Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Structural basis for tRNA decoding and aminoacylation sensing by T-box riboregulators

Nature Structural & Molecular Biology volume 26, pages 1106–1113 (2019)Cite this article

Subjects

Abstract

T-box riboregulators are a class of cis-regulatory RNAs that govern the bacterial response to amino acid starvation by binding, decoding and reading the aminoacylation status of specific transfer RNAs. Here we provide a high-resolution crystal structure of a full-length T-box from Mycobacterium tuberculosis that explains tRNA decoding and aminoacylation sensing by this riboregulator. Overall, the T-box consists of decoding and aminoacylation sensing modules bridged by a rigid pseudoknot structure formed by the mid-region domains. Stem-I and the Stem-II S-turn assemble a claw-like decoding module, while the antiterminator, Stem-III, and the adjacent linker form a tightly interwoven aminoacylation sensing module. The uncharged tRNA is selectively recognized by an unexpected set of favorable contacts from the linker region in the aminoacylation sensing module. A complex structure with a charged tRNA mimic shows that the extra moiety dislodges the linker, which is indicative of the possible chain of events that lead to alternative base-pairing and altered expression output.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Learn more

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Overall structure of Mtb-ileS with details of domain arrangement.
Fig. 2: tRNA decoding and pseudoknot formation.
Fig. 3: Details of the aminoacylation sensing module.
Fig. 4: Comparison of Mtb-ileS complex structures bound to tRNAIle-cP or tRNAIle-OH.
Fig. 5: T-box family portrait.

Data availability

Atomic coordinates and structure factors for Mtb-ileS_tRNA-OH_native and Mtb-ileS_tRNA-cP_native have been deposited in the Protein Data Bank with the accession codes 6UFG and 6UFH, respectively. Source data for Figs. 2b and 3f are available in Supplementary Table 3. Other data are available upon reasonable request.

References

  1. Mandal, M. & Breaker, R. R. Gene regulation by riboswitches. Nat. Rev. Mol. Cell Biol. 5, 451–463 (2004).

    Article  CAS  Google Scholar 

  2. Peselis, A. & Serganov, A. Themes and variations in riboswitch structure and function. Biochim. Biophys. Acta 1839, 908–918 (2014).

    Article  CAS  Google Scholar 

  3. Grundy, F. J. & Henkin, T. M. tRNA as a positive regulator of transcription antitermination in B. subtilis. Cell 74, 475–482 (1993).

    Article  CAS  Google Scholar 

  4. Grundy, F. J., Winkler, W. C. & Henkin, T. M. tRNA-mediated transcription antitermination in vitro: Codon-anticodon pairing independent of the ribosome. Proc. Natl Acad. Sci. USA 99, 11121–11126 (2002).

    Article  CAS  Google Scholar 

  5. Grigg, J. C. 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).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  7. Grigg, J. C. & Ke, A. Structural determinants for geometry and information decoding of tRNA by T Box Leader RNA. Structure 21, 2025–2032 (2013).

    Article  CAS  Google Scholar 

  8. Grundy, F. J., Rollins, S. M. & Henkin, T. M. Interaction between the acceptor end of tRNA and the T box stimulates antitermination in the Bacillus subtilis tyrS gene: a new role for the discriminator base. J. Bacteriol. 176, 4518–4526 (1994).

    Article  CAS  Google Scholar 

  9. Zhang, J. & Ferré-D’Amaré, A. R. Direct evaluation of tRNA aminoacylation status by the T-box riboswitch using tRNA-mRNA stacking and steric readout. Mol. Cell 55, 148–155 (2014).

    Article  CAS  Google Scholar 

  10. Grundy, F. J. & Henkin, T. M. Kinetic analysis of tRNA-directed transcription antitermination of the Bacillus subtilis glyQS gene in vitro. J. Bacteriol. 186, 5392–5399 (2004).

    Article  CAS  Google Scholar 

  11. Suddala, K. C. et al. Hierarchical mechanism of amino acid sensing by the T-box riboswitch. Nat. Commun. 9,1896 (2018).

  12. Zhang, J. et al. Specific structural elements of the T-box riboswitch drive the two-step binding of the tRNA ligand. eLife 7, e39518 (2018).

  13. Seliverstov, A. V., Putzer, H., Gelfand, M. S. & Lyubetsky, V. A. Comparative analysis of RNA regulatory elements of amino acid metabolism genes in Actinobacteria. BMC Microbiol. 5, 54 (2005).

    Article  Google Scholar 

  14. Sherwood, A. V., Grundy, F. J. & Henkin, T. M. T box riboswitches in Actinobacteria: translational regulation via novel tRNA interactions. Proc. Natl Acad. Sci. USA 112, 1113–1118 (2015).

