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The ribosome termination complex remodels release factor RF3 and ejects GDP

Nature Structural & Molecular Biology volume 31, pages 1909–1920 (2024)Cite this article

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Abstract

Translation termination involves release factors RF1, RF2 and the GTPase RF3 that recycles RF1 and RF2 from the ribosome. RF3 dissociates from the ribosome in the GDP-bound form and must then exchange GDP for GTP. The 70S ribosome termination complex (70S-TC) accelerates GDP exchange in RF3, suggesting that the 70S-TC can function as the guanine nucleotide exchange factor for RF3. Here, we use cryogenic-electron microscopy to elucidate the mechanism of GDP dissociation from RF3 catalyzed by the Escherichia coli 70S-TC. The non-rotated ribosome bound to RF1 remodels RF3 and induces a peptide flip in the phosphate-binding loop, efficiently ejecting GDP. Binding of GTP allows RF3 to dock at the GTPase center, promoting the dissociation of RF1 from the ribosome. The structures recapitulate the functional cycle of RF3 on the ribosome and uncover the mechanism by which the 70S-TC allosterically dismantles the phosphate-binding groove in RF3, a previously overlooked function of the ribosome.

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Fig. 1: Structure of the 70S ribosome termination complex bound to apo-RF3.
Fig. 2: Orientation of domain III in apo-RF3 triggers dissociation of GDP.
Fig. 3: Binding of GTP initiates domain rearrangements in RF3.
Fig. 4: Rotation of the 30S subunit positions the G-domain of RF3 near the SRL.
Fig. 5: Rolling-like motion of the 30S subunit activates RF3 for GTP hydrolysis.
Fig. 6: Model for the functional cycle of RF3 during translation termination.

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Data availability

The atomic coordinates have been deposited in the RCSB Protein Data Bank (PDB) under accession codes 8FZE (structure I-A; 70S–RF1), 8FZD (composite I-B; 70S–apo-RF3–RF1), 8FZF (composite I-C; 70S–RF3–ppGpp), 8FZG (composite II-A; 70S–RF3–GDPCP–RF1), 8FZI (composite II-B; 70S–RF3–GDPCP, non-activated conformation), 8FZJ (composite II-C; 70S–RF3–GDPCP, GTP-activated conformation) and 8FZH (structure II-D; 70S–RF1). The cryo-EM maps have been deposited in the Electron Microscopy Data Bank (EMDB) under accession codes EMD-29621 (structure I-A; 70S–RF1), EMD-29618 (structure I-B; 70S–apo-RF3–RF1 initial map), EMD-29619 (I-B: apo-RF3; from focused refinement), EMD-29620 (structure I-B; 70S–apo-RF3–RF1 composite), EMD-29622 (structure I-C; 70S–RF3–ppGpp initial map), EMD-29623 (I-C: RF3–ppGpp; from focused refinement), EMD-29624 (structure I-C; 70S–RF3–ppGpp composite), EMD-29625 (structure II-A; 70S–RF3–GDPCP–RF1 initial map), EMD-29626 (II-A: RF3–GDPCP; from focused refinement), EMD-29627 (structure II-A; 70S–RF3–GDPCP–RF1 composite), EMD-29629 (structure II-B; 70S–RF3–GDPCP initial map, non-activated conformation), EMD-29630 (II-B: RF3–GDPCP; from focused refinement, non-activated conformation), EMD-29631 (structure II-B; 70S–RF3–GDPCP composite, non-activated conformation), EMD-29632 (structure II-C; 70S–RF3–GDPCP initial map, GTP-activated conformation), EMD-29633 (II-C: RF3–GDPCP; from focused refinement, GTP-activated conformation), EMD-29634 (structure II-C; 70S–RF3–GDPCP composite, GTP-activated conformation), and EMD-29628 (structure II-D; 70S–RF1).

