binding has been postulated (14). Here, using single-molecule fluorescence energy transfer imaging, The consensus sequence and the secondary structure model we explore the folding dynamics of the preQ1 class II riboswitch, an for the preQ1-II motif (COG4708 RNA) (Fig. 1A) comprise upstream mRNA element that regulates downstream encoded mod- ∼80–100 nt (14). The minimal Streptococcus pneumoniae R6 fi i cation enzymes of queuosine biosynthesis. For reasons that are aptamer domain sequence binds preQ1 with submicromolar af- not presently understood, the classical pseudoknot fold of this sys- finity and consists of an RNA segment forming two stem–loops, tem harbors an extra stem–loop structure within its 3′-terminal re- P2 and P4, and a pseudoknot P3 (Fig. 1B). In-line probing gion immediately upstream of the Shine–Dalgarno sequence that studies suggest that the putative SD box (AGGAGA; Fig. 1) is contributes to formation of the ligand-bound state. By imaging li- sequestered by pseudoknot formation, which results in trans- gand-dependent preQ1 riboswitch folding from multiple structural lational-dependent gene regulation of the downstream gene (14). – fl perspectives, we reveal that the extra stem loop strongly in uences Here, we investigated folding and ligand recognition of the pseudoknot dynamics in a manner that decreases its propensity to S. pneumoniae R6 preQ1-II riboswitch, using complementary spontaneously fold and increases its responsiveness to ligand bind- chemical, biochemical, and biophysical methods including se- ing. We conclude that the extra stem–loop sensitizes this RNA to lective 2′-hydroxyl acylation analyzed by primer extension CHEMISTRY broaden the dynamic range of the ON/OFF regulatory switch. (SHAPE), mutational analysis experiments, 2-aminopurine fluorescence, and single-molecule fluorescence resonance energy SHAPE | single-molecule FRET | site-specific labeling | metabolite sensing transfer (smFRET) imaging. In so doing, we explored the struc- RNAs | ligand recognition tural and functional impact of the additional stem–loop element in the context of its otherwise “classical” H-type pseudoknot fold (29– variety of small metabolites have been found to regulate 32) (Fig. 1C). Our results reveal that the unique 3′-stem–loop el- Agene expression in bacteria, fungi, and plants via direct ement in the preQ -II riboswitch contributes to the process of interactions with distinct mRNA folds (1–4). In this form of 1 SD sequestration, and thus the regulation of gene expression, by BIOCHEMISTRY regulation, the target mRNA typically undergoes a structural – modulating both its intrinsic dynamics and its responsiveness to change in response to metabolite binding (5 9). These mRNA ligand binding. elements have thus been termed “riboswitches” and generally include both a metabolite-sensitive aptamer subdomain and an Results and Discussion expression platform. For riboswitches that regulate the process Temperature-Dependent SHAPE Indicates preQ -II Pseudoknot of translation, the expression platform minimally consists of 1 – Preorganization. Previous bioinformatics and in-line probing a ribosomal recognition site [Shine Dalgarno (SD)]. In the sim- studies on the preQ -II riboswitch sequence from S. pneumonia plest form, the SD sequence overlaps with the metabolite-sensitive 1 aptamer domain at its downstream end. Representative examples Significance include the S-adenosylmethionine class II (SAM-II) (10) and the S-adenosylhomocysteine (SAH) riboswitches (11, 12), as well as The three-dimensional fold and inherent structural dynamics of prequeuosine class I (preQ1-I) and II (preQ1-II) riboswitches (13, 14). The secondary structures of these four short RNA families RNA are critical determinants of function. Although the hier- contain a pseudoknot fold that is central to their gene regulation archical folding of RNA secondary structure followed by tertiary structure formation is well established, insights into how capacity. Although the SAM-II and preQ1-I riboswitches fold into classical pseudoknots (15, 16), the conformations of the SAH (17) secondary structure units impact tertiary structure formation and dynamics remain elusive. Using single-molecule FRET im- and preQ1-II counterparts are more complex and include a struc- aging of the preQ1 class II bacterial riboswitch, we reveal that tural extension that contributes to the pseudoknot architecture – (14). Importantly, the impact and evolutionary significance of RNA employs stem loop insertions into classical pseudoknots as a tool to tune ligand response and hence the outcome of these “extra” stem–loop elements on the function of the SAH and natural gene-regulatory elements. preQ1-II riboswitches remain unclear. PreQ riboswitches interact with the bacterial metabolite 1 Author contributions: M.F.S., S.C.B., and R.M. designed research; M.F.S., R.B.A., and V.S. 7-aminomethyl-7-deazaguanine (preQ1), a precursor molecule in performed research; A.H. contributed new reagents/analytic tools; M.F.S., R.B.A., V.S., the biosynthetic pathway of queuosine, a modified base en- A.H., S.C.B., and R.M. analyzed data; and M.F.S., S.C.B., and R.M. wrote the paper. countered at the wobble position of some transfer RNAs (14). The authors declare no conflict of interest. The general biological significance of studying the preQ1-II This article is a PNAS Direct Submission. system stems from the fact that this gene-regulatory element is Freely available online through the PNAS open access option. found almost exclusively in the Streptococcaceae bacterial family. 