    Article  CAS  Google Scholar 

  15. Rollins, S. M., Grundy, F. J. & Henkin, T. M. Analysis of cis-acting sequence and structural elements required for antitermination of the Bacillus subtilis tyrS gene. Mol. Microbiol. 25, 411–421 (1997).

    Article  CAS  Google Scholar 

  16. Saad, N. Y. et al. Two-codon T-box riboswitch binding two tRNAs. Proc. Natl Acad. Sci. USA 110, 12756–12761 (2013).

    Article  CAS  Google Scholar 

  17. Shepherd, J. & Ibba, M. Bacterial transfer RNAs. FEMS Microbiol. Rev 39, 280–300 (2015).

    Article  CAS  Google Scholar 

  18. Agris, P. F., Vendeix, F. A. P. & Graham, W. D. tRNA’s wobble decoding of the genome: 40 years of modification. J. Mol. Biol. 366, 1–13 (2007).

    Article  CAS  Google Scholar 

  19. Putzer, H., Condon, C., Brechemier-Baey, D., Brito, R. & Grunberg-Manago, M. Transfer RNA-mediated antitermination in vitro. Nucleic Acids Res. 30, 3026–3033 (2002).

    Article  CAS  Google Scholar 

  20. Ogle, J. M. et al. Recognition of cognate transfer RNA by the 30S ribosomal subunit. Science 292, 897–902 (2001).

    Article  CAS  Google Scholar 

  21. Ogle, J. M., Murphy, F. V., Tarry, M. J. & Ramakrishnan, V. Selection of tRNA by the ribosome requires a transition from an open to a closed form. Cell 111, 721–732 (2002).

    Article  CAS  Google Scholar 

  22. Demeshkina, N., Jenner, L., Westhof, E., Yusupov, M. & Yusupova, G. A new understanding of the decoding principle on the ribosome. Nature 484, 256–259 (2012).

    Article  CAS  Google Scholar 

  23. Vitreschak, A. G., Mironov, A. A., Lyubetsky, V. A. & Gelfand, M. S. Comparative genomic analysis of T-box regulatory systems in bacteria. RNA 14, 717–735 (2008).

    Article  CAS  Google Scholar 

  24. Kalvari, I. et al. Non-coding RNA analysis using the Rfam database. Curr. Protoc. Bioinformatics. 62, e51 (2018).

    Article  Google Scholar 

  25. Henkin, T. M., Glass, B. L. & Grundy, F. J. Analysis of the Bacillus subtilis tyrS gene: conservation of a regulatory sequence in multiple tRNA synthetase genes. J. Bacteriol. 174, 1299–1306 (1992).

    Article  CAS  Google Scholar 

  26. Grundy, F. J., Yousef, M. R. & Henkin, T. M. Monitoring uncharged tRNA during transcription of the Bacillus subtilis glyQS gene. J. Mol. Biol. 346, 73–81 (2005).

    Article  CAS  Google Scholar 

  27. Murakami, H., Ohta, A., Goto, Y., Sako, Y. & Suga, H. Flexizyme as a versatile tRNA acylation catalyst and the application for translation. Nucleic Acids Symp. Ser. (Oxf.) 50, 35–36 (2006).

    Article  Google Scholar 

  28. Golden, B. L., Chen, J. & Luptak, A. Ribozyme with tRNA synthetase activity and methods of manufacturing and using the same. US patent 20,160,348,108 (2016).

  29. Ke, A. & Doudna, J. A. Crystallization of RNA and RNA–protein complexes. Methods 34, 408–414 (2004).

    Article  CAS  Google Scholar 

  30. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  33. Sheldrick, G. M. et al. Experimental phasing with SHELXC/D/E: combining chain tracing with density modification. Acta Crystallogr. D 66, 479–485 (2010).

    Article  CAS  Google Scholar 

  34. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010).

    Article  CAS  Google Scholar 

  35. Emsley, P. et al. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010).

    Article  CAS  Google Scholar 

  36. Guérout-Fleury, A.-M., Frandsen, N. & Stragier, P. Plasmids for ectopic integration in Bacillus subtilis. Gene 180, 57–61 (1996).

    Article  Google Scholar 

  37. Anagnostopoulos, C. & Spizizen, J. Requirements for transformation in Bacillus subtilis. J. Bacteriol. 81, 741–746 (1961).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Chen, L., James, L. P. & Helmann, J. D. Metalloregulation in Bacillus subtilis: isolation and characterization of two genes differentially repressed by metal ions. J. Bacteriol. 175, 5428–5437 (1993).

