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Structural basis of amino acid surveillance by higher-order tRNA-mRNA interactions

Abstract

Amino acid availability in Gram-positive bacteria is monitored by T-box riboswitches. T-boxes directly bind tRNAs, assess their aminoacylation state, and regulate the transcription or translation of downstream genes to maintain nutritional homeostasis. Here, we report cocrystal and cryo-EM structures of Geobacillus kaustophilus and Bacillus subtilis T-box–tRNA complexes, detailing their multivalent, exquisitely selective interactions. The T-box forms a U-shaped molecular vise that clamps the tRNA, captures its 3′ end using an elaborate ‘discriminator’ structure, and interrogates its aminoacylation state using a steric filter fashioned from a wobble base pair. In the absence of aminoacylation, T-boxes clutch tRNAs and form a continuously stacked central spine, permitting transcriptional readthrough or translation initiation. A modeled aminoacyl disrupts tRNA-T-box stacking, severing the central spine and blocking gene expression. Our data establish a universal mechanism of amino acid sensing on tRNAs and gene regulation by T-box riboswitches and exemplify how higher-order RNA-RNA interactions achieve multivalency and specificity.

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Fig. 1: Overall structure of the T-box discriminator–tRNA complex.
Fig. 2: The A-minor latch and pseudohelix stabilize tRNA-T-box interactions.
Fig. 3: Structural basis of tRNA aminoacylation sensing by the T-box discriminator.
Fig. 4: Docking of stem III with helix A2 reinforces and rigidifies complex structure.
Fig. 5: 4.9-Å resolution cryo-EM map and structure of a full-length B. subtilis glyQS T-box-tRNAGly complex.
Fig. 6: The T-box central spine and structural comparisons.
Fig. 7: Mechanistic model of a cotranscriptionally acting T-box riboswitch.

Data availability

Atomic coordinates and structure factor amplitudes for the T-box discriminator in complex with tRNAGly have been deposited at the Protein Data Bank (PDB) under accession code PDB 6PMO. Cryo-EM structure of the full-length T-box–tRNA complex and map have been deposited to Electron Microscopy Data Bank under EMD-20416 and PDB 6POM. All other data are available upon reasonable request.

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Acknowledgements

We thank I. Botos for computational support, J.R. Hogg, S. Ranganathan, G. Piszczek, D. Wu, J.C. Lee and M. Watson for support in fluorescence analyses; Y. He and N. Tjandra for fermentation support; R. Levine and D.-Y. Lee for MS support; W. Zhang and J. W. Szostak for a gift of Ir(III) Hexammine; M. Apostolidi for β-galactosidase assay protocols; CAS-Shanghai Science Research Center High-End User Project for preliminary cryo-EM data collection; and S.K. Buchanan, A. Ferré-D’Amaré, N. Baird, M. Lau, K. Suddala, C. Bou Nader, and J. M. Gordon for insightful discussions. This work was supported by the intramural research programs of NIDDK and NCI, an NIH DDIR Innovation Award to J.Z. and Y-X.W., NIH U54GM103297 and U54AI150470 (Center for HIV RNA Studies, CRNA), P41GM103832, R01GM079429 and S10OD021600 to W.C., and Fondatión Santé E515 and project “INSPIRED” (MIS/5002550) implemented under the Action “Reinforcement of the Research and Innovation Infrastructure”, funded by the Operational Programme “Competitiveness, Entrepreneurship and Innovation” (NSRF 2014-2020) and co-financed by Greece and the European Union (European Regional Development Fund), and a Fulbright Scholarship to C.S. V. S. is a supported by an IKY-Siemens 2017 Excellence Postdoctoral Fellowship.

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Authors

Contributions

S.L. and J.Z. designed experiments. S.L. prepared RNA samples, cocrystals, and samples for cryo-EM and SAXS, with the help of F.E.H, and performed in vitro assays. S.L. and J.Z. collected X-ray diffraction data, solved and refined the crystal structure, and analyzed SAXS data. Z.S. collected cryo-EM data, Z.S., G.D.P., K.Z., M.C., S.J.L. and W.C. performed cryo-EM data processing and modeling. J.L. performed phylogenetic analyses. V.S., N.G. and C.S. carried out in vivo experiments. L.F. and Y.-X.W. collected and processed SAXS data. All authors contributed to the preparation of the manuscript.