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Acknowledgements

We thank the members of the Gagnon and Lin laboratories for critical reading of the manuscript and suggestions. We are thankful to M. Sherman for help with cryo-EM data acquisition, the Sealy Center for Structural Biology and Molecular Biophysics at the University of Texas Medical Branch for providing critical infrastructure and expertise, and K.-Y. (Clem) Wong and J. Perkyns for computational support. We also thank the Cryo-Electron Microscopy at the Multiscale Research Institute of Complex Systems and the School of Life Sciences at Fudan University. This work was supported by grants from the National Key R&D Program of China (2017YFA0504602 to J.L.), the National Natural Science Foundation of China (no. 31770784 to J.L.), the Innovation Program of Shanghai Municipal Education Commission (2021-01-07-00-07-E00074 to J.L.), the Starry Night Science Fund at Shanghai Institute for Advanced Study of Zhejiang University (SN-ZJU-SIAS-009 to J.L.), the National Institutes of Health (R01GM136936 to M.G.G.), the Welch Foundation (H-2032-20230405 to M.G.G.), startup funds from the University of Texas Medical Branch (to M.G.G.), a National Center for Research Resources NIH grant P41RR001081 (to the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco) for developing UCSF Chimera, and NIH grant R01GM129325 (to the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco) and the Office of Cyber Infrastructure and Computational Biology, National Institute of Allergy and Infectious Diseases for developing UCSF ChimeraX.

Author information

Author notes

  1. These authors contributed equally: Li Li, Mariia Yu. Rybak.

Authors and Affiliations

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

    Li Li & Jinzhong Lin

  2. Center for mRNA Translational Research, Fudan University, Shanghai, China

    Li Li & Jinzhong Lin

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

    Mariia Yu. Rybak & Matthieu G. Gagnon

  4. Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX, USA

    Matthieu G. Gagnon

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

    Matthieu G. Gagnon

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

    Matthieu G. Gagnon

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  1. Li Li
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  2. Mariia Yu. Rybak
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  4. Matthieu G. Gagnon
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Contributions

J.L. and M.G.G. designed the project; L.L. purified 70S ribosomes, generated wild-type and mutants RF3 and performed ribosome binding assays; M.Y.R. generated wild-type and mutant RF1, purified tRNAPhe, performed stopped-flow experiments, prepared the samples for structure determination and collected the cryo-EM data. M.Y.R. and M.G.G. processed the cryo-EM data and built the models. J.L. and M.G.G. wrote the manuscript and made the figures with inputs from all authors. All authors reviewed, commented on and approved the final manuscript.

Corresponding authors

Correspondence to Jinzhong Lin or Matthieu G. Gagnon.

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Nature Structural & Molecular Biology thanks the anonymous reviewers for their contribution to the peer review of this work. Primary Handling Editor: Sara Osman, in collaboration with the Nature Structural & Molecular Biology team. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Cryo-EM data processing and particle classification workflow (dataset I with RF3-GDP and no exogenous nucleotide).

Scheme of the data processing workflow. All steps were performed in cryoSPARC 3.3.278. 10,284 micrographs were collected, of which 8,923 were selected for further processing. After two rounds of reference-free 2D classification, the selected particles were used to generate ab-initio volumes. Particles from the 3D volumes that appeared to be the 70S ribosome based on their shape and size were separated according to their ratcheted state using heterogeneous refinement, yielding three main classes of particles (class averages I, II, and III). Among the non-ratcheted 326,675 particles (class average I), focused 3D variability analysis around RF3 and RF1 was utilized to separate 80,766 particles for containing solid density for RF3 and RF1 or for RF1 only (127,567 particles). The process discarded 40,336 particles containing non-rotated ribosomes and 12,965 particles of rotated ribosomes, both of which were not bound to any factor. The remaining 65,041 particles yielded highly noisy reconstructions and were not further analyzed. Non-uniform and CTF refinement of particles with RF1 and RF3, or with RF1 only, produced structures I-B and I-A, respectively. Local refinement was combined with particle subtraction to produce a higher-quality map of RF3 and RF1 in structure I-B. Similarly, the rotated ribosome particles (74,452 particles in class average II) were separated by focused 3D variability analysis around RF3, producing two defined volumes, one containing rotated ribosome with no factor bound (28,078 particles) and one with rotated ribosome bound to RF3 (33,348 particles). Non-uniform and CTF refinement of the latter volume yielded structure I-C. Local refinement combined with particle subtraction produced a higher-quality map of RF3 in structure I-C.

