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Co-crystal structure of a T-box riboswitch stem I domain in complex with its cognate tRNA


In Gram-positive bacteria, T-box riboswitches regulate the expression of aminoacyl-tRNA synthetases and other proteins in response to fluctuating transfer RNA aminoacylation levels under various nutritional states1. T-boxes reside in the 5′-untranslated regions of the messenger RNAs they regulate, and consist of two conserved domains. Stem I contains the specifier trinucleotide that base pairs with the anticodon of cognate tRNA. 3′ to stem I is the antiterminator domain, which base pairs with the tRNA acceptor end and evaluates its aminoacylation state2. Despite high phylogenetic conservation and widespread occurrence in pathogens, the structural basis of tRNA recognition3,4 by this riboswitch remains ill defined. Here we demonstrate that the 100-nucleotide T-box stem I is necessary and sufficient for specific, high-affinity (dissociation constant (Kd) 150 nM) tRNA binding, and report the structure of Oceanobacillus iheyensis glyQ stem I in complex with its cognate tRNA at 3.2 Å resolution. Stem I recognizes the overall architecture of tRNA in addition to its anticodon, something accomplished by large ribonucleoproteins such as the ribosome, or proteins such as aminoacyl-tRNA synthetases5, but is unprecedented for a compact mRNA domain. The C-shaped stem I cradles the L-shaped tRNA, forming an extended (1,604 Å2) intermolecular interface. In addition to the specifier–anticodon interaction, two interdigitated T-loops near the apex of stem I stack on the tRNA elbow in a manner analogous to those of the J11/12–J12/11 motif6 of RNase P and the L1 stalk7 of the ribosomal E-site. Because these ribonucleoproteins and T-boxes are unrelated, this strategy to recognize a universal tRNA feature probably evolved convergently. Mutually induced fit of stem I and the tRNA exploiting the intrinsic flexibility of tRNA and its conserved post-transcriptional modifications results in high shape complementarity, which in addition to providing specificity and affinity, globally organizes the T-box to orchestrate tRNA-dependent transcription regulation.

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Figure 1: Overall structure of the T-box stem I in complex with tRNA.
Figure 2: Interactions between stem I and tRNA.
Figure 3: Induced fit of tRNA by stem I binding.
Figure 4: Stem I reorganization by tRNA binding.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

Atomic coordinates and structure factor amplitudes for the T-box stem I in complex with tRNAGly and YbxF have been deposited at the Protein Data Bank under accession code 4LCK.


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We thank the staff at beamlines 24-ID-C and 24-ID-E of APS and 5.0.1 and 5.0.2 of the ALS for crystallographic data collection support, G. Piszczek for ITC support, R. Levine and D.-Y. Lee for help with mass spectrometry, K. Perry and K. R. Rajashankar for assistance with initial crystallographic phasing, and N. Baird, T. Hamma, C. Jones, M. Lau, J. Posakony, A. Roll-Mecak, O. Uhlenbeck and K. Warner for discussions. This work is partly based on research conducted at the APS on the Northeastern Collaborative Access Team (NE-CAT) beamlines, and at the ALS on the Berkeley Center for Structural Biology beamlines, which are both supported by the National Institute for General Medical Sciences, National Institutes of Health (NIH). Use of ALS and APS was supported by the US Department of Energy. This work was supported in part by the intramural program of the National Heart, Lung, and Blood Institute, NIH.

Author information

Authors and Affiliations



J.Z. and A.R.F.-D. designed experiments, J.Z. carried out all biochemistry and ITC experiments, J.Z. prepared co-crystals, J.Z. and A.R.F.-D. collected diffraction data, solved and refined the crystal structures, and wrote the manuscript.

Corresponding author

Correspondence to Adrian R. Ferré-D’Amaré.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Sequence of the B. subtilis glyQS T-box.

Unlike some other T-boxes, tRNAGly-responsive T-boxes (such as the B. subtilis glyQS T-box and the closely related O. iheyensis glyQ T-box) do not contain stems II and II A/B in the linker region. Conservation across known T-boxes is indicated in orange and yellow circles (from ref. 11). Base pairing between the specifier sequence in stem I and the antiterminator domain of the T-box and the anticodon and acceptor end, respectively, of tRNAGly is indicated. The functionally important K-turn8 is boxed. The sequences of tRNAGly of B. subtilis and O. iheyensis are identical.