1M.F.S. and R.B.A. contributed equally to this work. Moreover, the preQ1 metabolite is not generated in humans and 2To whom correspondence may be addressed. E-mail: [email protected] or has to be acquired from the environment (14). Correspondingly, [email protected]. the preQ1-II riboswitch represents a putative target for antibiotic This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. intervention. Although preQ1 class I (preQ1-I) riboswitches have 1073/pnas.1304585110/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1304585110 PNAS Early Edition | 1of9 Downloaded by guest on September 25, 2021 exhibited enhanced SHAPE reactivity in the presence of preQ1 ligand (see ref. 35 and Fig. S3), suggesting that its solvent ex- posure 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 2+ Mg 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. 1. PreQ1 class II riboswitch. (A) Chemical structure of 7-aminomethyl- 7-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 2+ and P4 are preformed in the absence of both Mg and preQ1 ligands, whereas the pseudoknot nucleotides (U15–U19, A50– Fig. 2. Temperature-dependent SHAPE analysis of the preQ1-II RNA. (A) G54) are disordered and highly sensitive to thermal dena- Representative gel for the SHAPE probing of the preQ1-II RNA structure with turation. The overall reactivity of the entire riboswitch decreases BzCN. Lanes from Left to Right: G and C base ladders, control in the absence in the presence of magnesium over the full range of temper- of probing reagent, probing in the presence of 5 mM Mg2+ ions and 10 μM atures examined, especially the nucleotides of the pseudoknot preQ1 across a temperature gradient from 25 to 70 °C in 5 °C increments. (B) – Representative gel analysis for the estimation of denaturation temperatures and J2 3 regions (Fig. 2C). These residues were shown to possess 2+ Td of individual nucleotides (here A50) without ligands, with Mg and with an even higher denaturation temperature than neighboring 2+ Mg and preQ1. Td values were determined by the inflection point of the pseudoknot nucleotides. With the addition of preQ1 ligand, plot of relative reactivities over increasing temperature. (C) The apparent a further increase in the denaturation temperature of the pseu- denaturation temperatures Td are colored as indicated onto the secondary doknot nucleotides was observed, with the exception of A50, structure for the three different conditions tested (free RNA, RNA with Mg2+, 2+ which showed a 5 °C-lower denaturation temperature. This base RNA with Mg and preQ1).

2of9 | www.pnas.org/cgi/doi/10.1073/pnas.1304585110 Soulière et al. Downloaded by guest on September 25, 2021 served in junction J2–3 with certain nucleosides exhibiting greater PNAS PLUS 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 rigidi- ﬁed 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 CHEMISTRY 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 BIOCHEMISTRY 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. Fig. 3. SHAPE analysis of preQ1-II RNA mutants. (A) Secondary structure and Strikingly, this construct retained the capacity to bind the preQ representative gel for the SHAPE probing of the preQ1-II C19U mutant with 1 BzCN. Lanes from Left to Right: control in the absence of probing reagent ligand (Fig. 3C). PreQ1-II RNAs with triple-uridine (ΔP4/U3)or (DMSO lane), probing in the absence of MgCl2 or ligand (BzCN) and presence triple-adenosine (ΔP4/A ) mutations of the bases surrounding 2+ 2+ 3 of 5 mM Mg ions, and 5 mM Mg ions plus 10 μM preQ1.(B) Same as A for the truncation (C36, G37, C49) also retained preQ1 binding, preQ1-II A11U/G12U mutant. (C) Same as A for a deletion mutant of preQ1-II suggesting that the contribution of the J2-4 junction to preQ1 RNA with ΔP4. The region that is highly indicative of preQ1 binding is binding is largely sequence independent (Fig. S3 and Table S1). highlighted by a red square. Secondary structures are color-coded to facili- To evaluate the relative ligand binding affinities of the WT tate correlation of nucleoside positions with gel electrophoresis band shifts. and truncated aptamers in parallel, binding experiments were Mutations and deletions highlighted by red lettering. performed using riboswitch constructs containing a 2-Ap substitution at position A11 (Fig. S4). These experiments revealed rearrangements of the RNA that report indirectly on the ligand that the truncated ΔP4 construct exhibited a Kd for preQ1 binding of 4.2 ± 0.1 μM, a roughly 10-fold decrease in affinity binding process, are in line with the parameters reported previously for a number of other riboswitch aptamer domains (for compared with the WT (A11Ap) aptamer construct (Kd = 0.43 ± 0.01 μM). Additional deletions in J2-4, which reduced the preQ -II comparison, see table S1 in ref. 22). Strikingly, the estimated kon 1 Δ ∼ ± × 6 −1· −1 riboswitch motif to 41 nt, were also tolerated without further for the truncated, P4/A3 construct was 1.6 0.1 10 M s (Fig. S5), more than an order of magnitude faster than the WT losses in preQ1 binding affinity (Table S1). We conclude that the P4 stem–loop is not essential but serves an important role of construct. Consequently, koff must also increase by an even greater −1 fi increasing the affinity of preQ1 binding. extent (ca. 5s ). These ndings show that the P4 helical element fi To reveal the kinetics of the preQ1–aptamer interaction, pre– serves to modulate ligand binding af nity by altering both the rate steady-state measurements were performed, where preQ1 bind- of productive preQ1-riboswitch aptamer domain interactions as ing was tracked by the increase in 2-Ap fluorescence over time. well as the stability of the ligand-bound state. Here, the WT A11Ap system exhibited an apparent on rate, kon, 4 −1 −1 of 2.9 ± 0.1 × 10 M ·s (Fig. S5). This rate, together with the Dynamics of the preQ1-II Riboswitch Revealed by smFRET Imaging. To estimated binding affinity, Kd, enabled us to estimate the dis- elucidate dynamic features of the preQ1-II riboswitch RNA un- −2 −1 sociation rate of the preQ1 ligand, koff,as1.2× 10 s (koff = derpinning the folding and ligand recognition process, three Kd × kon). These estimates, which are based on local structural distinct fluorescently labeled RNA constructs were created for

