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.

  • Letter
  • Published:

Structural basis for ArfA–RF2-mediated translation termination on mRNAs lacking stop codons

Abstract

In bacteria, ribosomes stalled on truncated mRNAs that lack a stop codon are rescued by the transfer-messenger RNA (tmRNA), alternative rescue factor A (ArfA) or ArfB systems1. Although tmRNA–ribosome and ArfB–ribosome structures have been determined2,3,4,5,6,7, how ArfA recognizes the presence of truncated mRNAs and recruits the canonical termination release factor RF2 to rescue the stalled ribosomes is unclear. Here we present a cryo-electron microscopy reconstruction of the Escherichia coli 70S ribosome stalled on a truncated mRNA in the presence of ArfA and RF2. The structure shows that the C terminus of ArfA binds within the mRNA entry channel on the small ribosomal subunit, and explains how ArfA distinguishes between ribosomes that bear truncated or full-length mRNAs. The N terminus of ArfA establishes several interactions with the decoding domain of RF2, and this finding illustrates how ArfA recruits RF2 to the stalled ribosome. Furthermore, ArfA is shown to stabilize a unique conformation of the switch loop of RF2, which mimics the canonical translation termination state by directing the catalytically important GGQ motif within domain 3 of RF2 towards the peptidyl-transferase centre of the ribosome. Thus, our structure reveals not only how ArfA recruits RF2 to the ribosome but also how it promotes an active conformation of RF2 to enable translation termination in the absence of a stop codon.

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

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

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

Figure 1: Cryo-EM structure of ArfA-RF2-SRC.
Figure 2: Interaction of ArfA with the small subunit.
Figure 3: Interaction of ArfA with RF2 on the ribosome.
Figure 4: ArfA stabilizes a unique conformation of the RF2 switch loop.

Similar content being viewed by others

Accession codes

Primary accessions

Electron Microscopy Data Bank

Protein Data Bank

References

  1. Keiler, K. C. Mechanisms of ribosome rescue in bacteria. Nat. Rev. Microbiol. 13, 285–297 (2015)

    Article  CAS  Google Scholar 

  2. Valle, M. et al. Visualizing tmRNA entry into a stalled ribosome. Science 300, 127–130 (2003)

    Article  ADS  CAS  Google Scholar 

  3. Fu, J. et al. Visualizing the transfer-messenger RNA as the ribosome resumes translation. EMBO J. 29, 3819–3825 (2010)

    Article  CAS  Google Scholar 

  4. Weis, F. et al. tmRNA-SmpB: a journey to the centre of the bacterial ribosome. EMBO J. 29, 3810–3818 (2010)

    Article  CAS  Google Scholar 

  5. Gagnon, M. G., Seetharaman, S. V., Bulkley, D. & Steitz, T. A. Structural basis for the rescue of stalled ribosomes: structure of YaeJ bound to the ribosome. Science 335, 1370–1372 (2012)

    Article  ADS  CAS  Google Scholar 

  6. Neubauer, C., Gillet, R., Kelley, A. C. & Ramakrishnan, V. Decoding in the absence of a codon by tmRNA and SmpB in the ribosome. Science 335, 1366–1369 (2012)

    Article  ADS  CAS  Google Scholar 

  7. Ramrath, D. J. et al. The complex of tmRNA–SmpB and EF-G on translocating ribosomes. Nature 485, 526–529 (2012)

    Article  ADS  CAS  Google Scholar 

  8. Chadani, Y. et al. Ribosome rescue by Escherichia coli ArfA (YhdL) in the absence of trans-translation system. Mol. Microbiol. 78, 796–808 (2010)

    Article  Google Scholar 

  9. Chadani, Y., Ono, K., Kutsukake, K. & Abo, T. Escherichia coli YaeJ protein mediates a novel ribosome-rescue pathway distinct from SsrA- and ArfA-mediated pathways. Mol. Microbiol. 80, 772–785 (2011)

    Article  CAS  Google Scholar 

  10. Chadani, Y. et al. trans-translation-mediated tight regulation of the expression of the alternative ribosome-rescue factor ArfA in Escherichia coli. Genes Genet. Syst. 86, 151–163 (2011)

    Article  CAS  Google Scholar 

  11. Garza-Sánchez, F., Schaub, R. E., Janssen, B. D. & Hayes, C. S. tmRNA regulates synthesis of the ArfA ribosome rescue factor. Mol. Microbiol. 80, 1204–1219 (2011)