Extended Data Fig. 2 Local resolution estimation and Fourier Shell Correlation (FSC) validation (dataset I with RF3-GDP and no exogenous nucleotide).

a, Local resolution heat maps on slices of density for structures I-A, I-B, and I-C shown in the range of 2.5 – 6.5 Å resolution, calculated with cryoSPARC 3.3.2 implementation of BlocRes87. b, Gold-standard FSC curves of each half-map (red), using a ‘soft mask’ excluding solvent and model-map (blue), are plotted across resolution. Map and model validation was performed in PHENIX 1.19.285. For structures I-B and I-C, for which composite maps were generated from EM maps of RF3-RF1 (I-B) or RF3 (I-C) obtained after focused refinement and the 70S ribosome, FSC curves for the composite EM map and the model vs composite map fit are shown. c, Local resolution maps shown in the range of 3.0 – 7.0 Å resolution and FSC curves of half-maps (masked with a ‘soft mask’) for RF3-RF1 (I-B) and for RF3 (I-C).

Extended Data Fig. 3 Cryo-EM data processing and particle classification workflow (dataset II with GDPCP).

All steps were performed in cryoSPARC 3.3.278. 9,810 micrographs were collected, of which 9,390 were selected for further processing. After two rounds of reference-free 2D classification, the selected particles were used to generate ab-initio volumes. Particles from the 3D volumes that appeared to be the 70S ribosome based on their shape and size were separated according to their ratcheted state using heterogeneous refinement, yielding three main classes of particles (class averages I, II, and III). Among the non-ratcheted 334,113 particles (class average I), focused 3D variability analysis around RF3 and RF1 was utilized to separate 31,256 particles for containing solid density for RF3 and RF1 or for RF1 only (146,778 particles). The process discarded 59,038 particles containing non-rotated ribosomes with weak density for RF3 and RF1, 58,392 particles containing non-rotated ribosomes with no factor bound, 8,460 particles of non-rotated ribosomes with only RF3 bound, and 30,189 particles that yielded noisy reconstructions. Non-uniform and CTF refinement of particles with RF1 and RF3, or with RF1 only, produced structures II-A and II-D, respectively. Local refinement was combined with particle subtraction to produce a higher-quality map of RF3 and RF1 in structure II-A. Similarly, the rotated ribosome particles (186,545 particles in class average II) were separated by focused 3D variability analysis around RF3, producing two defined volumes (56,202 and 116,992 particles), both of which containing RF3 in a distinct binding position. Non-uniform and CTF refinement of the latter two volumes yielded structures II-B and II-C. Local refinement combined with particle subtraction produced higher-quality maps of RF3 in structures II-B and II-C.

Extended Data Fig. 4 Local resolution estimation and Fourier Shell Correlation (FSC) validation (dataset II with GDPCP).

a, Local resolution heat maps on slices of density for structures II-A, II-B, II-C, and II-D shown in the range of 2.5 – 6.5 Å resolution, calculated with cryoSPARC 3.3.2 implementation of BlocRes87. b, Gold-standard FSC curves of each half-map (red), using a ‘soft mask’ excluding solvent and model-map (blue), are plotted across resolution. Map and model validation was performed in PHENIX 1.19.285. For structures II-A, II-B, and II-C, for which composite maps were generated from EM maps of RF3-RF1 (II-A), or RF3 (II-B and II-C) obtained after focused refinement and the 70S ribosome, FSC curves for the composite EM map and the model vs composite map fit are shown. c, Local resolution maps shown in the range of 3.0 – 7.0 Å resolution and FSC curves of half-maps (masked with a ‘soft mask’) for RF3-RF1 (II-A) and RF3 (II-B and II-C).