Soulière et al. PNAS Early Edition | 3of9 Downloaded by guest on September 25, 2021 smFRET imaging. These investigations focused on evaluating SHAPE probing data and mutational analysis, together with the features of the regulatory interaction, namely the sequestration 2-Ap data obtained here and elsewhere (35). We refer to these of the SD sequence through pairing with junction J2–3, as well as constructs as WT/11–57, WT/11–42, and ΔP4/11–57, where the the role of the “extra” P4 stem–loop element. Two WT preQ1-II acceptor fluorophore (Cy5) was linked at position 11 and the RNA constructs were synthesized carrying donor and acceptor donor fluorophore (Cy3) at position 57 or 42. In each case, the dye fluorophores within junction J2–3 and very close to the SD se- linkage to the RNA was engineered via an extended linker to a 5- quence (Fig. 4A and Table S2), and within junction J2–3 and at aminoallyluridine residue (Materials and Methods and Fig. S6). To the tip of P4, respectively (Fig. 4B and Table S2). To further enable surface immobilization of each construct for smFRET examine the role of the P4 stem–loop element, a ΔP4 preQ1-II imaging, a 5′-biotin moiety was linked to the 5′-terminus. The final RNA construct was prepared, where donor and acceptor fluo- products were confirmed by liquid chromatography–electrospray rophores were linked within the J2–3 and SD regions, re- ionization (LC-ESI) mass spectrometry (Fig. S6). spectively (Fig. 4C and Table S2). In each construct, the specific Single-molecule imaging was performed using a wide-field positions for fluorophore attachment were chosen based on our total internal reflection fluorescence microscope as previously

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 2+ 2+ 2+ (WT/11–57) in the absence of Mg and preQ1 ligand, in the presence of 2 mM Mg ions, and in the presence of 2 mM Mg 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.

4of9 | www.pnas.org/cgi/doi/10.1073/pnas.1304585110 Soulière et al. Downloaded by guest on September 25, 2021 described (36). The dynamics of hundreds of individual surface- by pseudoknot formation (Fig. 4B). In the absence of Mg2+ and PNAS PLUS immobilized preQ1-II riboswitch molecules were tracked simul- preQ1, this construct exhibited a dominant low-FRET (∼0.18) taneously over extended periods using an oxygen scavenging configuration (Fig. 4E, Top Left). Stem P4, like P2, is folded system in the presence of solution additives (37). Fluorescence under these conditions (see above), and therefore, the larger resonance energy transfer efficiency (FRET) was calculated average distance between the two dyes in construct WT/11–42 ratiometrically [FRET = ICy5/(ICy5 +ICy3)] to provide estimates (0.18 FRET) compared with WT/11–57 (0.3 FRET) can be ra- of time-dependent changes in distance between donor and tionalized by a conformation with stem P4 directed away from acceptor fluorophores (38, 39). Here, τFRET was ∼4.0 s, pre- J2–3 (Fig. 4B, left-side cartoons). Under these conditions, an dominantly limited by photobleaching of the Cy5 fluorophore. intermediate-FRET (around ∼0.5) configuration was also ob- The dynamic behaviors of individual molecules were assessed served, albeit at very low occupancy (less than 10%) (Fig. 4E, using hidden Markov modeling procedures (Materials and Top). Inspection of individual FRET trajectories revealed that − Methods), and ensemble information was obtained by combining such conformations arise from frequent (∼2s 1) excursions from FRET trajectories from individual molecules into population the predominant, low-FRET configuration to transient (∼40-ms FRET histograms. Measurements of each construct were perfor- lifetime) intermediate-FRET states. As this lifetime approaches med under three distinct conditions: (i) in the absence of Mg2+ the imaging frame rate (15-ms integration time), the estimated 2+ and preQ1;(ii)inpresenceof2mMMg ions and absence of lifetime of such configurations is likely an upper bound. 