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Insights into the ribosome function from the structures of non-arrested ribosome–nascent chain complexes

Nature Chemistry volume 15, pages 143–153 (2023)Cite this article

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Abstract

During protein synthesis, the growing polypeptide threads through the ribosomal exit tunnel and modulates ribosomal activity by itself or by sensing various small molecules, such as metabolites or antibiotics, appearing in the tunnel. While arrested ribosome–nascent chain complexes (RNCCs) have been extensively studied structurally, the lack of a simple procedure for the large-scale preparation of peptidyl-tRNAs, intermediates in polypeptide synthesis that carry the growing chain, means that little attention has been given to RNCCs representing functionally active states of the ribosome. Here we report the facile synthesis of stably linked peptidyl-tRNAs through a chemoenzymatic approach based on native chemical ligation and use them to determine several structures of RNCCs in the functional pre-attack state of the peptidyl transferase centre. These structures reveal that C-terminal parts of the growing peptides adopt the same uniform β-strand conformation stabilized by an intricate network of hydrogen bonds with the universally conserved 23S rRNA nucleotides, and explain how the ribosome synthesizes growing peptides containing various sequences with comparable efficiencies.

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Fig. 1: Synthesis of peptidyl-tRNA by NCL.
Fig. 2: Synthesis of full-length non-hydrolysable peptidyl-tRNAs by NCL.
Fig. 3: Structure of the 70S ribosome in complex with fMSEAC-peptidyl-tRNA.
Fig. 4: Tight coordination of the PTC-proximal part of the nascent peptide in the NPET.
Fig. 5: Structure of the 70S ribosome in complex with fMRC- and fMTHSMRC-peptidyl-tRNAs.

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Data Availability

Coordinates and structure factors were deposited in the RCSB Protein Data Bank with the following accession codes: 8CVJ for the T. thermophilus 70S ribosome in complex with mRNA, aminoacylated A-site Phe-NH-tRNAPhe, peptidyl P-site fMSEAC-NH-tRNAiMet and deacylated E-site tRNAPhe; 8CVK for the T. thermophilus 70S ribosome in complex with mRNA, aminoacylated A-site Phe-NH-tRNAPhe, peptidyl P-site fMRC-NH-tRNAiMet and deacylated E-site tRNAPhe; 8CVL for the T. thermophilus 70S ribosome in complex with mRNA, aminoacylated A-site Phe-NH-tRNAPhe, peptidyl P-site fMTHSMRC-NH-tRNAiMet and deacylated E-site tRNAPhe. All previously published structures that were used in this work for model building and structural comparisons were retrieved from the RCSB Protein Data Bank: PDB entries 6XHW, 6WDD, 6TC3, 5NWY and 5DGV. Source data are provided with this paper.

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Acknowledgements

We thank R. Micura, A. Mankin, N. Vazquez-Laslop and M. Svetlov for critical reading of the manuscript and valuable suggestions. We thank I. Kamyshko for providing expertise in reagent and material sourcing. We thank the staff at NE-CAT beamlines 24ID-C and 24ID-E for help with data collection and freezing of the crystals, especially M. Capel, F. Murphy, S. Banerjee, I. Kourinov, D. Neau, J. Schuermann, N. Sukumar, A. Lynch, J. Withrow, K. Perry, A. Kaya and C. Salbego. This work is based upon 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 (P30-GM124165 to NE-CAT). The Eiger 16M detector on the 24-ID-E beamline is funded by an NIH-ORIP HEI grant (S10-OD021527 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 under contract number DE-AC02-06CH11357. This work was supported by the National Institutes of Health (R01-GM132302 and R21-AI163466 to Y.S.P.), the National Science Foundation (MCB-1907273 to Y.S.P.) and Illinois State start-up funds (to Y.S.P.). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

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Authors and Affiliations

  1. Department of Biological Sciences, University of Illinois at Chicago, Chicago, IL, USA

    Egor A. Syroegin, Elena V. Aleksandrova & Yury S. Polikanov

  2. Department of Pharmaceutical Sciences, University of Illinois at Chicago, Chicago, IL, USA

    Yury S. Polikanov

  3. Center for Biomolecular Sciences, University of Illinois at Chicago, Chicago, IL, USA

    Yury S. Polikanov

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  1. Egor A. Syroegin
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Contributions

E.A.S. with help from E.V.A. developed the NCL-based method for the synthesis of non-hydrolysable full-length peptidyl-tRNAs. E.A.S., E.V.A. and Y.S.P. designed and performed X-ray crystallography experiments. Y.S.P. supervised the experiments. All authors interpreted the results. E.A.S., E.V.A. and Y.S.P. wrote the manuscript.