Extended Data Fig. 5 Interface between domain III of apo-RF3 and domain I of RF1 in structure I-B.

a, In apo-RF3, the residues for which mutation to alanine reduces the rate of GDP exchange are shown with yellow spheres. These residues form the interface with RF1. Mutation of the residues shown with light blue spheres has a mild effect on the rate of GDP exchange. Note that residue E516 does not interact with RF1. b, The Cα and Cβ atoms of residue N510 contribute to the interface with RF1, while the hydrophilic groups of N510 are not interacting with RF1.

Extended Data Fig. 6 Binding of RF3 to the 70S ribosome termination complex stabilizes domain I of RF1.

a, EM density of structure I-B bound to apo-RF3 and RF1 relative to that of structure I-A bound to RF1 only. b, Clear density for ribosomal proteins uL10 and uL11 in structure I-B bound to apo-RF3 and RF1 (mesh). In structure I-A, these proteins are more flexible as seen from the lack of density (gray surface). c, EM maps of RF1 in structures I-B (mesh) and I-A (gray surface). The presence of apo-RF3 in structure I-B stabilizes domain I of RF1. d, Additional density for the C-terminal domain of ribosomal protein bL12 is observed proximal to the α2-turn-α3 in RF1 and the G’ sub-domain in RF3. e, Relative to structure I-A, the tips of helices H43-H44 of the uL11-ribosomal stalk in structure I-B move toward domain I of RF1 by more than 3 Å. The α1-turn-α2 region in domain I of RF1 forms π-π-stacking interactions with the proline-rich 310-helix in the N-terminus of protein uL11, while nucleotides A1067 (H43) and A1095 (H44) stack along helix α2 of RF1.

Extended Data Fig. 7 Relative orientation of RF3-GDPCP in structure II-A versus that from a prior study (PDB 6GWT)3 (a), and potential steric clash between RF3-GDPCP in structure II-C with RF1 (taken from structure II-A) (b).

a, The structures are aligned based on the 16S rRNA. In the previous study, RF3-GDPCP (gray) is bound to a non-rotated ribosome with RF1 trapped by the antimicrobial peptide apidaecin (PDB 6GWT, state I)3. The orientation of domain III (gray RF3) is such that the closest distance to domain I of RF1 is ~9 Å, precluding the formation of any interaction3. b, The structures are aligned based on the 23S rRNA. On the rotated ribosome, RF3-GDPCP rotates by 27° as a whole between structures II-B (gray) and II-C (colored by domain) to dock against the sarcin-ricin loop (SRL) in the 50S subunit. In the GTP-activated state (II-C), domain III of RF3-GDPCP would clash with domain I of RF1 (red arrow), suggesting that complete accommodation of RF3 on the 50S subunit only occurs following dissociation of the class-I release factor.

Extended Data Fig. 8 Relative orientation of RF3 with GDPCP or GDPNP in previous structures versus that in structure II-B.

The structures are aligned based on the 23S rRNA. a, The conformation of RF3-GDPCP in structure II-B on the rotated ribosome (colored by domain) is highly similar to that of RF3-GDPCP reported in a previous study with the antimicrobial peptide apidaecin (state IV; rotated ribosome) (PDB 6GXO, gray)3. b, Same as in panel (a) relative to previous crystal structures of RF3-GDPNP bound to the rotated E. coli 70S ribosome (PDB 4V85)6 or RF3-GDPCP bound to the rotated Thermus thermophilus 70S ribosome (PDB 4V8O)7. c-f, Close-up views of the sarcin-ricin loop (SRL) and domain I of RF3 from (a) and (b). The catalytic histidine 92 in switch 2 (sw2) is located 9 to 13 Å away from the non-bridging oxygen of A2662 in the SRL, and RF3 is therefore in a GTP-inactive state on the ribosome. Switch 1 (sw1) is orange.

