Letter | Published:

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

Nature volume 500, pages 363366 (15 August 2013) | Download Citation

Abstract

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.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Accessions

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.

References

  1. 1.

    , & The T box mechanism: tRNA as a regulatory molecule. FEBS Lett. 584, 318–324 (2010)

  2. 2.

    & tRNA as a positive regulator of transcription antitermination in B. subtilis. Cell 74, 475–482 (1993)

  3. 3.

    , & Structural transitions induced by the interaction between tRNAGly and the Bacillus subtilis glyQS T box leader RNA. J. Mol. Biol. 349, 273–287 (2005)

  4. 4.

    , & tRNA requirements for glyQS antitermination: a new twist on tRNA. RNA 9, 1148–1156 (2003)

  5. 5.

    & Structural diversity and protein engineering of the aminoacyl-tRNA synthetases. Biochemistry 51, 8705–8729 (2012)

  6. 6.

    et al. Structure of a bacterial ribonuclease P holoenzyme in complex with tRNA. Nature 468, 784–789 (2010)

  7. 7.

    , , & Crystal structure of a 70S ribosome–tRNA complex reveals functional interactions and rearrangements. Cell 126, 1065–1077 (2006)

  8. 8.

    , , & The GA motif: an RNA element common to bacterial antitermination systems, rRNA, and eukaryotic RNAs. RNA 7, 1165–1172 (2001)

  9. 9.

    RNA Elements Required for T Box Antitermination. PhD thesis, Ohio State Univ. (2002)

  10. 10.

    , & A universal RNA structural motif docking the elbow of tRNA in the ribosome, RNase P and T-box leaders. Nucleic Acids Res. 41, 5494–5502 (2013)

  11. 11.

    , , , & Biochemical features and functional implications of the RNA-based T-Box regulatory mechanism. Microbiol. Mol. Biol. Rev. 73, 36–61 (2009)

  12. 12.

    , , & The kink-turn: a new RNA secondary structure motif. EMBO J. 20, 4214–4221 (2001)

  13. 13.

    , , & YbxF and YlxQ are bacterial homologs of L7Ae and bind K-turns but not K-loops. RNA 18, 759–770 (2012)

  14. 14.

    & The 5S rRNA loop E: chemical probing and phylogenetic data versus crystal structure. RNA 4, 1134–1153 (1998)

  15. 15.

    , & Structure and function of the T-loop structural motif in noncoding RNAs. Wiley Interdiscip Rev. RNA (23 June 2013)

  16. 16.

    , & Riboswitches: small-molecule recognition by gene regulatory RNAs. Curr. Opin. Struct. Biol. 17, 273–279 (2007)

  17. 17.

    et al. Structure of the 70S ribosome complexed with mRNA and tRNA. Science 313, 1935–1942 (2006)

  18. 18.

    et al. An antifungal agent inhibits an aminoacyl-tRNA synthetase by trapping tRNA in the editing eite. Science 316, 1759–1761 (2007)

  19. 19.

    et al. Structural basis for double-sieve discrimination of L-valine from L-isoleucine and L-threonine by the complex of tRNAVal and valyl-tRNA synthetase. Cell 103, 793–803 (2000)

  20. 20.

    , , , & Conformational flexibility in RNA: the role of dihydrouridine. Nucleic Acids Res. 24, 1073–1079 (1996)

  21. 21.

    et al. T box RNA decodes both the information content and geometry of tRNA to affect gene expression. Proc. Natl Acad. Sci. USA 110, 7240–7245 (2013)

  22. 22.

    , , & Crystal structure of the specificity domain of ribonuclease P. Nature 421, 760–764 (2003)

  23. 23.

    et al. Structure of the L1 protuberance in the ribosome. Nature Struct. Biol. 10, 104–108 (2003)

  24. 24.

    & Solution nuclear magnetic resonance analyses of the anticodon arms of proteinogenic and nonproteinogenic tRNAGly. Biochemistry 51, 3662–3674 (2012)

  25. 25.

    et al. Incorporation of aminoacyl-tRNA into the ribosome as seen by cryo-electron microscopy. Nature Struct. Biol. 10, 899–906 (2003)

  26. 26.

    et al. Structures of the bacterial ribosome in classical and hybrid states of tRNA binding. Science 332, 981–984 (2011)

  27. 27.

    et al. The crystal structure of the ribosome bound to EF-Tu and aminoacyl-tRNA. Science 326, 688–694 (2009)

  28. 28.