    Article  Google Scholar 

  12. Schaub, R. E., Poole, S. J., Garza-Sánchez, F., Benbow, S. & Hayes, C. S. Proteobacterial ArfA peptides are synthesized from non-stop messenger RNAs. J. Biol. Chem. 287, 29765–29775 (2012)

    Article  CAS  Google Scholar 

  13. Chadani, Y., Ito, K., Kutsukake, K. & Abo, T. ArfA recruits release factor 2 to rescue stalled ribosomes by peptidyl-tRNA hydrolysis in Escherichia coli. Mol. Microbiol. 86, 37–50 (2012)

    Article  CAS  Google Scholar 

  14. Shimizu, Y. ArfA recruits RF2 into stalled ribosomes. J. Mol. Biol. 423, 624–631 (2012)

    Article  CAS  Google Scholar 

  15. Kurita, D., Chadani, Y., Muto, A., Abo, T. & Himeno, H. ArfA recognizes the lack of mRNA in the mRNA channel after RF2 binding for ribosome rescue. Nucleic Acids Res. 42, 13339–13352 (2014)

    Article  CAS  Google Scholar 

  16. Zeng, F. & Jin, H. Peptide release promoted by methylated RF2 and ArfA in nonstop translation is achieved by an induced-fit mechanism. RNA 22, 49–60 (2016)

    Article  CAS  Google Scholar 

  17. Korostelev, A. et al. Crystal structure of a translation termination complex formed with release factor RF2. Proc. Natl Acad. Sci. USA 105, 19684–19689 (2008)

    Article  ADS  CAS  Google Scholar 

  18. Weixlbaumer, A. et al. Insights into translational termination from the structure of RF2 bound to the ribosome. Science 322, 953–956 (2008)

    Article  ADS  CAS  Google Scholar 

  19. Ito, K., Uno, M. & Nakamura, Y. A tripeptide ‘anticodon’ deciphers stop codons in messenger RNA. Nature 403, 680–684 (2000)

    Article  ADS  CAS  Google Scholar 

  20. Youngman, E. M., McDonald, M. E. & Green, R. Peptide release on the ribosome: mechanism and implications for translational control. Annu. Rev. Microbiol. 62, 353–373 (2008)

    Article  CAS  Google Scholar 

  21. Zhou, J., Korostelev, A., Lancaster, L. & Noller, H. F. Crystal structures of 70S ribosomes bound to release factors RF1, RF2 and RF3. Curr. Opin. Struct. Biol. 22, 733–742 (2012)

    Article  CAS  Google Scholar 

  22. Vestergaard, B. et al. Bacterial polypeptide release factor RF2 is structurally distinct from eukaryotic eRF1. Mol. Cell 8, 1375–1382 (2001)

    Article  CAS  Google Scholar 

  23. Franckenberg, S., Becker, T. & Beckmann, R. Structural view on recycling of archaeal and eukaryotic ribosomes after canonical termination and ribosome rescue. Curr. Opin. Struct. Biol. 22, 786–796 (2012)

    Article  CAS  Google Scholar 

  24. Simms, C. L., Thomas, E. N. & Zaher, H. S. Ribosome-based quality control of mRNA and nascent peptides. Wiley Interdiscip. Rev. RNA http://dx.doi.org/10.1002/wrna.1366 (2016)

  25. Jenner, L. B., Demeshkina, N., Yusupova, G. & Yusupov, M. Structural aspects of messenger RNA reading frame maintenance by the ribosome. Nat. Struct. Mol. Biol. 17, 555–50 (2010)

    Article  CAS  Google Scholar 

  26. Starosta, A. L. et al. Translational stalling at polyproline stretches is modulated by the sequence context upstream of the stall site. Nucleic Acids Res. 42, 10711–10719 (2014)

    Article  CAS  Google Scholar 

  27. Li, X. et al. Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM. Nat. Methods 10, 584–590 (2013)

    Article  CAS  Google Scholar 

  28. Rohou, A. & Grigorieff, N. CTFFIND4: Fast and accurate defocus estimation from electron micrographs. J. Struct. Biol. 192, 216–221 (2015)

    Article  Google Scholar 

  29. Chen, J. Z. & Grigorieff, N. SIGNATURE: a single-particle selection system for molecular electron microscopy. J. Struct. Biol. 157, 168–173 (2007)

    Article  CAS  Google Scholar 

  30. Scheres, S. H. RELION: implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180, 519–530 (2012)

    Article  CAS  Google Scholar 

  31. Arenz, S. et al. The stringent factor RelA adopts an open conformation on the ribosome to stimulate ppGpp synthesis. Nucleic Acids Res. 44, 6471–6481 (2016)

