High-affinity recognition of specific tRNAs by an mRNA anticodon-binding groove

Abstract

T-box riboswitches are modular bacterial noncoding RNAs that sense and regulate amino acid availability through direct interactions with tRNAs. Between the 5' anticodon-binding stem I domain and the 3' amino acid sensing domains of most T-boxes lies the stem II domain of unknown structure and function. Here, we report a 2.8-Å cocrystal structure of the Nocardia farcinica ileS T-box in complex with its cognate tRNA^Ile. The structure reveals a perpendicularly arranged ultrashort stem I containing a K-turn and an elongated stem II bearing an S-turn. Both stems rest against a compact pseudoknot, dock via an extended ribose zipper and jointly create a binding groove specific to the anticodon of its cognate tRNA. Contrary to proposed distal contacts to the tRNA elbow region, stem II locally reinforces the codon-anticodon interactions between stem I and tRNA, achieving low-nanomolar affinity. This study illustrates how mRNA junctions can create specific binding sites for interacting RNAs of prescribed sequence and structure.

Extended Data Fig. 1. Binding of tRNA^Ile by the N. farcinica ileS T-box riboswitch and effect of agarose on crystal morphology

a, Electrophoretic mobility shift assay (EMSA) using 10% non-denaturing polyacrylamide gel showing binding of T-box length variants to tRNA^Ile. T-box_1-29 (Stem I) bands were poorly stained by GelRed and are indicated by the dashed box. b, EMSA titration showing the binding of tRNA^Ile by N. farcinica ileS T-box_1-98. Lanes 1-5 (left-right): 0:1, 1:0, 1:0.5, 1:1 and 1:2 mixing ratios of T-box_1-98:tRNA^Ile. 10 μM of T-box_1-98 was used for the assay. c, Crystals grown in the absence of agarose appear as thin plates with parasitic growth from the corners. Crystals were grown at 21 °C by vapor diffusion by mixing an equal volume of reservoir solution containing 20% PEG 3350, 0.1 M Bis-Tris (HCl), pH 6.5 and 200 mM Li₂SO₄. d, Crystals grown under similar conditions as in c), but in the presence of ~0.1 % low-melting-point agarose. In the presence of agarose, crystals grew as thick rectangular prisms with sharp edges, and exhibited improved diffraction properties. Scale bar, ~100 μm.

Extended Data Fig. 2. Isothermal titration calorimetry (ITC) analysis of tRNA^Ile binding by the ileS T-box riboswitch length variants

a-d, Representative ITC isotherms for N. farcinica wt tRNA^Ile binding to (a) T-box_1-98, (b) T-box_1-89, (c) T-box_1-77, and (d) T-box_1-29. e, ITC isotherm of tRNA^Ile anticodon stem loop (ASL) binding to T-box_1-98 (wt). f-g, ITC isotherms for N. farcinica wt tRNA^Ile binding to (f) T-box_1-99 (with an extra 3'-G), and (g) T-box_1-89 A84C mutant. h-i, ITC isotherms of h) tRNA^Ile G19C mutant and i) M. smegmatis tRNA^Ile binding to T-box_1-98 (wt). The construct names and the K_d values (mean ± s.d., n = 4 for a, e; n = 3 for b, d, f; n = 2 for c, g, h, i) are reported in the top and bottom panels of the ITC isotherms.

Extended Data Fig. 3. ITC analysis of the effects of single nucleobase substitutions on the ileS T-box and tRNA^Ile

a-d, Representative ITC isotherms for wt tRNA^Ile binding by T-box_1-98 Stem I mutants (a) A8G, (b) A16U, (c) A16G, (d) U17A, (d) C18G. e, T-box_1-98 binding to tRNA^Ile tA35G mutant. f-g, ITC isotherms for wt tRNA^Ile binding by T-box_1-98 Stem I mutants (f) C18G and (g) C18U. h, T-box_1-98 U17A mutant binding to tRNA^Ile tA35U mutant. i) ITC isotherms for wt tRNA^Ile binding by T-box_1-98 Stem I mutant A19U. j-s, tRNA^Ile binding to T-box_1-98 Stem II mutants (j) G37U, (k) A38U, (l) A39U, (m) A66U, (n) G67U, (o) A69U, (p) G70U, (q) A71U, (r) G85C and (s) U90C. (t) Sequence and secondary structure of T-box_1-98 for reference. The construct names and the K_d values (mean ± s.d., n = 3 for a, f, h, n, o, q, s; n = 2 for b, c, d, e, g, i, j, k, l, m, p, r) are reported in the top and bottom panels of the ITC isotherms.

Extended Data Fig. 4. Representative X-ray crystallographic electron density maps

a, Composite simulated anneal-omit 2|F_o|-|F_c| electron density calculated using the final model (1.0 s.d.) superimposed with the final refined model. b-c, Portions of the map showing the tRNA anticodon - T-box specifier duplex region (b) and the characteristic “S”-like backbone bend of the Stem II S-turn (c).

