Structural analysis of a class III preQ1 riboswitch reveals an aptamer distant from a ribosome-binding site regulated by fast dynamics

Abstract

PreQ1-III riboswitches are newly identified RNA elements that control bacterial genes in response to preQ1 (7-aminomethyl-7-deazaguanine), a precursor to the essential hypermodified tRNA base queuosine. Although numerous riboswitches fold as H-type or HLout-type pseudoknots that integrate ligand-binding and regulatory sequences within a single folded domain, the preQ1-III riboswitch aptamer forms a HLout-type pseudoknot that does not appear to incorporate its ribosome-binding site (RBS). To understand how this unusual organization confers function, we determined the crystal structure of the class III preQ1 riboswitch from Faecalibacterium prausnitzii at 2.75 Å resolution. PreQ1 binds tightly (KD,app 6.5 ± 0.5 nM) between helices P1 and P2 of a three-way helical junction wherein the third helix, P4, projects orthogonally from the ligand-binding pocket, exposing its stem-loop to base pair with the 3' RBS. Biochemical analysis, computational modeling, and single-molecule FRET imaging demonstrated that preQ1 enhances P4 reorientation toward P1-P2, promoting a partially nested, H-type pseudoknot in which the RBS undergoes rapid docking (kdock ∼ 0.6 s(-1)) and undocking (kundock ∼ 1.1 s(-1)). Discovery of such dynamic conformational switching provides insight into how a riboswitch with bipartite architecture uses dynamics to modulate expression platform accessibility, thus expanding the known repertoire of gene control strategies used by regulatory RNAs.

Keywords: crystal structure; gene regulation; molecular dynamics; preQ1 riboswitch; single-molecule FRET.

Fig. 1.

Queuosine biosynthesis, secondary structure, and overall fold of the ligand-bound preQ₁-III riboswitch. (A) Biosynthesis of the hypermodified nucleotide queuosine (Q) begins with GTP and leads to the intermediate preQ₁ via enzymes of the queCDEF operon, which is regulated by a preQ₁-I riboswitch in some bacteria (33). PreQ₁ is then inserted at the wobble position of specific tRNAs with additional modifications added in situ (the complete pathway is reviewed in ref. 52). A scavenging pathway has been proposed wherein Q-related molecules are imported by the queT (COG4708) gene product (32), which is regulated in some bacteria by class I, II, and III preQ₁ riboswitches. (B) Secondary structure of the wild-type F. prausnitzii riboswitch based on the crystal structure. PreQ₁ is green, junctions are labeled J, and pairing regions (P) are color-coded; long-range interactions are indicated by dashed lines. A boxed sequence (gray) near P3 indicates the sequence used to produce a phasing module (PM) (53). The RBS sequence 5′-CGGAG-3′ is highlighted (yellow). The assigned secondary structure differs subtly from comparative sequence analysis (23) because the U17•A84 interaction is not a canonical pair. The sym label indicates the crystallographic domain-swapping interaction that resembles bioinformatically predicted helix P5. (C) Cartoon of the preQ₁-bound crystal structure preserving colors from B; preQ₁ is depicted as a semitransparent surface model. The RBS sugar and base rings are yellow. (Inset) View from C rotated 90° about the y axis. (D) Coaxial stacking of P1 with the proximal end of helix P3, and depiction of P3 to J3-4 tertiary contacts.

Fig. S1.

Representative ITC experiments for preQ₁ binding to preQ₁-III riboswitches. Apparent K_D, stoichiometry values (n), and c values for individual experiments are shown; average values are provided in Table S1. (A) PreQ₁ binding to the wild-type Fpr preQ₁-III riboswitch 101-mer (Fig. 1B) in a buffer containing 0.050 M Na–Hepes (pH 7.0), 0.10 M NaCl, 0.006 M MgCl₂; Methods. (B) PreQ₁ binding to the split 74 env preQ₁-III riboswitch (Fig. S4A). (C) PreQ₁ binding to the 74 env preQ₁-III riboswitch in which the 3′ end was removed after P2 (s2Δ30–43). (D) PreQ₁ binding to the wild-type Fpr preQ₁-III riboswitch, except 0.5 mM EDTA was substituted for MgCl₂ in the buffer used in A. (E) PreQ₁ binding to the C7U mutant in the context of the Fpr wild-type riboswitch under conditions described in A. (F) PreQ₁ binding to the Fpr U17C mutant as in A. (G) PreQ₁ binding to the Fpr A52G mutant as in A. (H) PreQ₁ binding to the Fpr A84G mutant as in A. (I) IXP does not bind appreciably to the wild-type preQ₁-III riboswitch under conditions in A. (J) 2AP does not bind appreciably to the preQ₁-III riboswitch under buffer conditions described in A. The slightly positive slope of the curve fit could be the result of very weak binding consistent with observations on the wild-type preQ₁-II riboswitch, which shows an apparent K_D for 2AP that is ∼3 log units poorer than that for preQ₁ (32).

