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Structural basis for ribosome recycling by RRF and tRNA

Nature Structural & Molecular Biology volume 27, pages 25–32 (2020)Cite this article

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Abstract

The bacterial ribosome is recycled into subunits by two conserved proteins, elongation factor G (EF-G) and the ribosome recycling factor (RRF). The molecular basis for ribosome recycling by RRF and EF-G remains unclear. Here, we report the crystal structure of a posttermination Thermus thermophilus 70S ribosome complexed with EF-G, RRF and two transfer RNAs at a resolution of 3.5 Å. The deacylated tRNA in the peptidyl (P) site moves into a previously unsuspected state of binding (peptidyl/recycling, p/R) that is analogous to that seen during initiation. The terminal end of the p/R-tRNA forms nonfavorable contacts with the 50S subunit while RRF wedges next to central inter-subunit bridges, illuminating the active roles of tRNA and RRF in dissociation of ribosomal subunits. The structure uncovers a missing snapshot of tRNA as it transits between the P and exit (E) sites, providing insights into the mechanisms of ribosome recycling and tRNA translocation.

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Fig. 1: Structure of the 70S ribosome recycling complex.
Fig. 2: Interactions between EF-G, RRF and the ribosome.
Fig. 3: Interactions between the p/R-tRNA and the ribosome.
Fig. 4: Interactions between the TΨC loop of the p/R-tRNA and ribosomal protein uL5.
Fig. 5: Conformation of the p/R-tRNA compared to that of the p/I state during initiation.
Fig. 6: Implications for tRNA translocation on the 50S subunit during the elongation cycle.

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Data availability

Atomic coordinates and structure factors have been deposited in the PDB under the accession code 6UCQ.

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Acknowledgements

We thank M. Garcia-Blanco and H. Lander for critical reading of the manuscript and valuable suggestions. We are grateful to Y. Polikanov and M. White for their kind help and advice with the Oxford 700 series cryostream cooler. We also thank the staff of the Advanced Photon Source NE-CAT beamline 24-ID-C and of the Shanghai Synchrotron Radiation Facility beamline BL17U for help with data collection. This work was supported by grants from the National Key R&D Program of China (no. 2017YFA0504602) and the National Natural Science Foundation of China (no. 31770784) (to J.L.), and by startup funds from the University of Texas Medical Branch (to M.G.G.), by a McLaughlin Fellowship from the Institute for Human Infections and Immunity at the University of Texas Medical Branch (to M.G.G.), by a Rising Science and Technology Acquisition and Retention Program award (to M.G.G.) and by an endowment from Sealy and Smith Foundation to Sealy Center for Structural Biology at the University of Texas Medical Branch. 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 National Institutes of Health (grant no. P41-GM103403 to NE-CAT). The Pilatus 6M detector on 24-ID-C beamline is funded by the NIH–ORIP HEI Grant (no. S10-RR029205 to NE-CAT). This research used resources of the Advanced Photon Source, a US Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory (contract no. DE-AC02-06CH11357).

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Author notes

  1. These authors contributed equally: Dejian Zhou, Takehito Tanzawa.

Authors and Affiliations

  1. State Key Laboratory of Genetic Engineering, School of Life Sciences, Zhongshan Hospital, Fudan University, Shanghai, China

    Dejian Zhou & Jinzhong Lin

  2. Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas, USA

    Takehito Tanzawa & Matthieu G. Gagnon

  3. Sealy Center for Structural Biology and Molecular Biophysics, University of Texas Medical Branch, Galveston, Texas, USA

    Matthieu G. Gagnon

  4. Institute for Human Infections and Immunity, University of Texas Medical Branch, Galveston, Texas, USA

    Matthieu G. Gagnon

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  1. Dejian Zhou
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Contributions

D.Z. and T.T. performed experiments, analyzed the data and made the figures. J.L. and M.G.G. designed the study; purified ribosomes, protein factors and tRNAPhe; performed experiments, determined the structure, analyzed the data and wrote the manuscript with input from all authors. All authors discussed and commented on the final manuscript.

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Correspondence to Jinzhong Lin or Matthieu G. Gagnon.

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Extended data

Extended Data Fig. 1 Difference Fourier map of the 70S ribosome recycling complex and conformation of the ribosome.

a, Unbiased Fo – Fc difference electron density map contoured at 2.0 σ following the first round of refinement. Both deacylated tRNAs in the P and E sites, together with the mRNA, RRF, and EF-G were clearly visible. b, In the 70S ribosome recycling complex (orange), the 30S subunit rotates counter-clockwise by ~2° compared to the classical state ribosome (blue).

Extended Data Fig. 2 Conformation of RRF on the ribosome in the recycling complex.

a, Interactions between domain I of RRF and the ribosomal RNA. Shown are parts of H74 and H80 of the 23S rRNA (white), the CCA-end of the p/R-tRNA (pink), and domain I of RRF (blue). Key interacting residues in RRF are shown. b, Compared with the E. coli 70S ribosome bound to RRF (PDB 4V5413), domain II of RRF in the current structure rotates by ~28° and becomes wedged between the 23S rRNA helix H69 (orange) and ribosomal protein uS12 (magenta).

Extended Data Fig. 3 Difference Fourier map of the CCA-end of the p/R-tRNA.

a, Unbiased Fo – Fc map contoured at 2.0 σ of the 3′ end of the p/R-tRNA showing its location relative to the 23S rRNA helices H74 and H80. b, Rotated view of panel a with ribosomal elements omitted for clarity.

Extended Data Fig. 4 Conformation of anticodon stem loop (ASL) of tRNAs in the recycling complex.

a, Unstable state of binding of the p/R-tRNA ASL in the P site of the ribosome recycling complex. The unbiased Fo – Fc difference Fourier map contoured at 2.0 σ of the ASL of the p/R-tRNA shows that the electron density is much clearer for the nucleotide bases than for the phosphate backbone. b, Average Wilson B-factor values for the phosphate backbone (blue bars) and the nucleotide bases (orange bars) of the p/R-tRNA ASL. c, Movements of ASLs in the P and E site in the recycling complex compared to tRNAs bound in the classical state (gray).

Extended Data Fig. 5 Conformation of the p/R-tRNA compared to that of the p/I state during initiation.

Superimposition of the current recycling complex with that of a eukaryotic initiation 80S complex bound to eIF5B (orange) (PDB 4V8Z42) shows that like the IF2-70S initiation complex (Fig. 5), the conformation of the p/R-tRNA (magenta) resembles that of the p/I-tRNA (yellow). The same molecular mimic is observed between RRF/EF-G and eIF5B as that seen with IF2.

Supplementary information

Reporting Summary

Supplementary Video 1

Structure of the bacterial ribosome recycling complex. Animation showing the interactions between EF-G and RRF with the ribosome and the p/R-tRNA. The conformation of tRNAs in the recycling complex is compared with that of tRNAs bound in the canonical e/E and p/P states. The position of domain II of RRF is compared to a previous ribosome-bound RRF structure in the absence of EF-G (PDB 4V54, ref. 13). The RRF-mediated displacement of the P-site tRNA from the p/P to the p/R state is guided by ribosomal protein uL5 shown in green.

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Zhou, D., Tanzawa, T., Lin, J. et al. Structural basis for ribosome recycling by RRF and tRNA. Nat Struct Mol Biol 27, 25–32 (2020). https://doi.org/10.1038/s41594-019-0350-7

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