    Article  CAS  Google Scholar 

  39. Miller, J. H. Experiments in Molecular Genetics (Cold Spring Harbor Laboratory, 1972).

Download references

Acknowledgements

The authors thank A.J. Sachla and J.D. Helmann for advice on the β-galactosidase assay, the Bacillus Genetic Stock Center, the Nicholson Lab for the use of their spectrophotometer and the Fromme Lab for the use of their microscope. This work is supported by the National Institutes of Health (NIH) (grant nos. GM118174 and GM116632 to A.K.). This work is based on research conducted at the Northeastern Collaborative Access Team beamlines, which are funded by the National Institute of General Medical Sciences from the NIH (grant no. P30 GM124165). The Pilatus 6 M detector on the 24-ID-C beamline is funded by a NIH-ORIP HEI grant (no. S10 RR029205). This research used the resources of the Advanced Photon Source, a US Department of Energy Office of Science User Facility operated for the US Department of Energy Office of Science by Argonne National Laboratory (contract no. DE-AC02-06CH11357).

Author information

Authors and Affiliations

  1. Department of Molecular Biology and Genetics, Ithaca, NY, USA

    Robert A. Battaglia & Ailong Ke

  2. Department of Microbiology and Immunology, Life Sciences Institute, The University of British Columbia, Vancouver, British Columbia, Canada

    Jason C. Grigg

Authors
  1. Robert A. Battaglia
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jason C. Grigg
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ailong Ke
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

R.A.B., J.C.G. and A.K. designed the research. R.A.B. was the main contributor to the structure-function analysis. J.C.G. contributed to experimental design and structural refinement. R.A.B. and A.K. wrote the manuscript.

Corresponding author

Correspondence to Ailong Ke.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

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.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data 1 Size exclusion chromatographic analysis and mutagenesis guide.

a, Size exclusion chromatogram of Mtb-ileS folded with (blue) and without (orange) tRNAIle. b, Urea denaturing PAGE gel of peak fractions from T-box + tRNA peak. c, Secondary structure model of tyrS T-box from Bacillus subtilis (Bsub-tyrS). Boxes highlight location and identity of each mutation. d, Conversion table for residue notation in Mtb-ileS and Bsub-tyrS. Fold difference from WT is the difference between the average of three induction measurements at 1 hour for WT versus mutation of indicated residue.

Extended Data 2 Details of AntiS-tRNA and AntiS-Stem-III interactions.

a, Detail of G132-U160 wobble pair in the aminoacylation sensing pocket showing AntiS (cyan), tRNA (pale blue) and linker (yellow). Hydrogen bonds represented by black dashes. Magnesium ion represented by green sphere. b, View of AntiS bound to tRNA showing tRNA 3′-end exposed in a large opening. c, Stem-III (pink) and AntiS minor groove interactions. d, Details of G98 interactions with AntiS. e, Details of A118 type-I A-minor interaction with AntiS.

Extended Data 3 Structural details and size exclusion chromatography of Mtb-ileS with tRNAIle-cP.

a, Tertiary structure model of Mtb-ileS in complex with tRNAIle-cP. b, Detail of AntiS interaction with tRNAIle-cP NCCA sequence. Hydrogen bonds represented by black dashes. Asterisk indicates residues mutated from wildtype sequence. c, Size exclusion chromatograms of Mtb-ileS folded with tRNAIle-cP (blue) and with tRNAIle-OH (orange). d, Urea denaturing PAGE gel of peak fractions from T-box + tRNAIle-cP peak.

Extended Data 4 Mechanistic diagram of atypical T-box translational regulation.

a, tRNA recruitment and decoding. b, Pseudoknot formation positions AntiS to interact with tRNA NCCA sequence. c, Transient intermediate where aminoacylation sensing module interacts with uncharged (top) and charged (bottom) tRNA. d, Favorable interactions between uncharged tRNA and linker locks T-box into ON conformation. Exposed Shine–Dalgarno (SDS) allows translation initiation. e, Steric clashing between charged tRNA and linker unravels the aminoacylation sensing module leading to alternative base pairing. Sequestrator formation prevents ribosome access to SDS and prevents translation.

Supplementary information

Supplementary Information

Supplementary Tables 1 and 2

Reporting Summary

Supplementary Table 3

Source data for Figs. 2b and 3f

Rights and permissions

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Battaglia, R.A., Grigg, J.C. & Ke, A. Structural basis for tRNA decoding and aminoacylation sensing by T-box riboregulators. Nat Struct Mol Biol 26, 1106–1113 (2019). https://doi.org/10.1038/s41594-019-0327-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41594-019-0327-6

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing