Letter | Published:

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

Nature volume 541, pages 546549 (26 January 2017) | Download Citation

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.

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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

Author notes

    • Paul Huter
    •  & Claudia Müller

    These authors contributed equally to this work.

Affiliations

  1. Gene Center, Department of Biochemistry and Center for integrated Protein Science Munich (CiPSM), Ludwig-Maximilians-Universität München, Feodor-Lynen-Strasse 25, 81377 Munich, Germany

    • Paul Huter
    • , Claudia Müller
    • , Bertrand Beckert
    • , Stefan Arenz
    • , Otto Berninghausen
    • , Roland Beckmann
    •  & Daniel N. Wilson
  2. Institute for Biochemistry and Molecular Biology, University of Hamburg, Martin-Luther-King-Pl. 6, 20146 Hamburg, Germany

    • Bertrand Beckert
    •  & Daniel N. Wilson

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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.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Daniel N. Wilson.

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

Supplementary information

Videos

  1. 1.

    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).

  2. 2.

    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).

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DOI

https://doi.org/10.1038/nature20821

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