    & Solution structure of the K-turn and specifier loop domains from the Bacillus subtilis tyrS T-Box leader RNA. J. Mol. Biol. 408, 99–117 (2011)

  29. 29.

    , , & Comparative genomic analysis of T-box regulatory systems in bacteria. RNA 14, 717–735 (2008)

  30. 30.

    & Geometric nomenclature and classification of RNA base pairs. RNA 7, 499–512 (2001)

  31. 31.

    , & Structural basis for specific, high-affinity tetracycline binding by an in vitro evolved aptamer and artificial riboswitch. Chem. Biol. 15, 1125–1137 (2008)

  32. 32.

    & Idiosyncratically tuned switching behavior of riboswitch aptamer domains revealed by comparative small-angle X-ray scattering analysis. RNA 16, 598–609 (2010)

  33. 33.

    , & Thermodynamic analysis of ligand binding and ligand binding-induced tertiary structure formation by the thiamine pyrophosphate riboswitch. RNA 16, 186–196 (2010)

  34. 34.

    & Modulation of quaternary structure and enhancement of ligand binding by the K-turn of tandem glycine riboswitches. RNA 19, 167–176 (2013)

  35. 35.

    Size distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and Lamm equation modeling. Biophys. J. 78, 1606–1619 (2000)

  36. 36.

    et al. High-precision isothermal titration calorimetry with automated peak-shape analysis. Anal. Chem. 84, 5066–5073 (2012)

  37. 37.

    XDS. Acta Crystallogr. D 66, 125–132 (2010)

  38. 38.

    Scaling and assessment of data quality. Acta Crystallogr. D 62, 72–82 (2006)

  39. 39.

    & Processing of diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997)

  40. 40.

    A short history of SHELX. Acta Crystallogr. A 64, 112–122 (2008)

  41. 41.

    & HKL2MAP: a graphical user interface for macromolecular phasing with SHELX programs. J. Appl. Crystallogr. 37, 843–844 (2004)

  42. 42.

    , , & The crystal structure of unmodified tRNAPhe from Escherichia coli. Nucleic Acids Res. 38, 4154–4162 (2010)

  43. 43.

    & MOLREP: an automated program for molecular replacement. J. Appl. Crystallogr. 30, 1022–1025 (1997)

  44. 44.

    , , & Structural basis of specific tRNA aminoacylation by a small in vitro selected ribozyme. Nature 454, 358–361 (2008)

  45. 45.

    et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007)

  46. 46.

    et al. Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr. D 68, 352–367 (2012)

  47. 47.

    Maximum-likelihood density modification. Acta Crystallogr. D 56, 965–972 (2000)

  48. 48.

    , , & Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010)

  49. 49.

    & The crystal structure of yeast phenylalanine tRNA at 1.93 Å resolution: a classic structure revisited. RNA 6, 1091–1105 (2000)

  50. 50.

    , , & Metals, motifs, and recognition in the crystal structure of a 5S rRNA domain. Cell 91, 705–712 (1997)

  51. 51.

    The PyMOL Molecular Graphics System (DeLano Scientific, 2002)

  52. 52.

    , , & Recurrent structural RNA motifs, isostericity matrices and sequence alignments. Nucleic Acids Res. 33, 2395–2409 (2005)

Download references

Acknowledgements

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

Affiliations

  1. National Heart, Lung and Blood Institute, 50 South Drive, MSC 8012, Bethesda, Maryland 20892-8012, USA

    • Jinwei Zhang
    •  & Adrian R. Ferré-D’Amaré

Authors

  1. Search for Jinwei Zhang in:

  2. Search for Adrian R. Ferré-D’Amaré in:

Contributions

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.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Adrian R. Ferré-D’Amaré.

Extended data

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    This file contains a Supplementary Discussion and additional references.

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nature12440

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.