    Article  CAS  Google Scholar 

  32. Kucukelbir, A., Sigworth, F. J. & Tagare, H. D. Quantifying the local resolution of cryo-EM density maps. Nat. Methods 11, 63–65 (2014)

    Article  CAS  Google Scholar 

  33. Fischer, N. et al. Structure of the E. coli ribosome-EF-Tu complex at <3 Å resolution by Cs-corrected cryo-EM. Nature 520, 567–570 (2015)

    Article  ADS  Google Scholar 

  34. Pettersen, E. F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004)

    Article  CAS  Google Scholar 

  35. Söding, J., Biegert, A. & Lupas, A. N. The HHpred interactive server for protein homology detection and structure prediction. Nucleic Acids Res. 33, W244–W248 (2005)

    Article  Google Scholar 

  36. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004)

    Article  Google Scholar 

  37. 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 

  38. Vagin, A. A. et al. REFMAC5 dictionary: organization of prior chemical knowledge and guidelines for its use. Acta Crystallogr. D 60, 2184–2195 (2004)

    Article  Google Scholar 

  39. Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D 66, 12–21 (2010)

    Article  CAS  Google Scholar 

  40. Brown, A. et al. Tools for macromolecular model building and refinement into electron cryo-microscopy reconstructions. Acta Crystallogr. D 71, 136–153 (2015)

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank H. Sieber, S. Rieder and C. Ungewickell for technical assistance and L. Bischoff and R. Green for providing expression plasmids for E. coli ArfAΔ17 and RF2, respectively. This research was supported by grants from the Deutsche Forschungsgemeinschaft WI3285/4-1, SPP-1879 (to D.N.W.), GRK 1721 and FOR1805 (to R.B. and D.N.W.).

Author information

Authors and Affiliations

Authors

Contributions

D.N.W. designed the study. C.M. and P.H. prepared the cryo-EM sample. P.H., C.M. and B.B. processed the cryo-EM data. P.H., S.A. and D.N.W. built and refined the molecular models. O.B. collected the cryo-EM data. P.H., C.M., R.B. and D.N.W. interpreted the results and D.N.W. wrote the paper.

Corresponding author

Correspondence to Daniel N. Wilson.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Additional information

Reviewer Information Nature thanks T. Abo, Y. Hashem, K. Keiler and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Figure 1 Recycling of ribosomes stalled on truncated mRNA by ArfA and RF2.

In vitro translation assay of the truncated nlpD template was performed in the presence of ArfA, RF2 or RF2-GAQ, revealing a peptidyl-tRNA band (nlpD_ns*P-tRNA), whereas the peptidyl-tRNA was absent and free nlpD peptide (nlpD_ns) was observed when the reaction was performed with ArfA and RF2. Replacing wild-type RF2 with the inactive RF2-GAQ mutant led to the reappearance of the peptidyl-tRNA band and loss of the free nlpD peptide.

Extended Data Figure 2 Classification of the ArfA-RF2-SRC.

The complete dataset of 227,608 particles was initially aligned against a vacant E. coli 70S ribosome, refined with RELION using 3D auto-refine and the movie particles were extracted. The polished particles were then subjected to a 3D refinement and 3D classification using FREALIGN. The class 2 (138,582 particles) resulting from the 100 rounds of 3D classification with 3× binned images using a ribosomal mask was then further refined and classified with 2× binned images. The remaining 69,089 particles containing ArfA-RF2-SRC were then 3D-refined, resulting in a final reconstruction of 3.1 Å (0.143 Fourier shell correlation (FSC)) average resolution.

Extended Data Figure 3 Resolution of the ArfA-RF2-SRC.

a, Overview of the final refined cryo-EM map of the ArfA-RF2-SRC with separated densities for small (yellow) and large (grey) ribosomal subunit, as well as ArfA (red), RF2 (orange) and P-tRNA (green). b, Same view as in a but coloured according to local resolution. c, Transverse section of b showing local resolution in the core of the ribosomal subunits. d, FSC curve of the refined final map, indicating that the average resolution of the ArfA-RF2-SRC is 3.1 Å (at 0.143). e, Fit of models to maps. FSC curves calculated between the refined model and the final map (blue), with the self- and cross-validated correlations in orange and black, respectively. Information beyond 3.2 Å was not used during refinement and preserved for validation. f, g, Selected examples illustrating the quality of fit of the molecular models to the unsegmented cryo-EM map (grey mesh) for the ArfA (red) interaction with S12 (blue), related to Fig. 2b (f), and with RF2 (orange), related to Fig. 3c (g).

