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Structural basis for interaction of a cotranslational chaperone with the eukaryotic ribosome

Nature Structural & Molecular Biology volume 21, pages 10421046 (2014) | Download Citation

Abstract

Cotranslational chaperones, ubiquitous in all living organisms, protect nascent polypeptides from aggregation and facilitate their de novo folding. Importantly, emerging data have also suggested that ribosome-associated cotranslational chaperones have active regulatory roles in modulating protein translation. By characterizing the structure of a type of eukaryotic cotranslational chaperone, the ribosome-associated complex (RAC) from Saccharomyces cerevisiae, we show that RAC cross-links two ribosomal subunits, through a single long α-helix, to limit the predominant intersubunit rotation required for peptide elongation. We further demonstrate that any changes in the continuity, length or rigidity of this middle α-helix impair RAC function in vivo. Our results suggest a new mechanism in which RAC directly regulates protein translation by mechanically coupling cotranslational folding with the peptide-elongation cycle, and they lay the foundation for further exploration of regulatory roles of RAC in translation control.

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Acknowledgements

We thank J. Frank and H. Wang for helpful discussion and critical reading of the manuscript. We acknowledge the China National Center for Protein Sciences (Beijing) and the Explorer 100 cluster system of the Tsinghua National Laboratory for Information Science and Technology for providing computation resources. This work was supported by grants from the National Natural Science Foundation of China (31422016 and 31470722 to N.G.), the Ministry of Science and Technology of China (2010CB912402 and 2013CB910404 to N.G.; 2010CB912401 to J.L.) and the Beijing Higher Education Young Elite Teacher Project (YETP0131 to N.G.).

Author information

Affiliations

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

    • Yixiao Zhang
    • , Chengying Ma
    • , Yi Yuan
    • , Ningning Li
    • , Chu Chen
    • , Shan Wu
    • , Jianlin Lei
    •  & Ning Gao
  2. State Key Laboratory of Biomembrane and Membrane Biotechnology, Tsinghua University–Peking University Center for Life Sciences, Tsinghua University, Beijing, China.

    • Jing Zhu
    •  & Li Yu

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Contributions

Y.Z., J.L. and N.G. designed experiments and analyzed data. Y.Z. performed protein preparation, ribosome purification (together with C.M. and S.W.), data collection (together with Y.Y.), image processing (together with N.L.) and spot assays. J.Z., C.C. and L.Y. contributed to yeast-strain and plasmid construction. Y.Z. and N.G. wrote the manuscript; L.Y. commented on the manuscript; and all authors approved the final manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Jianlin Lei or Ning Gao.

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Supplementary information

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  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–6 and Supplementary Table 1

Videos

  1. 1.

    Cryo-EM structure of the yeast 80S–RAC complex in 10.2-Å resolution.

    The density map is from the dataset RAC6, which has relatively highest occupancies for both Zuotin and Ssz. The map is displayed in surface representation with segmented parts separately colored. The movie shows the overall orientation of Zuotin and Ssz on the ribosome.

  2. 2.

    The 4.9-Å cryo-EM map of the yeast 80S–RAC complex in nonrotated state.

    The density map is obtained by structural refinement of multiple datasets based on three-dimensional classification. The map shows the quality of the reconstruction. Due to substoichiometric binding and flexibility of factors, which results in fragmentation of factors in high-resolution structures, segmented map of RAC is presented in a lower contour level. After two rounds of RELION-based multiple-reference refinement, a 7.2 Å cryo-EM map of the yeast 80S-RAC complex in nonrotated state was obtained. Three ribosomal contacts (C1, C2 and C3) of RAC are shown, with five ribosomal components highlighted.

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DOI

https://doi.org/10.1038/nsmb.2908

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