Extended Data Fig. 5. Similarity between ileS T-box Stem I distal loop and tRNA^Ile anticodon stem-loop (ASL)

Superposition of the ileS Stem I distal loop (orange) with tRNA^Ile ASL (green) showing structural similarities.

Extended Data Fig. 6. Comparison of S-turn structures and S-turn-mediated RNA-RNA interactions

a-d, The S-turn (or loop E) motif from a) N. farcinica ileS T-box riboswitch structure, b) glyQ T-box Stem I from Oceanobacillus iheyensis (PDB ID: 4lck) c) 23S rRNA from the Haloarcula marismortui large ribosomal subunit (PDB ID: 4v9f), and d) glutamine riboswitch (PDB ID: 5ddp). e, Overlay of the S-turn structures in a-d. f, Stabilization of codon-anticodon helix by the Stem II S-turn in ileS T-box riboswitch. g, Interactions between an S-turn and a neighboring helix in 23S rRNA from H. marismortui. h, Interactions between an atypical S-turn motif in helix 6 of the Varkud satellite (VS) ribozyme (PDB ID: 4r4v) and A652 of helix 2. Red and green dashes represent H-bonds involving nucleotides in the backbone-turn-containing strand and the opposite strand, respectively.

Extended Data Fig. 7. Examples of the recurring inclined tandem A-minor (ITAM) motifs in RNA structures

a, The inclined tandem A-minor (ITAM) motif observed in the ileS T-box-tRNA^Ile structure. b-d, Similar ITAM motifs observed in the b) TPP riboswitch (PDB ID: 2gdi), c) SAM-II riboswitch (PDB ID: 2qwy) and d) preQ₁-I riboswitch (PDB ID: 3fu2) crystal structures. For comparison, the motifs shown in b-d are formed by adjacent adenosines on the same strand, whereas the ITAM motif in a) uses cross-strand stacked adenosines from opposite strands. This configuration allows both the ribose and the nucleobases from both adenosines to contact the C-G base pair. The inclined adenosine motif in c) is unusual in that three adjacent, stacked adenosines span the width of a single C-G base pair and form multiple contacts with it. The motif in d) (also termed the A-amino kissing motif) involves the Watson-Crick edges of the adenosines and does not involve interactions with the ribose. These interactions are formed by L3 loop adenosines of the preQ₁-I riboswitch and are common in many other H-type pseudoknots.

Extended Data Fig. 8. Three different strategies to stabilize the codon-anticodon duplex.

a, The minor groove side of the N. farcinica ileS T-box Stem II S-turn interacts extensively with the minor groove of the anticodon-specifier duplex. In particular, the A38-A69 inclined tandem A-minor (ITAM) motif stabilizes the C18-tG34 base pair. G70 and A71 stack with the top layer of the S-turn (G37•A69) and stabilize the U17-tA35 and A16-tU36 pairs, respectively (see also Fig. 3d). b, The conserved A1492 and A1493 of ribosomal RNA in the ribosome A-site interact with the codon-anticodon duplex via tandem, stacked A-minor interactions (similar to G70 and A71 in panel a) in conjunction with the G530 latch (similar to A38 and A69 in panel a) to ensure decoding fidelity^-. c, Similarly, in the adenovirus virus-associated RNA I (VA-I RNA), A36 stabilizes a 3-bp pseudoknot formed between residues 103-105 in Loop 8 (5'-ACC-3', anticodon-like) and residues 124-126 (5'-GGU-3', codon-like) of Loop 10, through extensive A-minor interactions between A36 and the minor groove.

Extended Data Fig. 9. Intermolecular interface of the N. farcinica T-box -tRNA^Ile complex

a, Solvent-accessible surface colored according to area buried from light blue or white (no burial) to red (>125 Ų per residue). b, Open-book view of the binding interface. c and d, Plots of solvent-accessible surface area buried per residue (Ų) on the tRNA (c) and T-box (d).

Extended Data Fig. 10. Structural model of a canonical, feature-complete T-box riboswitch Stem I and II domains in complex with its cognate tRNA

Model of a canonical, feature-complete T-box riboswitch, such as the originally described B. subtilis tyrS T-box containing a long Stem I (salmon), Stem II (marine) and Stem IIA/B pseudoknot (cyan) elements bound to its cognate tRNA (green). The composite structural model is derived by combining the cocrystal structures of the ileS T-box riboswitch bound to tRNA^Ile and the cocrystal structure of the Oceanobacillus iheyensis glyQ T-box Stem I bound to tRNA^Gly (PDB: 4LCK). tRNAs from both structures were superimposed as shown in Fig. 6b. The distal loop of the ultrashort Stem I from the ileS T-box was then replaced with the long, canonical Stem I from the glyQ T-box.