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Correspondence to Yury S. Polikanov.

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Extended data

Extended Data Fig. 1 Native chemical ligation of Cys-NH-tRNAiMet with TBZ-activated peptides of various lengths and sequences.

(a) General information about the model peptides chosen for this study: macrolide-sensing ErmDL stalling peptide11,32; macrolide-resistance peptide MFLRC75; chloramphenicol-sensitive protein H-NS76; proline-rich antimicrobial peptide apidaecin77. ERY, erythromycin or other macrolides; CHL, chloramphenicol; RF, release factor. Peptidyl-tRNA products used in the structural studies are highlighted in grey. (b) Electrophoretic separation of crude NCL reaction mixtures. Indicated TBZ-activated peptides and Cys-NH-tRNAiMet were used as N- and C-terminal reactants, respectively. Electrophoresis was performed in 20-cm long 8% PAAG with 7 M urea and stained with ethidium bromide. Note the slower mobility of tRNA after the NCL that is also proportional to the ligated peptide length (lanes 2–8 vs. 1).

Source data

Extended Data Fig. 2 Native chemical ligation of non-hydrolyzable or native Cys-tRNACys with TBZ-activated fMSEA-peptide.

(a, b) Electrophoretic separations of deacylated, aminoacyl- and peptidyl-tRNACys before and after NCL reactions. Amino-tailed 3’-NH2-tRNACys (a, lane 1) or native 3’-HO-tRNACys (b, lane 1) were first aminoacylated using cysteine-specific aminoacyl-tRNA-synthetase (lanes 2) and then ligated with activated fMSEA-TBZ peptide for various times (lanes 3–5 in a and 3–7 in b) to yield either non-hydrolyzable fMSEAC-NH-tRNACys (a) or native ester-linked fMSEAC-O-tRNACys (b) peptidyl-tRNAs.

Source data

Extended Data Fig. 3 Comparison of the structures of fMSEAC-peptidyl-tRNA with aminoacylated full-length tRNAs.

(a, b) Superpositioning of our 70S ribosome structure carrying Phe-NH-tRNAPhe (green) and fMSEAC-NH-tRNAiMet (blue) in the A and P sites, respectively, with the previously reported structures of ribosome-bound full-length aminoacyl-tRNAs featuring either non-hydrolyzable amide linkages (a, PDB entry 6XHW28) or native ester bonds (b, PDB entry 6WDD19) between the amino acid moieties and the ribose of nucleotide A76 of A- and P-site tRNAs. All structures were aligned based on domain V of the 23S rRNA. (c, d) Comparisons of the positions of key 23S rRNA nucleotides around the PTC in the same structures. Note that there are no significant differences in the positions of A- or P-site substrates or the PTC nucleotides indicating that the amide-linked aminoacyl and peptidyl-tRNAs represent functionally meaningful analogs of native ester-linked tRNAs.

Extended Data Fig. 4 Tightly coordinated water molecules in the pre-attack state of the peptidyl transferase center.

(a-c) Close-up views of the 2Fo-Fc electron difference Fourier map (black mesh) for water molecules W1, W2, and W3 (yellow; nomenclature from20) in the pre-peptidyl-transfer complex structures featuring Phe-NH-tRNAPhe in the A site (omitted for clarity) and fMSEAC-NH-tRNAiMet (a, yellow), fMRC-NH-tRNAiMet (b, orange), or fMTHSMRC-NH-tRNAiMet (c, crimson) in the P site. H-bonds are shown by black dotted lines.

Extended Data Fig. 5 Ramachandran plots for the peptide moieties of the ribosome-bound peptidyl-tRNAs.

(a-c) Diagrams of the phi vs. psi angles for amino acid residues in fMSEAC-NH-tRNAiMet (a, yellow), fMRC-NH-tRNAiMet (b, orange), or fMTHSMRC-NH-tRNAiMet (c, crimson) in the P site. Favored regions are shown in green; allowed regions are light green. Due to the absence of fMet1 residue in the structure of fMSEAC-peptidyl-tRNA, phi angle for the subsequent Ser2 residue cannot be determined.

Extended Data Fig. 6 Comparison of the structures of fMSEAC-peptidyl-tRNA with the structures of other ribosome-bound peptidyl-tRNAs.