2+ 2+ preQ1;and(iii) in presence of 2 mM Mg ions and saturating In the presence of Mg , intermediate-FRET (0.54) config- concentrations of the preQ1 ligand (100 μM) (Fig. 4). urations exhibited a fivefold increase in average lifetime (∼190 In the absence of both ligands, the WT/11–57 preQ1-II con- ms; Table S3). Correspondingly, the intermediate-FRET config- struct exhibited a dominant low-FRET (0.3) configuration (Fig. uration became significantly more populated on a time-averaged 4D, Top Left). This FRET value is consistent with a relatively basis (∼20%) (Fig. 4E, Middle). Addition of saturating concen- open conformation in which the pseudoknot is not formed and trations of preQ1 (100 μM) stabilized the intermediate-FRET the dyes are separated by ∼60–70 Å [assuming an R0 of ∼56 Å configuration by an additional eightfold (∼1.6-s lifetime; Table (40, 41)]. Under these conditions, this construct also exhibited S3). However, under these conditions, where our 2-Ap data roughly 20% high-FRET state occupancy (∼0.84 FRET) on (Fig. S4) suggest that the preQ1 binding site is saturated, the in- a time-averaged basis, consistent with an interdye distance of termediate-FRET state exhibited only partial occupancy (roughly <20 Å (Fig. 4D, Top Left). Inspection of individual FRET tra- 50%) (Fig. 4E, Bottom). Although direct comparisons between CHEMISTRY jectories revealed that occupancy of the high-FRET state arose these experiments must be interpreted with caution (e.g., dif- − from frequent (∼4s 1) excursions out of the predominant low- ferent modifications, variations in Mg2+ concentrations, etc.), FRET configuration that were transient in nature (∼90-ms life- taken together with the distinct kinetic signatures of this con- time; Table S3 and Fig. 4D, Top Right). These data suggest that struct relative to the WT/11–57 system, this observation suggests the WT preQ1-II riboswitch can spontaneously achieve a pseu- that the P4 element remains flexible in the context of an other- 2+ doknot-like fold in the absence of Mg and preQ1 ligands that is wise compacted riboswitch fold (Fig. 4 A and B). We speculate intrinsically unstable. that the preQ1-II riboswitch undergoes conformational changes The high-FRET state was stabilized ∼10-fold in the presence in the P4 stem–loop region that place the tip of P4 closer (∼55 of Mg2+ (2 mM), resulting in a 20:80 distribution of open (low- Å) and further away (∼65 Å) from J2–3. Rearrangements in this BIOCHEMISTRY FRET) and compacted (high-FRET) riboswitch conformations region may conceivably arise if a rigid P4 stem undergoes a hinge- (Fig. 4D, Middle Left). Visual inspection of individual smFRET like motion relative to stem P2 (and the putatively coaxially trajectories revealed that this distribution could be attributed in stacked P3 stem) without making extensive tertiary contacts to the part to a shift in the relative stability of the high-FRET state pseudoknot core. Such conformational plasticity is indeed con- (ca. ∼1-s lifetime) and residual dynamics where the low-FRET, sistent with our SHAPE analysis data (Figs. 2 and 3). In light of open configuration was transiently sampled (ca. ∼350-ms aver- our mutational and 2-Ap data (Fig. 3 and Fig. S4), we conclude age lifetime). Notably, roughly two-thirds of the low-FRET that these structural and dynamic features of the P4 stem con- population (∼15% of all molecules) appeared unaffected by the tribute in some manner to preQ1 binding. presence of Mg2+. The absolute value of the low-FRET state was observed to increase by 0.05 (whereas the high-FRET value Truncation of P4-L4 Impacts the Dynamics and Stability of the remained unchanged), suggesting that Mg2+ binding promotes Pseudoknot Fold. Similar to the WT/11–57 system, in the ab- 2+ Δ – compaction or rigidification of the open conformation of the sence of both Mg and preQ1,the P4/11 57 construct exhibited 2+ ∼ preQ1-II riboswitch fold. In the presence of Mg and preQ1-II, a two-state behavior, where low-FRET ( 0.3) and high-FRET the high-FRET state was further stabilized (approximately two- (∼0.8) states were present at a ratio of 30:70 in favor of the lower- fold), whereas the lifetime of the low FRET state and number of FRET, open riboswitch fold (Fig. 4F, Top). The modest increase in molecules apparently unaffected by the presence of ligands high-FRET state occupancy observed for this system (∼30% vs. remained largely unchanged. Interestingly, the mean FRET ∼20% for the WT/11–57 construct) could be principally attributed value of the high-FRET state decreased by ∼0.05 to a value of to a roughly threefold increase in the high-FRET state lifetime 0.8. This change is consistent with the observations from SHAPE relative to the WT system (270 ms vs. 90 ms). This finding suggests and 2-Ap measurements (see ref. 35 and Fig. S3), which sug- that the P4 stem–loop destabilizes the compacted pseudoknot gested that the nucleobase at position 11 (to which Cy5 is at- configuration in the absence of ligand (Fig. 5 and Table S3). – tached), adopts an unstacked, extrahelical position upon preQ1 Collectively, these data suggest that truncation of the P4 stem binding, a configuration that is expected to modestly increase the loop entropically favors pseudoknot formation. interdye distance. Taken together, these data suggest that the Similar to the WT system, the high-FRET configuration be- high-FRET, compacted state observed for this construct repre- came dominant (75%) in the presence of Mg2+ (Fig. 4F, Middle). sents a fully folded riboswitch configuration. However, two distinctions from the WT system could be dis- cerned. First, all molecules appeared to be Mg2+ responsive. Uncoupled Dynamic Behavior of P4 Positioning and Pseudoknot Second, the lifetime of a high-FRET, compacted preQ1-II fold, Folding. We next set out to investigate the preQ1-II variant was reduced nearly twofold compared with the WT system (510 WT/11–42, whose labeling pattern directly reports on P4 move- ms for ΔP4/11–57 vs. 940 ms for WT/11–57; Table S3). This ments relative to junction J2–3. Like the WT/11–57 construct, trend was even more pronounced in the presence of saturating the dynamic behavior of this system is expected to be influenced (100 μM) concentrations of the preQ1 ligand (Fig. 4F, Bottom).