Extended Data Figure 4 Hydroxyl radical probing of ArfA on the ribosome.

ad, Hydroxyl-radical probing data15 of ArfA in complex with RF2 on the ribosome reveal that tethers linked to the N-terminal region of ArfA, for example, residues S2 and R3 (magenta), cleave the 16S rRNA within the vicinity of helices h18, whereas tethers linked to the C-terminal region of ArfA, such as residues 33–34/38–39 and 46 (teal), cleave the 16S rRNA within the vicinity of helices h34 (ref. 15). These findings are in excellent agreement with the position of ArfA (red) within the ArfA-RF2-SRC structure reported here. In the overview panels a and c, P-tRNA (green) is shown for reference.

Extended Data Figure 5 Sequence alignment of E. coli RF1 and RF2 with secondary structure assignments.

Sequence alignment of E. coli RF1 and RF2 generated using ClustalX with secondary structure (helices and strands) and domain (I–IV) assignments based on the crystal structures of E. coli RF2 (ref. 22), except for the switch loop (yellow) and extension to helix α7 (purple), which was based on the ArfA-RF2-SRC structure. The pink boxes indicate regions of RF2 that form an interface with ArfA, with residues in bold predicted to prevent interaction of RF1 with ArfA. Asterisk (*) or colon (:) and full stop (.) indicate a single, fully conserved residue or residues with strong (>0.5 in the Gonnet PAM 250 matrix) and weakly (>0.5) similar properties, respectively.

Extended Data Figure 6 Potential specificity determinants for ArfA-mediated ribosome recycling.

a, b, ArfA (red) and E. coli RF2 (orange) compared to homology model of E. coli RF1 (blue) aligned to RF2 in the ArfA-RF2-SRC. a, The ArfA interface with β4 and β5 strands of E. coli RF2 (orange) consists of hydrophobic residues V198, F217 and F221, which are mutated to Gly, Ala and Ala, respectively, in RF1 (blue). b, The ArfA interface with α-helix α7 of RF2 (orange). Replacing negatively charged residues such as E311 and D312 in RF2 with Arg in RF1 is also likely to disrupt the interaction with ArfA. c, d, Sequence alignments for the regions of RF1 and RF2 corresponding to a and b, respectively. The pink boxes indicate regions of RF2 that form an interface with ArfA, including residues in bold predicted to prevent interaction of RF1 with ArfA and therefore could provide the basis for RF2-specificity of ArfA action. Organisms in bold contain ArfA, whereas others have no detectable ArfA homologue. Asterisk (*), colon (:) or full stop (.) indicate a single, fully conserved residue or residues with strong (>0.5 in the Gonnet PAM 250 matrix) and weakly (>0.5) similar properties, respectively.

Extended Data Figure 7 Location of the ArfA-A18T mutation relative to RF2.

a, Overview of ArfA (red) and RF2 (gold) on the ribosome (30S, grey; 50S, slate). b, c, Zoom of boxed region in a showing the environment of A18 (teal) of ArfA in close proximity to I11 and K8 in the N terminus of ArfA (red) (b), and A18T (teal) of ArfA in sterically clashing with I11 and K8 in the N terminus of ArfA (red) (c).

Extended Data Figure 8 Comparison of ArfA with other ribosome rescue systems.

ac, Relative orientation on the ribosome with truncated mRNAs and ArfA (red) and RF2 (orange) (a), ArfB (purple, PDB code 4V95)5 (b) or tmRNA (brown) and SmpB (yellow) (PDB code 4V8Q)6 (c). In all cases, the mRNA and P-tRNA are coloured cyan and green, respectively.

Extended Data Table 1 Data collection and refinement statistics

Supplementary information

Comparison of free and ArfA-bound conformations of RF2

Animation showing the conformation change in RF2 when comparing the crystal structure of the free (closed) form of RF2 (PDB ID 1GQE) with that when ArfA (red) is bound. RF2 is coloured orange except for the switch loop (purple). (MP4 5790 kb)

Comparison of decoding and ArfA-bound conformations of RF2

Animation showing the conformation change in RF2 when comparing the canonical termination form of RF2 (PDB ID 4V5E) with that when ArfA (red) is bound. RF2 is coloured orange except for the switch loop (purple). (MP4 7093 kb)

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Huter, P., Müller, C., Beckert, B. et al. Structural basis for ArfA–RF2-mediated translation termination on mRNAs lacking stop codons. Nature 541, 546–549 (2017). https://doi.org/10.1038/nature20821

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature20821

This article is cited by

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.

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