Superpositioning of the 70S ribosome structure containing A-site Phe-NH-tRNAPhe (green) and P-site fMSEAC-NH-tRNAiMet (blue) with the previously reported structures of stalled RNCCs carrying full-length peptidyl-tRNAs (a-c) or non-stalled RNCCs carrying short non-hydrolyzable tripeptidyl-tRNA analogs (d-f). Individual panels show comparisons of the fMSEAC-tripeptidyl-tRNA with the following peptides: (a) SpeFL (dark blue, PDB entry 6TC312); (b) ErmDL (teal, PDB entry 7NSO11), (c) VemP (cyan, PDB entry 5NWY13); (d) MAI-tripeptide (yellow, PDB entry 7RQB44); (e) MTI-tripeptide (orange, PDB entry 7RQA44), (f) MFI-tripeptide (red, PDB entry 7RQC44). All structures were aligned based on domain V of the 23S rRNA. Note that the overall path of the fMSEAC-peptide in our structure is similar to the trajectories of the other peptides in the NPET.

Extended Data Fig. 7 Additional interactions of the side chains of fMRC- and fMTHSMRC-peptidyl-tRNAs with the ribosome.

(a, b) Close-up views of the electrostatic interactions between the side chain of the penultimate Arg residue of the P-site fMRC-NH-tRNAiMet (a, blue with peptide highlighted in orange) or fMTHSMRC-NH-tRNAiMet (b, navy with peptide highlighted in crimson) and the phosphate of nucleotide G2505 of the 23S rRNA. H-bonds are shown by black dotted lines. (c) Stacking interactions between the aromatic side chain of His3 of fMTHSMRC-peptidyl-tRNA and A2062 nucleobase of the 23S rRNA.

Extended Data Fig. 8 Proline residues in the nascent peptide are unable to form stabilizing H-bonds in the NPET.

(a-c) In silico modeling of proline residues at ultimate (a, Cys5Pro), penultimate (b, Ala4Pro), or pen-penultimate (c, Glu3Pro) positions of the fMSEAC-peptide chain. Note that besides its inability to form most of the peptide-stabilizing H-bonds, proline in the penultimate and pen-penultimate positions clashes with nucleotides A2062 and U2506 of the 23S rRNA, respectively. Geometrically possible and impossible H-bonds are shown by black and white dotted lines, respectively.

Extended Data Fig. 9 Proline residues alter the path of the nascent peptide in the NPET.

(a) In silico modeling of the two consecutive proline residues at ultimate (Cys5Pro) and penultimate (Ala4Pro) positions of the fMSEAC-peptide chain. Note that, due to the side chains, the diproline-containing peptide cannot adopt a conformation possible for other peptides in the NPET and must re-orient. (b) Comparison of the previous structure of ribosome-bound short diprolyl-tRNA analog (green, PDB entry 5DGV53) with the in silico-modeled diprolyl-containing tRNA based on the structure of fMSEAC-peptidyl-tRNA (red). Note that in order to avoid a steric clash with the A2062, the diprolyl moiety of the nascent peptide deviates to the side (black dashed arrows) and, thus, has an alternative path in the NPET.

Extended Data Fig. 10 Formylation of the first methionine residue provides additional stability to the initiator tRNA substrate in the P site.

Superpositioning of the previous structure of ribosome-bound initiator fMet-NH-tRNAiMet (navy with the fMet moiety in red, PDB entry 6XHW28) with the new structure of fMSEAC-peptidyl-tRNA (blue with the peptide moiety highlighted in yellow) viewed from two opposite sides (a, b). H-bonds are shown by black dotted lines. Note that the positions of carbon and oxygen atoms in the formyl group and those in the carbonyl group of the penultimate residue in the nascent peptide chain are nearly identical, ensuring formation of the same H-bond with the exocyclic amino group of the G2061 residue.

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Unprocessed gels for Fig. 2b and Fig. 2c

Source Data Extended Data Fig. 1

Unprocessed gel for Extended Data Fig. 1b

Source Data Extended Data Fig. 2

Unprocessed gels for Extended Data Fig. 2a and Extended Data Fig. 2b

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Syroegin, E.A., Aleksandrova, E.V. & Polikanov, Y.S. Insights into the ribosome function from the structures of non-arrested ribosome–nascent chain complexes. Nat. Chem. 15, 143–153 (2023). https://doi.org/10.1038/s41557-022-01073-1

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