Soulière et al. PNAS Early Edition | 5of9 Downloaded by guest on September 25, 2021 lifetime of the fully folded structure can reach the order of seconds or even tens of seconds (42). Although the preQ1 class II riboswitch falls into a similar pseudoknot category as the preQ1-I and SAM-II riboswitches, it differs in that it contains an internal stem–loop extension immediately 5′ to the actual pseudoknot pairing interaction, the extra P4 stem–loop. When this second class of preQ1 riboswitches was discovered, this extension had been described as an essential element as mutations in this region abrogated ligand binding (14). By imaging this system from multiple structural perspectives using smFRET, the present investigation has shed important light on the functional role of this additional structural element. The first unexpected observation was that pseudoknot conformations of the preQ1-II riboswitch were significantly more populated in the absence of the cognate ligand (∼85% occupancy in the presence of Mg2+ alone) compared with the SAM-II system (42). Nevertheless, like the SAM-II riboswitch, the preQ1-II system exhibited pronounced “breathing” with average lifetimes Fig. 5. Model for ligand recognition and folding of the preQ1-II riboswitch. For direct comparison, lifetimes of closed pseudoknot conformations τ(P3) in for the closed pseudoknot that were only increased roughly 2+ the absence and presence of ligands (Mg , preQ1) are depicted (in red) next twofold compared with SAM-II. The second unexpected obser- to each structure cartoon. Lifetimes of compacted P4/J2–3 conformations vation was that, although the pseudoknot interaction that is τ (P4) are indicated in green. The pseudoknot becomes prefolded in the crucial to riboswitch function became stabilized upon preQ1 2+ presence of Mg ions and further stabilized in the presence of preQ1. The extra binding, persistent flexibility was observed in what should be the stem–loop P4 behaves rather uncoupled from pseudoknot formation and 2+ 2+ preQ1-bound state (200 times the apparent Kd). Such dynamics retains higher dynamics throughout all three conditions (no ligands; Mg ;Mg were particularly apparent in the position of the extra P4 stem– and preQ1) investigated here, even in the fully folded, preQ1-bound state. loop. Consistent with our SHAPE analysis, two configurations of P4 with roughly similar stability remained in dynamic ex- fi change. This distinct dynamic signature suggests that pseudoknot The lifetime of the high-FRET pseudoknot con guration was – approximately threefold lower for the ΔP4/11–57 construct dynamics and P4 stem loop motions are structurally only – ∼ loosely coupled. compared with the WT/11 57 system (approximately 850 ms vs. – 2.2 s) (Table S3). Similar to the WT construct, the absolute value Our investigations into the role of the P4 stem loop extension fi ∼ using a truncated ΔP4/11–57 construct surprisingly revealed only of the high-FRET con guration decreased by about 0.05 to fi a value of 0.8 in the ligand-bound state, supporting the notion a10-folddecreaseinpreQ1 ligand af nity. This observation suggests that the truncated system may retain a significant degree that WT and truncated versions behave comparably with respect of signaling functionality. However, the kinetic features of preQ to conformational changes toward the fully folded pseudoknot 1 binding of the truncated preQ -II riboswitch were even more structure. The distinct dynamic behaviors of the ΔP4/11–57 1 substantially altered: both the on- and off-rates were estimated construct shed light on the altered ligand binding affinity of this to have increased by more than an orders of magnitude. Such system. Here, the increased ligand on rate observed in bulk (Fig. disparities were manifested in the structural dynamics observed S5) may relate to the increased propensity of this construct to through smFRET, but the relationship was highly nonlinear. adopt a pseudoknot fold, whereas the increased ligand dissoci- Here, the lifetimes of the pseudoknot-like conformation, both in ation rate may stem from the reduced stability of the ligand- the absence and presence of ligands, differed only modestly (Fig. bound state. 5) by comparison with the kinetic parameters of ligand binding deduced from our bulk studies (Fig. S5). Although a deeper Model for Folding and Ligand Recognition of the preQ -II Riboswitch. 