Letter | Published:

Mechanistic insights into the alternative translation termination by ArfA and RF2

Nature volume 541, pages 550553 (26 January 2017) | Download Citation

Abstract

During cellular translation of messenger RNAs by ribosomes, the translation apparatus sometimes pauses or stalls at the elongation and termination steps1,2,3,4,5,6. With the exception of programmed stalling, which is usually used by cells for regulatory purposes5,7,8, ribosomes stalled on mRNAs need to be terminated and recycled to maintain adequate translation capacity9. Much ribosome stalling originates in aberrant mRNAs that lack a stop codon. Transcriptional errors, misprocessing of primary transcripts, and undesired mRNA cleavage all contribute to the formation of non-stop mRNAs. Ribosomes stalled at the 3′ end of non-stop mRNAs do not undergo normal termination owing to the lack of specific stop-codon recognition by canonical peptide release factors at the A-site decoding centre. In bacteria, the transfer-messenger RNA (tmRNA)–SmpB-mediated trans-translation rescue system reroutes stalled ribosomes to the normal elongation cycle and translation termination3,4,10,11,12. Two additional rescue systems, ArfA–RF2 (refs 13, 14, 15, 16) and ArfB (formerly known as YaeJ)17,18, are also present in many bacterial species, but their mechanisms are not fully understood. Here, using cryo-electron microscopy, we characterize the structure of the Escherichia coli 70S ribosome bound with ArfA, the release factor RF2, a short non-stop mRNA and a cognate P-site tRNA. The C-terminal loop of ArfA occupies the mRNA entry channel on the 30S subunit, whereas its N terminus is sandwiched between the decoding centre and the switch loop of RF2, leading to marked conformational changes in both the decoding centre and RF2. Despite the distinct conformation of RF2, its conserved catalytic GGQ motif is precisely positioned next to the CCA-end of the P-site tRNA. These data illustrate a stop-codon surrogate mechanism for ArfA in facilitating the termination of non-stop ribosomal complexes by RF2.

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Acknowledgements

We thank the National Center for Protein Sciences (Beijing, China) for providing resources for data collection and computation. Part of the computation was performed on the Computing Platform of the Center for Life Science, Peking Univesity. We also thank S. Goto for providing expression vectors and the staff of Gene Research Center of Hirosaki University for the use of the facility. This work was supported by the National Natural Science Foundation of China (31422016, 31470722 and 31630087 to N.G.), the Ministry of Science and Technology of China (2016YFA0500700 and 2013CB910404 to N.G.) and by the Japan Society for the Promotion of Science (16K18684 to D.K.). N.L. is supported by a postdoctoral fellowship from the Peking-Tsinghua Center for Life Sciences.

Author information

Author notes

    • Chengying Ma
    •  & Daisuke Kurita

    These authors contributed equally to this work.

Affiliations

  1. Ministry of Education Key Laboratory of Protein Sciences, Beijing Advanced Innovation Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing 100084, China

    • Chengying Ma
    • , Yan Chen
    •  & Ning Gao
  2. Department of Biochemistry and Molecular Biology, Faculty of Agriculture and Life Science, Hirosaki University, Hirosaki 036-8561, Japan

    • Daisuke Kurita
    •  & Hyouta Himeno
  3. Peking-Tsinghua Center for Life Sciences, School of Life Sciences, Peking University, Beijing 100871, China

    • Ningning Li

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Contributions

H.H. and N.G. conceived the study; D.K. prepared proteins and performed peptide release assays; C.M., N.L., and Y.C. performed ribosome purification, sample preparation, data collection and image processing; N.G. wrote the paper, and all authors edited the paper.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Hyouta Himeno or Ning Gao.

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

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https://doi.org/10.1038/nature20822

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