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 tRNAIle. 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.
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Atomic coordinates and structure factor amplitudes for the RNA have been deposited at the Protein Data Bank under accession code PDB 6UFM. Source data for Figs. 2d, 3e and 5d are available online. All other data generated or analyzed during this study are included in this published article or available upon request.
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We thank I. Botos for computational support, G. Piszczek and D. Wu for support in biophysical analyses, F. Dyda for suggesting the use of zero-dose correction, and S. Li, C. Bou-Nader, J.M. Gordon, C. Stathopoulos, and A. Ferré-D’Amaré for discussions. Data were collected at Southeast Regional Collaborative Access Team (SER-CAT) 22-ID beamline at the Advanced Photon Source of the Argonne National Laboratory, supported by the US Department of Energy under Contract No. W-31–109-Eng-38. This work was supported by the intramural research program of NIDDK, NIH, an NIH Deputy Director for Intramural Research (DDIR) Innovation Award and a DDIR Challenge Award to J.Z.
The authors declare no competing interests.
Peer review information Anke Sparmann was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.
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Extended Data Fig. 1 Binding of tRNAIle 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 tRNAIle. T-box1–29 (stem I) bands were poorly stained by GelRed and are indicated by the dashed box. b, EMSA titration showing the binding of tRNAIle by N. farcinica ileS T-box1–98. Lanes 1–5 (left to right): 0:1, 1:0, 1:0.5, 1:1 and 1:2 mixing ratios of T-box1–98:tRNAIle. 10 μM of T-box1–98 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 tRNAIle binding by the ileS T-box riboswitch length variants.
a−d, Representative ITC isotherms for N. farcinica WT RNAIle binding to (a) T-box1–98, (b) T-box1–89, (c) T-box1–77, and (d) T-box1–29. e, ITC isotherm of tRNAIle anticodon stem loop (ASL) binding to T-box1–98 (WT). f,g, ITC isotherms for N. farcinica WT RNAIle binding to (f) T-box1–99 (with an extra 3′-G), and (g) T-box1–89 A84C mutant. h,i, ITC isotherms of (h) tRNAIle G19C mutant and (i) M. smegmatis tRNAIle binding to T-box1–98 (WT). The construct names and the Kd values (mean ± s.d., n = 4 for a,e; n = 3 for b,d,f; n = 2 for c,g−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 tRNAIle.
a−d, Representative ITC isotherms for WT tRNAIle binding by T-box1–98 stem I mutants (a) A8G, (b) A16U, (c) A16G, (d) U17A, (d) C18G. e, T-box1–98 binding to tRNAIle tA35G mutant. f,g, ITC isotherms for WT tRNAIle binding by T-box1–98 stem I mutants (f) C18G and (g) C18U. h, T-box1–98 U17A mutant binding to tRNAIle tA35U mutant. i, ITC isotherms for WT tRNAIle binding by T-box1–98 stem I mutant A19U. j−s, tRNAIle binding to T-box1–98 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-box1–98 for reference. The construct names and the Kd values (mean ± s.d., n = 3 for a, f, h, n, o, q, s; n = 2 for b−e, g, i−m, p, r) are reported in the top and bottom panels of the ITC isotherms.
a, Composite simulated anneal-omit 2Fo – Fc 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 tRNAIle anticodon stem loop (ASL).
Superposition of the ileS stem I distal loop (orange) with tRNAIle ASL (green) showing structural similarities.
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 4LCK)9 (c) 23S rRNA from the Haloarcula marismortui large ribosomal subunit (PDB 4V9F)26, and (d) glutamine riboswitch (PDB 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 4R4V)25 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−tRNAIle structure. b−d, Similar ITAM motifs observed in the (b) TPP riboswitch (PDB 2GDI)27, (c) SAM-II riboswitch (PDB 2QWY)29 and (d) preQ1-I riboswitch (PDB 3FU2)30 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.
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 (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 a) in conjunction with the G530 latch (similar to A38 and A69 in a) to ensure decoding fidelity35,36,37. c, Similarly, in the adenovirus virus-associated RNA I (VA-I RNA), A36 stabilizes a 3-bp kissing 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 groove38.
a, Solvent-accessible surface colored according to area buried from light blue or white (no burial) to red (>125 Å2 per residue). b, Open-book view of the binding interface. c,d, Plots of solvent-accessible surface area buried per residue (Å2) 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 (blue) 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 tRNAIle and the cocrystal structure of the Oceanobacillus iheyensis glyQ T-box stem I bound to tRNAGly (PDB 4LCK)9. 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.
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Suddala, K.C., Zhang, J. High-affinity recognition of specific tRNAs by an mRNA anticodon-binding groove. Nat Struct Mol Biol 26, 1114–1122 (2019). https://doi.org/10.1038/s41594-019-0335-6
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