Extended Data Fig. 9 Structure of the ribosome bound to RF3-ppGpp.

a, Relative to the non-rotated ribosome in structure II-A (light gray), the ribosome has the rotated conformation in structure I-C bound to RF3-ppGpp (beige), similar to the ribosome bound to RF3-GDPCP in structure II-B (black). The structures are aligned by the 23S rRNA. b, Close-up view of ribosome-free RF3-ppGpp (PDB 3VR1, beige)26 and RF3-GDP (PDB 3VQT ref. 26, black; PDB 2H5E ref. 10, gray) superimposed with ribosome-bound RF3-ppGpp in structure I-C (colored domains). c, Orthogonal view of (a), showing that RF3-ppGpp is bound proximal to the sarcin-ricin loop (SRL, light green) in the 50S subunit. d, Cryo-EM map of the nucleotide-binding pocket. The density of ppGpp is shown as teal mesh, and that of the Mg2+ ions is shown as black mesh. e, The catalytic histidine 92 in switch 2 (sw2) is located within interaction distance from the β-phosphate non-bridging oxygen. The δ-phosphate oxygen of ppGpp forms a Mg2+-mediated hydrogen bond with A2662 in the SRL and the ε-phosphate interacts with a Mg2+ ion.

Supplementary information

Supplementary Information

Supplementary Figs. 1 and 2, Supplementary Tables 1–4, Uncropped gels for Supplementary Fig. 1a,b,d and Supplementary References

Reporting Summary

Peer Review File

Supplementary Data 1

List and sequences of DNA oligonucleotides used in this study.

Supplementary Data 2

Representative micrograph for apo-RF3 bound to the 70S ribosome termination complex.

Supplementary Data 3

Representative micrograph for RF3–GDPCP bound to the 70S ribosome termination complex.

Supplementary Video 1

Structural basis for the GDP-to-GTP exchange in RF3 catalyzed by the ribosomal termination complex. The animation is a morph between structures I-A, I-B, and II-A. The comparison of apo-RF3 in structure I-B with the structure of ribosome-free RF3-GDP (PDB 2H5E)10 illustrates the conformational changes in RF3 that lead to remodeling of the sw2-helix and of the phosphate-binding loop (P-loop). The flip of the carbonyl oxygen at P22 encroaches into the nucleotide-binding pocket and would collide with the β-phosphate of GDP, promoting its dissociation from RF3. Further comparison between apo-RF3 (I-B) and RF3-GDPCP in structure II-A shows the rotation of domain I of RF3 toward the 50S subunit SRL. Note the absence of interaction between RF3 and the 50S subunit in structures I-B and II-A.

Supplementary Video 2

Dissociation of RF1 catalyzed by RF3–GDPCP and activation of RF3 for GTP hydrolysis. The animation is a morph between structures II-A, II-B, and II-C. The movement of domain I in RF3 toward the SRL is facilitated by the ratchet-like rotation of the 30S subunit. In structure II-B, the catalytic histidine H92 in domain I of RF3 remains 11 Å away from the SRL, indicating that RF3–GDPCP in structure II-B is in a non-activated state. The rolling-like motion of the 30S subunit and the rotation of RF3–GDPCP in structure II-C bring domain I of RF3 proximal to the SRL. The catalytic H92 is within interaction distance from A2662 in the SRL, suggesting that RF3 is in the activated state primed for GTP hydrolysis.

Supplementary Data 4

Uncropped gels for Supplementary Fig. 1a,b,d.

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Li, L., Rybak, M.Y., Lin, J. et al. The ribosome termination complex remodels release factor RF3 and ejects GDP. Nat Struct Mol Biol 31, 1909–1920 (2024). https://doi.org/10.1038/s41594-024-01360-0

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