Fig. S2.

Pseudoknot classification of various preQ₁ riboswitches. The diagrams and classifications are based on established nomenclature (70) in which stem (S) or loop (L) regions are depicted as paired nucleotides (open circles) joined by a short black line, or single-stranded segments (closed circles). A green oval indicates the location of preQ₁ binding, which pairs with L1 and L2 and can generate an L2 = 0 configuration that yields coaxial helical stacking of S1 and S2. Bases of the RBS are highlighted (yellow). (A) The preQ₁-III riboswitch 5′ HL_out-type pseudoknot based on the crystal structure (Fig. 1C). The organization is comparatively atypical because of the extended nature of the L3 loop, which encompasses (i) the stem-loop subsequent to S1 (i.e., P3 in Fig. 1 B and C), (ii) a loop of four nucleotides, (iii) a second stem-loop (i.e., P4 of Fig. 1 B and C), and (iv) an unpaired loop of two nucleotides that precedes the second strand of stem S2. (B) The preQ₁-I riboswitch folds as a simple H-type pseudoknot; base pairing was derived from the crystal structures (9, 13). (C) The preQ₁-II riboswitch forms a classical HL_out-type pseudoknot in which the entire RBS is sequestered in stem S2 (12, 18).

Fig. 2.

Details of the preQ₁-binding pocket within a three-helix junction. (A) Close-up view of the P1–P2–P4 helical junction that binds preQ₁. The “ceiling” exhibits tandem purine base pairs emanating from P1. PreQ₁ resides in the center of a base triple-flanked by C7 and U17. Stacked bases A84 and A52 make respective N1-imino to 2′-hydroxyl group interactions with U17 and A85; the latter base stacks below preQ₁ as part of the U•A-U triplex that composes the pocket “floor.” (B) Close-up view of the preQ₁-binding site depicting the final refined ligand bathed in unbiased (average kicked) mF_o–DF_c electron density at the 3.0 σ level. Ligand-specific readout by C7 and U17 is shown in the context of the U8•A85 Hoogsteen pair that forms the floor. The A85 phosphate group and O2 keto of U8 make complementary interactions to the 7-aminomethyl moiety of preQ₁, providing additional specificity. ΔΔG (kcal⋅mol^-1) values relative to wild type are shown for various mutations tested for ligand binding (Table S1). The view is rotated ∼180° about the y axis relative to Fig. 1C. (C) Major-groove base-triple pairing of J1-2 with P2 under the preQ₁-binding pocket; tandem U•A-U triples are flanked by a single A10•A87-U14 triplex.

Fig. S3.

Representative electron-density maps and crystallographic symmetry interactions of the Fpr preQ₁-III riboswitch. (A) Stereoview of the preQ₁-binding site with the final refined structure shown within unbiased, composite iterative-build omit electron density (71) at 2.75 Å resolution; the contour levels are 1.0 σ (blue) and 7.0 σ (green). (B) The 3.0-Å resolution experimental electron density map contoured at 1.0 σ based on the initial density-modified SIRAS phases; a cartoon of the final refined coordinates (Fig. 1C) is included. (C) The final 2.75-Å resolution, reduced-bias (σ_A-weighted) 2mF_o–DF_c electron density map contoured at 1.0 σ using phases from the final refined model included as a ribbon diagram (Fig. 1C). Nucleotides 34 and 35 located in the P3 loop (Top) exhibited poor electron density and were not included in the final model. (D) Ribbon diagram depicting the dyad (filled oval) crystallographic contacts that compose the intermolecular, “domain swapped” region of helix P5. The RBS is labeled and comprises the last five nucleotides of the 3′ terminus. (E) Magnified view of suboptimal, intermolecular helix P5 from D (dashed box). Only the first three bases of the RBS are depicted for clarity (yellow base and ribose planes). Base pairing begins with a ⁷¹U•⁹²U noncanonical pair, followed by five Watson–Crick base pairs ending at the ⁶⁶G-⁹⁷C pair, which encompasses the first base of the RBS. (Lower) The suboptimal pairing of P5 is depicted as a secondary structure with the RBS highlighted (yellow). (F) Stereoview showing three symmetry-related molecules in the lattice (blue, purple, and green) emphasizing the absence of crystal contacts at the binding site for preQ₁, depicted as a space-filling model (green). All cartoon diagrams of the structure were produced using PyMOL (Schrödinger, LLC).