1 understanding of the precise relationship between ligand binding Gene-regulating mRNA riboswitches often use pseudoknots as and riboswitch conformation requires further investigation, we scaffolding for selective recognition of small molecules. The fi – — – speculate based on our ndings that the P4 stem loop element structural organization of a pseudoknot namely, a stem loop serves to tune the structural stabilities of open and compacted with a short sequence overhang that folds back onto the loop fi — con gurations, and therefore the ligand responsiveness of the region provides the platform for folding and biological func- system, in a manner that is critical for proper regulation of the tion. Sequestration versus liberation of functional sequence translation apparatus. Here, the extra stem–loop in preQ1-II elements within the pseudoknot thereby directly impacts tran- indirectly affords submicromolar ligand affinity and tunes the scriptional, translational or RNA processing machinery. dynamic features of the riboswitch folding landscape as well as Recent high-resolution structures of pseudoknot-forming SAH, the kinetic features underpinning ligand binding. By contrast, the – SAM-II, and preQ1-I riboswitch aptamers (15 17, 19, 20) have extra P2/P2b stem–loop in the SAH riboswitch is essential be- revealed how this fundamental RNA fold can create a high- cause it directly contributes to ligand recognition and binding (17). affinity ligand binding pocket. Additionally, a detailed smFRET investigation of the SAM-II riboswitch has shed much-needed Large, Structurally Complex Riboswitches with Pseudoknots and light on the principles of folding dynamics in a ligand-responsive Extensions. Although the relatively small size of the preQ1-II, pseudoknot that harbors the decision-making regulatory in- SAM-II, and SAH riboswitches (∼50 nt) may significantly reduce teraction (42). The SAM-II RNA pseudoknot employs a defined the complexity of the folding landscape, the present inves- two-state behavior, where the closed, pseudoknot-like confor- tigations demonstrating the impact of the P4 stem–loop element mation is transiently sampled in the absence of ligand. Under insertion on the kinetic and structural features of the preQ1-II physiological buffer conditions containing Mg2+ ions, the life- riboswitch argue that substantial challenges remain toward gaining time of the closed pseudoknot conformation increases to on the a complete understanding of even the most compact aptamer order of hundreds of milliseconds and becomes even more dra- folds. Larger (100- to 200-nt) riboswitches such as SAM-I and matically stabilized when the cognate SAM ligand is bound. The B12 riboswitches, and the glmS riboswitch-ribozyme also contain

6of9 | www.pnas.org/cgi/doi/10.1073/pnas.1304585110 Soulière et al. Downloaded by guest on September 25, 2021 pseudoknots as integral structural components (Table S4). In the nized roughly perpendicular to the long axis of aptamer PNAS PLUS case of the SAM-I riboswitch, a helical extension (P4) is located pseudoknot. immediately 3′ to the pseudoknot and its deletion reduces ligand binding affinity (43). However, to the best of our knowledge, the Materials and Methods effects of this extension on the dynamics of the SAM-I pseu- Preparation of Cy3/Cy5-Labeled RNA. Solid-phase RNA synthesis (54, 55) was doknot and the kinetic features of ligand binding have yet to be performed as described in the SI Materials and Methods. (Sulfo-) Cy3 and determined. In the glmS system (44), a large extension (P4-P4.1) (sulfo-) Cy5 NHS ester were purchased from GE Healthcare or Lumiprobe. impacts pseudoknot formation (P3.1) and is crucial for self- DMSO was dried over activated molecular sieves. Dye-NHS ester (1 mg; ∼1,200 nmol) was dissolved in anhydrous DMSO (500 μL, dried over activated cleavage activity (45). In this system, the extension is involved in molecular sieves). Lyophilized RNA (20 nmol) containing a 5-aminoallyl- a series of tertiary interactions with the pseudoknot core and uridine modification was dissolved in labeling buffer (50–100 mM phosphate may be anticipated to affect both the stability of the overall fold buffer, pH 8.0), and nanopure water was added to reach a fraction of 55% as well the kinetics of ligand binding. For the B12 riboswitch, (vol/vol) (49 μL) of the intended final reaction volume (89 μL) with a final a large structural extension (P6 versus P6-P11) in the pseudoknot concentration of cRNA of 225 μM. The corresponding volume of the dye-NHS core aids in the ligand discrimination process (46, 47). Again, the ester solution [45% (vol/vol)] (40 μL) was added to the RNA solution (to reach μ fi influence of this extension on the dynamics of folding and ligand a concentration of cDye = 1,124 Minthe nal reaction volume). The re- binding have yet to be explored. action mixture was gently tumbled on a shaker overnight at room temperature in the dark. The reaction was stopped by precipitation with absolute ethanol and sodium acetate for 30 min at –20 °C followed by centrifugation Comparison with Other Riboswitches Analyzed by smFRET. Detailed for 30 min at 4 °C at 13,000 rpm Eppendorf 5430R, rotor F-45-30-11. The smFRET studies have been recently performed on a number colored pellets were dried, resuspended in water, and purified by anion- of RNA systems, including the c-di-GMP, SAM-I, and lysine exchange chromatography on a Dionex DNAPac100 column (9 × 250 mm) at riboswitches, to elucidate the dynamics underlying the riboswitch 60 °C. Flow rate was 2 mL/min; gradient was Δ12–22% B in A within 20 min; folding process. In the case of the