Corresponding authors

Correspondence to Wah Chiu or Jinwei Zhang.

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Authors declare no competing interests.

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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

Extended Data Fig. 1 Secondary structures and conservation analyses of glycyl T-box riboswitches.

a,b, Secondary structures of G. kaustophilus glyQ and B. subtilis glyQS T-box riboswitch. Glycine-specific T-boxes lack the stem II and stem IIA/B pseudoknot structures. Conserved nucleotides are highlighted, based on previous reports supplemented by new phylogenetic analysis (Fig. 1; Methods). Previous sequence annotations of the G. kaustophilus glyQ T-box had omitted a 5′ ssRNA leader that precedes stem I in all validated T-boxes, which is now restored. The probable transcription start site, 17 nts upstream of stem I, was identified using prokaryotic promoter prediction algorithms. Nucleotide numbering is thus offset by +17 relative to previous reports. c, Sequence conservation of the T-box discriminator region based on G. kaustophilus glyQ T-box. The split patterns show that the intercalating G130 is at the center of a 5′-AR(U/A)-3′ motif (middle). This motif is shifted 1 nt to the left when there is no G in position 130 (bottom). In this case, a moderately conserved G is predominant in position 129, and two pyrimidines are present in positions 130 and 131. Assuming that G129 is the intercalating nucleotide equivalent to G130 in the middle panel, one of these pyrimidines (nt 130 or 131) may adopt an extrahelical conformation to account for the motif shift.

Extended Data Fig. 2 Mutational analysis of T-box discriminator-tRNA interactions.

a, Secondary structures of wild-type, mutant, and truncated T-box discriminators. Deletions are indicated by red boxes. b, Electrophoretic mobility shift assay (EMSA) analysis of the constructs shown in a, showing the requirement of stem III and flanking purines for tRNA binding. The antiterminator (discriminator without stem III and its flanking purines; Δ3 mutant) is prone to dimerization. c, tRNA variants used that carry various 3′ chemical modifications. Only the terminal tA76 is shown. d, EMSA analysis of constructs in c, showing that binding is selective for uncharged tRNA. e, Quantitation of d and comparison with previously reported in vitro transcription readthrough data of the same tRNA variants. The values and error bars represent mean and s.d., n = 3 biologically independent samples.

Extended Data Fig. 3 Representative X-ray crystallographic electron density maps.

a, Composite simulated anneal-omit 2FoFc electron density calculated using the final model (1.0 s.d.) superimposed with the final refined model. bd, Portions of the map showing tRNA-T-box discriminator coaxial stacking (b), encapsulation of tRNA 3′-end by the discriminator (c), and long-range interactions between stem III 5′ flanking purines and the T-box bulge (d). Note the density fusion as a result of nucleobase-ribose packing interactions between A129 and G161.

Extended Data Fig. 4 In vitro transcription termination-readthrough assay and in vivo β-gal assay.

a, Representative raw data of in vitro transcription termination-readthrough assay using wild-type B. subtilis T-box riboswitch. The rates of fluorescence increase between 34 and 180 min (segments with trendlines) report the production of readthrough transcripts. b, Quantitation of data in a. Rates of fluorescence increase (slopes) were subsequently normalized to that of the reference in the presence of NTP but absence of tRNA (green data points) and reported in Figs. 2h, 3e and 4f. c, Validation of fluorescence-based readthrough assay in a and b with subsequent, conventional gel-based analysis of the same samples. Addition of the uncharged tRNA led to significantly increased transcription readthrough. d, Scheme of in vivo gene expression assay using the G. kaustophilus glyQ T-box riboswitch transcriptionally fused with lacZ. e, Relative β-gal activity of wild-type and mutant T-boxes under glycine-replete and glycine-starvation conditions, normalized to wild-type T-box-containing strain grown in minimal media supplemented with glycine. The values and error bars represent mean and s.d., n = 3 biologically independent samples.