Figure 1.. Overall structure of the N. farcinica ileS T-box in complex with tRNA^Ile.

a) Schematic of the proposed mechanism of translation regulation by the N. farcinica ileS T-box riboswitch. b-d) ITC analysis of tRNA^Ile binding by b) T-box_1-98 (residues 1-98) consisting of Stems I, II and IIA/B, c) T-box_1-77 containing Stems I and II and d) T-box_1-29 Stem I only. K_d values are mean ± s.d. (n = 4 independent experiments for T-box_1-98, n = 2 for T-box_1-77, n = 3 for T-box_1-29). e) Sequence and secondary structure of the cocrystallized ileS T-box_1-98 and tRNA^Ile, with non-canonical base-pairing interactions indicated using the Leontis-Westhof symbols. The 77-nt long tRNA^Ile is numbered (with a ‘t’ prefix) according to standard convention, with t76 as the last nucleotide. f) Structure of the ileS T-box-tRNA^Ile complex colored as in e). Sequence changes to facilitate crystallization are shown in gray.

Figure 2.. Recognition of tRNA anticodon by the ileS T-box riboswitch.

a) Specifier-anticodon interactions in the ileS T-box-tRNA^Ile complex. Dashed lines indicate hydrogen bonds. b) Specifier-anticodon interactions in the glyQ T-box riboswitch (PDB ID: 4LCK). c) Superposition of the Specifier-anticodon interactions in the ileS (colored as in a) and glyQ (gray) T-box riboswitches. d) Mutagenesis and ITC analysis of selected Stem I mutations in T-box_1-98 (orange columns) compared to the wt T-box_1-98 (gray column). The fold change in the K_d value, compared to wt T-box_1-98 is shown on top of each column. Data shown are mean ± s.d. (n = 4 independent experiments for WT, n = 3 for A8G, tA35G, C18G, U17A:tA35U, n = 2 for A16U, A16G, U17A, C18U, A19U). Source data for panel d are available online.

Figure 3.. Stabilization of specifier-anticodon interaction by the S-turn motif.

a) The S-turn motif of Stem II showing the characteristic ‘S’ shape of the backbone bend near the bulged G67. Stem I and Stem II dock via extensive ribose-zipper interactions, shown as red dashed lines. b) Hydrogen-bonding patterns of each of the three layers of the S-turn motif. The curved lines indicate stacking between the layers and the spheres indicate backbone phosphate atoms. c) The S-turn and neighboring residues make multiple hydrogen bonds to the specifier (red dashes) and the anticodon (green dashes) to stabilize the 3-bp anticodon-specifier helix. d) Interactions from the S-turn and adjacent residues to each layer of the 3-bp specifier-anticodon duplex. e) Mutagenesis and ITC analysis of selected Stem II mutations in T-box_1-98 (blue columns) compared to the wt T-box_1-98 (gray column). The fold change in the K_d value compared to wt T-box_1-98 is shown on top of each column. Data shown are mean ± s.d. (n = 4 independent experiments for WT, n = 3 for G67U, A69U, A71U, n = 2 for G37U, A38U, A39U, G70U, A66U). Source data for panel e are available online.

Figure 4.. tRNA recognition by the T-box anticodon-binding groove.

a) Surface representation of the T-box riboswitch showing the anticodon-binding groove (red dashed oval) formed between Stem I and Stem II. tRNA is omitted for clarity b) same view as a) with tRNA bound. See also Extended Data Fig. 9 for detailed surface burial analysis.

Figure 5.. Stem IIA/B forms a compact pseudoknot to position Stems I and II.

a) Structure of the Stem IIA/B pseudoknot. The base-pairing interactions within Stems IIA and IIB and key tertiary interactions with loops L1 and L3 are shown. A single hydrogen bond between the 2'-OH of U90 in L3 and the U30 OP2 (red sphere) is indicated. A pseudoknot-bound sulfate ion is shown. b) Tertiary interactions of loop L1 residues (light blue) with Stem IIB (cyan). c) L3 (teal) interactions with Stem IIA (light cyan). d) ITC analysis of T-box length variants (gray), pseudoknot mutants (cyan), tRNA tG19C mutant and ASL (green). The fold change in K_d value compared to wt T-box_1-98 is shown on top of each column. Data shown are mean ± s.d. (n = 4 independent experiments for ASL, 1-98, n = 3 for 1-99, 1-89, U90C, n = 2 for 1-77, 1-89 UGCG, G85C, tG19C). Source data for panel d are available online.

Figure 6.. Structural comparison with T-boxes containing canonical long Stem I

a) Cocrystal structure of N. farcinica ileS T-box_1-98 bound to tRNA^Ile. The K-turn motif in Stem I is in light blue. b) Superposition of cocrystal structures of the ileS T-box_1-98-tRNA^Ile in a) and glyQ Stem I-tRNA^Gly complexes in c). The structures are aligned using tRNAs from both models. The superposition shows that the K-turn motifs in both the structures occupy similar locations and the long Stem I of glyQ T-box is compatible with the Stem II and Stem IIA/B pseudoknot elements of ileS T-box, providing a plausible model for canonical T-boxes containing all these elements c) Cocrystal structure of the O. iheyensis glyQ T-box Stem I bound to tRNA^Gly (PDB ID: 4LCK). K-turn in the proximal region of Stem I is shown in dark brown.