c-di-GMP riboswitch (48), UV detection was at a wavelength λ of 260 nm (RNA), 548 nm (Cy3), and 646 such studies revealed that the free RNA occupies four distinct (or 595) nm (Cy5). Fractions containing labeled oligonucleotide were loaded + − configurations that appeared to differ dramatically in their dy- on a C18 SepPak cartridge (Waters/Millipore), washed with 0.1 M (Et3NH) HCO3 namic properties and apparent stabilities. Here, evidence was and H2O, eluted with H2O/CH3CN(1/1),andlyophilizedtodryness. Enzymatic ligation. PreQ1-II RNA aptamers containing 2-aminopurine or presented that all four states interconvert over very long (ca. ′ minutes) timescales and that allosteric interactions accelerate 5 -biotinylated, and Cy3/Cy5 labels were prepared by splinted enzymatic ligation of two chemically synthesized fragments (Fig. S6) using T4 DNA ligase CHEMISTRY ligand recognition by aiding preorganization of the RNA aptamer (Fermentas) (56). Briefly, 10 μM of each RNA fragment, 15 μM of a 25-nt DNA fold to favor rapid c-di-GMP binding. For the SAM-I riboswitch, splint oligonucleotide (IDT), and a final T4 DNA ligase (Fermentas) concen- a dynamic three-state exchange process was uncovered where the tration of 0.5 U/μL in a total volume of 1 mL were incubated for 6–20 h at 37 °C ligand-free state exhibited unfolding dynamics in the milli- in the provided buffer system. Analysis of the ligation reaction and purification seconds regime and the most compacted fold was frozen in the of the ligation products were performed by anion exchange chromatography presence of ligand (43). For the lysine riboswitch, smFRET was and the final product confirmed by LC-ESI mass spectrometry. used to measure the opening/closing rate constants for the smFRET experiments. smFRET data were acquired using a prism-based total fl aptamer domain as well as the lysine dissociation constant (9). internal re ection microscope, where the biotinylated preQ1 riboswitch was

surface immobilized within PEG-passivated, streptavidin-coated quartz micro- BIOCHEMISTRY Although these studies have provided an important foundation fluidic devices (36). The Cy3 fluorophore was directly illuminated under 1.5 kW for understanding the relationship between riboswitch dynamics, cm−2 intensity at 532 nm (Laser Quantum). Photons emitted from both Cy3 ligand binding and gene expression regulation, continued, in- and Cy5 were collected using a 1.2 N.A. 60× Plan-APO water-immersion depth investigations along these lines of pursuit are greatly objective (Nikon), where optical treatments were used to spatially separate needed. For instance, in most smFRET studies, only a single Cy3 and Cy5 frequencies onto two synchronized EMCCD devices (Evolve 512; structural perspective on riboswitch dynamics has been obtained Photometrics). Fluorescence data were acquired using MetaMorph acquisi- (e.g., from just one FRET pair). The importance of applying tion software 13 (Universal Imaging Corporation) at a rate of 66.7 frames per multiple structural perspectives in smFRET imaging experiments second (15-ms integration). Fluorescence trajectories were selected from the fi is exemplified by the present investigation, prior studies of the movie les for analysis using automated image analysis software coded in MATLAB (The MathWorks). Fluorescence trajectories were selected on the TPP riboswitch aptamer (49), as well as more complex systems basis of the following criteria: a single catastrophic photobleaching event, at such as DNA helicase (50) and the bacterial ribosome (51, 52). least 6:1 signal-to-background noise ratio calculated from the total fluo- Such studies have the potential to uncover unexpected com- rescence intensity, and a FRET lifetime of at least 30 frames (450 ms) in any plexities in the structure and dynamics that would be difficult or FRET state ≥0.15. smFRET trajectories were calculated from the acquired fl impossible to discern through bulk techniques. This argument is uorescence data using the formula FRET = ICy5/(ICy3 +ICy5), where ICy3 and fl particularly relevant to systems that exhibit asynchronous be- ICy5 represent the Cy3 and Cy5 uorescence intensities, respectively. Equi- haviors as well as static and dynamic heterogeneities where the librium smFRET experiments were performed in 50 mM 3-(N-morpholino) relationship between structural dynamics and ligand binding is propanesulfonate-KOH (KMOPS), 100 mM KCl, pH 7.5, buffer in the presence obscured. The present study on the preQ -II riboswitch high- of an optimized oxygen scavenging and triplet state quenching mixture in 1 the presence of an oxygen scavenging environment (1 unit protocatchuate- lights this point given the nonlinear relationship between ligand 3,4-dioxygenase, 2 mM protocatechuic acid; 1 mM Trolox, 1 mM cyclo- binding kinetics and structural dynamics of the aptamer fold. octatetraene, 1 mM nitrobenzyl-alcohol) (37). All smFRET experiments were fi Here, further clari cation of the precise relationship between performed at 25 °C. Concentrations