Fig. S4.

Sequences of the preQ₁-III riboswitch used for ITC, SHAPE, and smFRET. (A) The 74 env (environmental sequence) used for benchmarking ligand binding by a split sequence construct (Table S1 and Fig. S1 B and C). Strand 1 is black and strand 2 is red. (B) The wild-type Fpr riboswitch sequence used for SHAPE. The black sequences designated Fpr indicate wild-type 5′ and 3′ genomic extensions added between the riboswitch and the SHAPE cassette. The 5′ linker (green), spacer (pink), and reverse-transcription primer binding site (blue) are as described (41). All other sequences are color-coded as in Fig. 1B. The AUG start codon of the queT (COG4708) gene is highlighted (cyan). (C) smFRET split sequence (split seq) construct of the wild-type Fpr riboswitch that reports on helix P5 formation. P3 was extended to base pair with a 5′-biotinylated DNA oligonucleotide (maroon), allowing attachment to the quartz slide. The break in the P3 stem-loop was based on its poor phylogenetic conservation (23) and the feasibility of using a split seq construct was validated by ITC (Table S1 and Fig. S1B). (Inset) DNA sequence of an anti-P5, 11-mer oligonucleotide used in control experiments to block intramolecular helix P5 formation.

Fig. 3.

Comparison of the preQ₁-III riboswitch to other regulatory RNAs that use triplexes to recognize nucleobase ligands. (A) Overlay of the preQ₁-III riboswitch base triples (gold) upon the preQ₁-II riboswitch (deep purple; PDB ID code 2MIY) (12). The superposition is based on the major-groove base triples and preQ₁, which yielded an average rmsd of 1.1 Å. Here and elsewhere, the preQ₁-III riboswitch ligand is green, and the superimposed ligand is magenta. (B) Overlay of the preQ₁-III riboswitch base triples upon those of the SAM-II riboswitch (deep purple; PDB ID code 2QWY) (8). The superposition is based on shared major-groove base triplex nucleotides (excluding ligand), which yielded an average rmsd of 0.82 Å. (C) Overlay of the preQ₁-III riboswitch base triples with those of the c-di-GMP-II riboswitch (deep purple; PDB ID code 3Q3Z) (34). The superposition is based on shared nucleotide atoms (excluding ligand), which yielded an average rmsd of 2.0 Å.

Fig. 4.

Representative ligand dependence of 2′-OH chemical modification for the wild-type preQ₁-III riboswitch. (A) Electrophoretic SHAPE analysis conducted in the absence and presence of preQ₁ using the chemical modification reagent NAI; DMSO represents a control without NAI; U and G indicate reference nucleotide sequences. P1(s2) represents the 3′-most strand of helix P1; anti-RBS represents the P4 loop predicted to pair within helix P5; (−)preQ₁ indicates no ligand and added NAI; (+)preQ₁ indicates 100 μM ligand and added NAI. (B) High-resolution analysis of the 5′-riboswitch sequence similar to A, but emphasizing changes in P1 and P2 in the absence and presence of ligand. (C) Differential SHAPE reactivity as a function of nucleotide position. Reactivity is shown as a heat map; dark blue indicates little or no change; red indicates large differences between ligand bound and free states. (D) The crystal structure of the preQ₁-III riboswitch (Fig. 1C) showing the spatial distribution of differential SHAPE reactivity using the heat map from C.

Fig. 5.

Model of the preQ₁-III riboswitch showing intramolecular base pairing of the RBS in helix P5. (A) Cartoon diagram of a representative all-atom computational model demonstrating the feasibility of loop P4 engagement in an H-type pseudoknot that sequesters a portion of the RBS within helix P5, consistent with a “gene off” conformation. (B) Close-up view of P5 showing explicit base pairs between the anti-RBS of the P4 loop and the 3′ RBS, representing the “docked” state. The view is rotated −90° about the z axis, and +45° about the x axis relative to A. (Inset) The model accounts for the proposed P5 secondary structure from bioinformatic analysis (23). Base-paired nucleotides of P5 are >93% conserved. Unpaired base 71 of J4-5 is present in only 75% of sequences as any base, and unpaired nucleotides 91 and 92 of J2-5 indicate a 75% preference for purine followed by any base in 95% of sequences.