Extended Data Figure 2 Representative isothermal calorimetric titrations.

af, Full-length tRNAGly (ae) and an isolated tRNAGly ASL (f) were titrated into various truncated B. subtilis glyQS T-box stem I constructs (Methods). The specifier trinucleotide GGC is in bold. g, Superposition of the experimental data and fits for the experiments shown in Fig. 1a–c, colour-coded to correspond to schematic RNA construct depictions. h, ITC analysis of binding by the engineered tRNAGly construct used for co-crystallization. Results of all fits (Methods) are in Extended Data Table 1.

Extended Data Figure 3 Intermolecular interface of the stem-I–tRNA complex and phylogenetic conservation.

a, The residue 29–30 bulge allows the O. iheyensis stem I to bend and track the tRNA backbone. b, Structure21 of the equivalent region of the Geobacillus kaustophilus T-box, which folds as a C-loop52. c, Solvent-accessible surface coloured according to area buried from blue or white (no burial) to red (>75 Å2 per residue). d, Open-book view. e, Surfaces coloured by phylogenetic conservation of stem I residues (see Extended Data Fig. 1). Conservation of tRNA is not indicated. f, Open-book view. For comparison, RNase P binding to pre-tRNA buries6 2,959 Å2.

Extended Data Figure 4 T-box can accommodate bulky post-transcriptional modifications at tRNA position 37.

A86 of the T-box stem I is unstacked from the duplex formed by pairing of the tRNA anticodon and the T-box specifier by its participation in the sheared A•A pair at the base of the loop E motif. This results in a pocket (dotted line) that could accommodate the large, modified nucleobases (for example, the three-ring heterocycle wybutine in yeast tRNAPhe) often present at position 37 of tRNAs (Fig. 2a). Note the single hydrogen bond between tU32 and tA38.

Extended Data Figure 5 Structural organization of the interdigitated T-loop motifs of the T-box stem I.

a, Six base-paired layers form the stem I distal domain core (view as in Fig. 2b). Dashed black lines denote base–base hydrogen bonding. Dashed yellow lines denote Sr2+-ion–water (green and red spheres, respectively) interactions. Leontis–Westhof symbols30 depict non-canonical pairing. b, Orthogonal view. c, View from the tRNA elbow. di, Detail of layers 1–6. The position of each nucleotide within the pentaloop (1–5) or outside of it (negative and positive denote 5′ and 3′ directions, respectively) is in parentheses (Supplementary Discussion).

Extended Data Figure 6 Comparison of the interdigitated T-loops of RNase P, the L1 stalk and the T-box.

a, b, RNase P (a) and the L1 stalk (b) use equivalent surfaces formed by two purines—namely, nucleotide −2 of T-loop 1 and nucleotide 3 of T-loop 2 (asterisk)—to stack on the elbows of pre-tRNA and the E-site tRNA, respectively. c, By contrast, the T-box uses the opposite surface, using a triple (Fig. 2c) formed by nucleotide 3 of T-loop 1 (dagger symbol), nucleotide −2 from T-loop 2, and nucleotide +1 from T-loop 2 to stack on the tRNA elbow.

Extended Data Figure 7 Displacement of the tRNA elbow induced by T-box binding.

Although the anticodon stem of the free tRNAPhe (orange) superimposes well on that of the T-box-bound tRNAGly (T-loop and acceptor stem in violet; D-loop and ASL in pale green; Fig. 3b, c), the elbow (tG19–tC56) clashes with the stem I distal domain. T-box binding induces bending about the t26•t44 hinge (Fig. 3c) that displaces the tRNA elbow and acceptor stem away from the distal region of stem I by 11 Å (arrows), allowing the tRNA elbow to associate with the T-box. a, Side view. b, Top view.

Extended Data Figure 8 Representative electron density maps.

Portions of electron density maps corresponding to the interdigitated stem I T-loops and the tRNA elbow (oriented like Fig. 2b). a, Unbiased, 3.2-Å-resolution density-modified SAD electron density (1.0 s.d. above mean peak height) shown as grey mesh superimposed on the refined model. b, Electron density resulting from combination of SAD and molecular replacement (using a search model consisting of two copies each of tRNAPhe and a K-turn in complex with YbxF; Methods) phases followed by density modification (1.5 s.d.) c, Composite simulated anneal-omit 2|Fo| − |Fc| electron density calculated using the final model (2.0 s.d.).

Extended Data Table 1 Thermodynamic parameters from ITC analysis
Extended Data Table 2 Data collection and refinement statistics

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Zhang, J., Ferré-D’Amaré, A. Co-crystal structure of a T-box riboswitch stem I domain in complex with its cognate tRNA. Nature 500, 363–366 (2013).

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