of MgCl2 and preQ1 were as specified in structure, dynamics and function would be greatly aided by the individual figure captions. FRET state occupancies and transition rates multicolor smFRET studies where ligand binding and aptamer were estimated by idealization to a two-state Markov chain model using the fold could be measured simultaneously. Ultimately, efforts of segmental k-means algorithm implemented in QuB (57). this kind are essential to enabling the rational design of artificial – riboswitch systems as efficient tools for precise gene regulation in RNA Transcription for Chemical Probing. PreQ1-II RNAs of 74 85 nt were diverse cellular contexts. synthesized using a pair of complementary oligonucleotides (IDT) including a T7 RNA promoter followed by the sequence of the RNA with flanking 5′ and 3′ linkers for reverse transcription. Following transcription at 37 °C Concluding Remarks. During review and revision of the present for 2 h, phenol/chloroform extraction, and isopropanol precipitation, the investigation, the molecular details of the preQ1-RNA class II fi RNA substrates were separated on a denaturing 8% (vol/vol) polyacrylamide interaction were revealed at high-resolution for the rst time gel (90 min, 28 W) and visualized by UV shadowing. The corresponding (53). The results presented are entirely consistent with the bands were excised and eluted from the gel by an overnight incubation structure presented in this study, which show the P4 helix orga- in 0.1% SDS/0.5 M ammonium acetate. The RNAs were then precipitated

Soulière et al. PNAS Early Edition | 7of9 Downloaded by guest on September 25, 2021 with isopropanol, and the pellets were resuspended in nanopure water. The addition of 0.4 μL of SuperScript III Reverse Transcriptase (Invitrogen), and RNA substrates were then quantitated by spectrophotometry and stored further incubation at 61 °C for 10 min. Reactions were then stopped by at –20 °C. addition of 1 μL of 4 M NaOH and incubation at 95 °C for 5 min. Radio- labeled cDNA strands were recovered by ethanol precipitation with sodium RNA 2′-Hydroxyl Acylation by Benzoyl Cyanide. Reaction mixtures containing acetate and glycogen. The samples were resuspended in 8 μL of migration T7-transcribed unlabeled RNA (5 pmol) with a 3′-end flanking sequence and buffer [xylene cyanol, 97% (vol/vol) formamide, 10 mM EDTA] after centri- 50 mM KMOPS, pH 7.5, 100 mM KCl, in the presence or absence of 5 mM fugation. Sequencing ladders were produced by adding 1 μLof10mMddGTP MgCl2 and 10 μM preQ1 were heated at 65 °C for 2 min, cooled to 4 °C for or ddCTP in addition to the 8-μL reaction mixture of unmodified RNA samples, 5 min, and incubated at 37 °C for 25 min in an Eppendorf Mastercycler before incubation at 61 °C. Electrophoresis on a 10% (vol/vol) polyacrylamide (VWR). Following incubation, the control background reaction was treated gel for 95 min at 45 W was used to separate 300–500 cpm of the generated with anhydrous DMSO, while the probing reagent benzoyl cyanide (BzCN), cDNA fragments. The gel was dried using a Vacuum Gel Dryer (VWR) at 75 °C fi dissolved in DMSO, was added to the probing reaction mixtures for a nal for 45–60 min. Following overnight exposition on a 32P-sensitive phosphor- concentration of 55 mM. The RNA was recovered by ethanol precipitation screen, the primer extension labeling was revealed by autoradiography. Band with sodium acetate and glycogen. The RNA samples were resuspended in intensities visualized by gel electrophoresis were quantified using SAFA, ver- μ – 8 L of sterile water after centrifugation and stored at 20 °C. sion 1.1 [Semi-Automated Footprinting Analysis (34)]. Datasets were normal- ized for loading variations and RT efficiency by dividing all intensities by the ′– γ 32 Primer Extension. DNA primers (18 nt) were 5 end-labeled with [ - P]ATP intensity of the last bases of primer extension. Final results for graphical rep- (Hartmann Analytic) using T4 polynucleotide kinase (Fermentas) according resentation were obtained by subtracting the DMSO control background from ’ to the manufacturer s instructions. Three microliters of labeled DNA primer the BzCN-probed reaction intensities. was added to 8 μL of RNA from BzCN 2′-hydroxyl acylation and allowed to anneal at 65 °C for 5 min, then incubated at 35 °C for 5 min, and cooled at 4 °C ACKNOWLEDGMENTS. This work was funded by Austrian Science Founda- for 1 min in an Eppendorf Mastercycler. Eight microliters of a mix containing tion (Fonds zur Förderung der Wissenschaftlichen Forschung) Projects I1040, μ × fi · 4 Lof5 rst strand buffer (250 mM Tris HCl, pH 8.3, 375 mM KCl, 15 mM P21641 (to R.M.), and M1449 (to M.F.S.), National Science Foundation Project μ μ μ MgCl2), 1 L of 0.1 M DTT, 1 L of 10 mM dNTPs mixture, and 2 L of DMSO 1223732 (to S.C.B.), and The Irma T. Hirschl/Monique Weill Caulier Trust (S.C.B. were then added to the reactions, followed by incubation at 61 °C for 1 min, and R.B.A.).

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