Fig. S5.

Pseudoknot classification of the Fpr preQ₁-III riboswitch expression platform model, and interhelical orientation of the crystal structure vs. a representative model with accompanying molecular dynamics trajectories. (A) The preQ₁-III riboswitch model folds as a 5′ HL_out-type pseudoknot (black) as described in Fig. S2A, but also exhibits a partially nested 3′ H-type pseudoknot (red) in which L1 = 0, allowing coaxial stacking of S1 and S2; the RBS at the 3′ terminus is highlighted (yellow). (B) Crystal structure of the preQ₁-III riboswitch from Fig. 1C. The angle between P2—J4-2—P4 (yellow cone) is calculated as 61° based on the phosphorus atoms at C90, A84, and G63. (C) Representative model of the preQ₁-III riboswitch after 2 μs of MD. The model is bound to preQ₁ and maintains the HL_out-type pseudoknot as observed in the crystal structure (Fig. 1 B and C); however, the model indicates P4 bending to an angle of 27° (yellow cone) to form P5. (D) Mass-weighted rmsd of all heavy atoms as a function of molecular dynamics simulation time for the model. Atoms for the P3 loop were excluded from the calculation. (E) Mass-weighted rmsd of atoms in P5 as function of simulation time based on the trajectories in D. P5 is defined by nucleotides 64–70 and 93–99 (Fig. 5B), and includes the RBS from position 97–101. (F) Intramolecular distance between atom C5 of U77 in helix P4 and atom O3′ of G101 in the RBS plotted as a function of simulation time. (G) Histogram distribution analysis showing the probability of encountering various U77–G101 distances, denoted (r), plotted for the atom pair in F. The distance of each peak is labeled. Analysis of trajectories was performed by Ptraj and Cpptraj from AmberTools (67).

Fig. 6.

smFRET analysis of the preQ₁-III riboswitch revealing the ligand dependence of dynamic RBS sequestration via P5 helix formation. (A) Schematic of the prism-based TIRF microscopy setup used to probe docking of helix P5 of the preQ₁-III riboswitch by smFRET. Position U77 is labeled with Cy5 (red star); the 3′ terminus is labeled with Dy547 (green star). The mid-FRET distance of ∼55 Å corresponds to the length of the flexible self-avoiding polymer extending from position A91 to G101 (i.e., ∼26.0 Å) (54) added to the C90–U77 distance from the computational model (Fig. 5). In this conformation, helix P5 is not formed or undocked. The high-FRET state is consistent with the ∼38 Å distance between U77 and G101, observed in the preponderance of riboswitch models (Fig. S5 F and G); in this conformation, helix P5 is formed as a double-stranded RNA duplex or docked. As a basis for comparison, the intramolecular U77–G101 distance in the crystal structure is 66 Å. (B) Representative smFRET traces for each condition in the presence of no preQ₁ (Top), 25 nM preQ₁ (Middle), and 1 μM preQ₁ (Bottom). Green, Dy547 intensity; red, Cy5 intensity; black, FRET efficiency; cyan, hidden Markov model fit. (C) FRET efficiency histograms of the preQ₁-III riboswitch under the ligand concentrations in B. N indicates the number of molecules included in each histogram. Percentages in blue correspond to the high-FRET population. The mean FRET values are shown as fractional numbers in red and blue. (D) TODPs depicting heat-map contours corresponding to static, on-diagonal molecules and dynamic, off-diagonal molecules; here the fraction of static and dynamic molecules is shown for each condition in B. The percentage of dynamic molecules, represented by off-diagonal contours (dashed boxes), is indicated in gold.

Fig. S6.

Representative smFRET trajectories and exponential fits to extract life times (τ) for the docked and undocked states in the absence or presence of preQ₁. (A) Trajectories in the absence of preQ₁ in 1× smFRET buffer containing 1 mM Mg²⁺. (B) Trajectories in 25 nM preQ₁ under conditions in A. (C) Trajectories in 1 μM preQ₁ under conditions in A. Here and elsewhere, arrows indicate single-step photobleaching events of a fluorophore that results in loss of measureable FRET, and is indicative of a single intramolecular FRET pair. (D–F) Exponential fits to extract dwell times under ligand conditions in A–C. The smFRET construct used here and elsewhere is described in Fig. S4C.