Extended Data Fig. 5 Comparison of the T-box tandem A-minor latch with the A1492-A1493-G530 latch in the ribosome A site.

a, The A128-A129 latch reinforces the functionally important tRNA (green)-helix A1 (blue) stacking interface. A128 and A129 form a continuous adenosine stack. b and c, The stacked A128 and A129 engage extensive hydrogen bonds with the minor groove, reinforce tRNA-T-box base-pairing and stacking, and “staple” the two RNAs together. d, In the ribosome A site, A1492 and A1493 similarly reinforce the intermolecular codon-anticodon duplex via tandem, stacked A-minor interactions in conjunction with G530. e and f, Hydrogen-bond patterns in the ribosome A site resemble those in the T-box (b and c).

Extended Data Fig. 6 Intermolecular interface of the T-box discriminator-tRNA complex.

a, Solvent-accessible surface colored according to area buried from light blue or white (no burial) to red (>25 Å2 per atom). b, Open-book view of the binding interface. The lower inset shows the extensive burial of tRNA tA76, particularly its Watson−Crick edge (N6-N1-C2) and both 2′-OH and 3′-OH. c,d, Solvent-accessible surface area buried per residue for tRNA (c) and discriminator (d).

Extended Data Fig. 7 Effect of the G•U wobble pair on tRNA aminoacylation sensing and comparison of steric sieves in the T-box and ribosome-RelA complex.

a, A modeled tRNA 3′-glycyl moiety strongly clashes with the U185 nucleobase of the G•U wobble pair. b, Modeled Watson–Crick pair (C185, white) still clashes with the tRNA 3′-glycyl moiety, albeit to a lesser extent than the G•U wobble pair (a). c,d, Comparison of the steric sieves in the T-box (c) snd RelA–ribosome complex (d). Solid green lines indicate inter-atomic distances in Å. The RelA–ribosome complex structure is based on PDB 5IQR.

Extended Data Fig. 8 A conserved G•U wobble pair enhances stacking with its neighboring base pair both in the T-box discriminator and in the tRNA T-loop.

a, Through local helix underwinding, helix A2 terminal G•U wobble pair produces exceptionally large nucleobase overlap areas and enhances stacking with the penultimate C-G pair. b, Reduced nucleobase overlap areas between a modeled G-C pair and the penultimate C-G pair. c, The G•U wobble pair is reminiscent of the conserved G49•U65 wobble pair found in the tRNAGly T-loop in the same complex. d, For comparison, the penultimate C-G pair stacks with its neighboring G-C pair with less than half of the total overlap area (5.5 Å2 versus 13.9 Å2). Overlap areas (in Å2) between stacked nucleobases were calculated with 3DNA.

Extended Data Fig. 9 Cryo-EM single particle analysis (SPA) workflow of full-length B. subtilis T-box−tRNA complex.

a, 3D classification yielded two major classes (black boxes) that were combined for auto refinement. b, Final reconstruction. c, FSC curve showing 4.9-Å resolution at 0.143 cut-off. d, Euler angle distribution of the final reconstruction.

Extended Data Fig. 10 Relion 3D classification and local resolution of the full-length B. subtilis T-box−tRNA complex.

3D classification of the complex converged to three maps. Superposition of the tRNA density in three maps revealed motions of the T-box relative to tRNA as indicated by the arrows. This flexibility of the T-box RNA is the major limitation that prevented cryo-EM reconstruction from achieving higher resolutions. Resmap analysis shows that the tRNA was better resolved at ~4-Å resolution (upper right).

Supplementary information

Reporting Summary

Supplementary Video 1

360° view of the 2.66-Å resolution cocrystal structure of the T-box discriminator in complex with an uncharged tRNA.

Supplementary Video 2

Animation of the 4.9-Å resolution Cryo-EM structure of the full-length T-box riboswitch-tRNA complex. Multiple zoom-in views display the match between the model and the densities including the three separate tRNA-mRNA interfaces and the better resolved regions of the map in the tRNA.

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Li, S., Su, Z., Lehmann, J. et al. Structural basis of amino acid surveillance by higher-order tRNA-mRNA interactions. Nat Struct Mol Biol 26, 1094–1105 (2019). https://doi.org/10.1038/s41594-019-0326-7

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