Fig. S7.

Representative smFRET histograms recorded in the absence or presence of ligand in buffer containing Mg²⁺. Combined data are presented in Fig. 6. (A–C) Three independent experiments conducted in 1× smFRET buffer with 1 mM Mg²⁺ and various amounts of ligand as indicated. Red and blue peaks correspond to the mid- and high-FRET states representing the respective undocked and docked P5 conformations of Fig. 6A. The percentage of the high-FRET population in each condition is shown in blue text. (D–F) Difference histograms between the minus (−)preQ₁ vs. 1 µM preQ₁ conditions for each experiment. These plots demonstrate the small, but reproducible, differences in the FRET histograms upon addition of preQ₁.

Fig. S8.

smFRET control experiments for an anti-P5 DNA oligonucleotide and nonbinding ligands. (A) FRET histogram in the absence of preQ₁ and the presence of 10 µM anti-P5 DNA oligonucleotide (Fig. S4C, Inset). (B) TODP for the experiment in A. (C) FRET histogram in the presence of 1 µM preQ₁ and 10 µM anti-P5 DNA oligonucleotide. (D) TODP for the experiment in C. (E) Comparison of the histograms for the experiments in A (black) and C (red), as well as in the absence of the anti-P5 DNA oligonucleotide (gray), demonstrating a clear decrease in FRET in the presence of DNA. (F) Representative trace for the experiments in A and C, showing a typical static ∼0.4 mid-FRET state. (G–J) smFRET histograms under conditions of no ligand (G), 1 µM IXP (H), 1 µM 2AP (I), and 1 µM preQ₁ (J). (K and L) Comparative and difference FRET histograms for the experiments in G–J, indicating the compaction and increase in high-FRET population specific to preQ₁. (M–P) TODPs for the corresponding experiments to the left in G–J, showing a significant increase in the fraction of dynamic molecules only in the presence of the cognate preQ₁ ligand. (Q) Exemplary trace showing both dynamic and static behavior in the absence of ligand. (R) smFRET trace showing switching from the static to the dynamic regime upon addition of 1 µM preQ₁ at ∼50 s as indicated. After preQ₁ addition at ∼50 s, the molecule was incubated in the dark for 2 min, as indicated by the x-axis break, before commencing imaging. All experiments were performed in the presence of 1 mM Mg²⁺.

Fig. S9.

smFRET histograms, trajectories, and TODPs for smFRET experiments recorded in the absence or presence of ligand in buffer without Mg²⁺. (A) smFRET histograms compiled from trajectories under various preQ₁ conditions. Histograms are shown for the mid-FRET population (red) and high-FRET population (blue); percentage values for the high-FRET state are in blue text. (B) Representative trajectories in the absence of preQ₁. (C) Representative trajectories in 25 nM preQ₁. (D) Representative TODPs for samples recorded under ligand conditions in B (Upper) and C (Lower).

Fig. 7.

Stereo diagrams depicting inclined A-minor bases of the preQ₁-II and preQ₁-III riboswitches mediating stacking interactions between the ligand and a nearby helix. (A) The preQ₁-III riboswitch crystal structure depicting nucleotides that compose the binding pocket and flank the ligand (rendered as transparent surfaces covering ball-and-stick models); helix P4 is drawn as a ribbon with nucleotides depicted as sticks. The pyrrole ring of preQ₁ forms an edge-to-face interaction with A-minor base A84 that is integral to formation of the binding pocket. On its opposite face, A84 stacks upon A52, forming a cross-strand interaction. A52 also stacks against neighboring purine A53, establishing a continuous base stack through helix P4 that culminates in the anti-RBS loop. In this manner, preQ₁ occupancy within the binding pocket influences the orientation of P4. The view is similar to Fig. 2B. (B) Representative computational model of the preQ₁-III riboswitch bound to preQ₁ as described in A. The inclined A-minor interactions of the crystal structure (Fig. 2B) are preserved in the model, and base stacking is still continuous from A84 to the anti-RBS, despite formation of the P5 helix that sequesters the RBS. (C) Analogous view of the preQ₁-II riboswitch (PDB ID code 2MIY) (12) illustrating preQ₁ packing against structurally homologous inclined A-minor bases A50 and A35. Like the preQ₁-III riboswitch, these bases form a continuous stacking interaction that influences the conformation of helix P4. Here, the pocket floor contains the first base of the RBS (yellow) located directly beneath preQ₁.