Termination of protein synthesis occurs when a translating ribosome encounters one of three universally conserved stop codons: UAA, UAG or UGA. Release factors recognize stop codons in the ribosomal A-site to mediate release of the nascent chain and recycling of the ribosome. Bacteria decode stop codons using two separate release factors with differing specificities for the second and third bases1. By contrast, eukaryotes rely on an evolutionarily unrelated omnipotent release factor (eRF1) to recognize all three stop codons2. The molecular basis of eRF1 discrimination for stop codons over sense codons is not known. Here we present cryo-electron microscopy (cryo-EM) structures at 3.5–3.8 Å resolution of mammalian ribosomal complexes containing eRF1 interacting with each of the three stop codons in the A-site. Binding of eRF1 flips nucleotide A1825 of 18S ribosomal RNA so that it stacks on the second and third stop codon bases. This configuration pulls the fourth position base into the A-site, where it is stabilized by stacking against G626 of 18S rRNA. Thus, eRF1 exploits two rRNA nucleotides also used during transfer RNA selection to drive messenger RNA compaction. In this compacted mRNA conformation, stop codons are favoured by a hydrogen-bonding network formed between rRNA and essential eRF1 residues that constrains the identity of the bases. These results provide a molecular framework for eukaryotic stop codon recognition and have implications for future studies on the mechanisms of canonical and premature translation termination3,4.
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Human mtRF1 terminates COX1 translation and its ablation induces mitochondrial ribosome-associated quality control
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We thank C. Savva, F. de Haas, and S. Welsch for assisting with cryo-EM data collection, J. Grimmett and T. Darling for computing support, D. Barford for critically reading the manuscript, and I. Fernández, J. Llácer, G. Murshudov, S. Scheres, and R. Voorhees for useful discussions. Gctf is available on request from K. Zhang (email@example.com). This work was supported by the UK Medical Research Council (MC_UP_A022_1007 to R.S.H. and MC_U105184332 to V.R.). A.B. was supported by a Career Development Fellowship. S.S. was supported by a St John’s College Title A fellowship. J.M. thanks T. Dever, NICHD, and the NIH Oxford-Cambridge Scholars’ Program for support. V.R. was supported by a Wellcome Trust Senior Investigator award (WT096570), the Agouron Institute, and the Jeantet Foundation.
The authors declare no competing financial interests.
Extended data figures and tables
a, Line diagrams of mRNA encoding nascent chain (NC) substrates used in this study. The cytosolic portion of human Sec61β (residues 1–68, orange) was modified to contain an N-terminal 3 × Flag tag (green) for affinity purification and the autonomously-folding villin headpiece (VHP, blue) domain. The three stop codons were individually inserted after Val68 of Sec61β to generate substrates for eRF1(AAQ)-mediated stalling, or the mRNA was truncated after the same residue to generate an independently-stalling substrate. b, In vitro translation reactions of NC-stop substrates containing the indicated stop codon (see panel a) in the presence of [35S]methionine without or with excess eRF1 WT or eRF1(AAQ). Reactions were for 25 min at 32 °C and directly analyzed by SDS–PAGE and auto-radiography. The terminated (NC) and tRNA-associated (NC-tRNA) nascent chain products are indicated. Addition of eRF1(AAQ) selectively prevents peptide hydrolysis when the stop codon is reached. c, Anti-Flag affinity purifications of ribosome-nascent chains (RNCs) stalled either by mRNA truncation or at the UAA stop codon with eRF1(AAQ) (see a) were immunoblotted for the splitting factors Hbs1 and ABCE1. The different amounts of Hbs1 and ABCE1 co-purified despite identical nascent chain sequences in each RNC complex suggest that eRF1(AAQ) selectively traps ABCE1 on pre-termination complexes. d, SDS–PAGE and Coomassie staining of affinity-purified eRF1(AAQ)-stalled ribosome-nascent chains containing the UGA stop codon used for cryo-EM analysis. Bands corresponding to ribosomal proteins, ABCE1, and eRF1(AAQ), which were verified by immunoblotting and mass spectrometry (data not shown), are indicated.
Particles extracted from automated particle picking in RELION were subjected to 2D classification. Non-ribosomal particles were discarded and the remaining particles were combined for a 3D refinement. The resulting map was used as a reference for 3D classification, which typically isolated 5 distinct classes of ribosomal complexes with the indicated distributions. Classes containing 80S ribosomes with canonical P- and E-site tRNAs and weak factor density in the A-site (∼40%) were combined and subjected to another round of 3D classification for A-site occupancy. Approximately one-third of this population contained strong density for eRF1(AAQ) and ABCE1. These particles were combined for subsequent 3D refinement and movie processing. All four data sets (two for the UAA stop codon and one each for the UAG and UGA stop codons) were processed similarly. The eRF1(AAQ)–ABCE1-containing particles of the two UAA data sets after the two rounds of classification were combined for refinement to yield the final map.
a, Fourier shell correlation (FSC) curves for the electron microscopy maps of each termination complex containing the indicated stop codon. b, Isolated eRF1(AAQ)–ABCE1 density from the UAA termination complex map coloured by local resolution. c, Fit of models to maps. FSC curves calculated between the refined model and the final map (black), and with the self- (blue) and cross-validated (magenta) correlations for each stop codon complex. The electron microscopy map of each termination complex coloured by local resolution (as in b) is displayed next to the corresponding curves.
a, Comparison of ribosome-bound eRF1(AAQ) (coloured by domain) with the crystal structure of eRF1 (PDB accession code 1DT9, grey) superposed on the C domain. Both the N and M domains of eRF1 rotate upon stop codon recognition on the ribosome. The P-site tRNA (green) and nascent chain (teal) are shown for orientation. b, Interaction of helix α2 of the N domain of eRF1(AAQ) (purple) with the anticodon stem loop (ASL) of the P-site tRNA (green). c, Superposition of the eRF1(AAQ) M domain (purple) with the eRF1 crystal structure (PDB accession code 1DT9) showing a 10 Å movement of the GGQ-loop to accommodate within the peptidyl transferase centre.
a, Density (from the UAG-containing termination complex) for the nascent chain (teal) attached to the CCA end of the P-site tRNA (green) is of sufficient resolution to model the defined sequence of the C-terminal end of the programmed nascent chain. This provides additional verification that the termination complexes are stalled at Val68 of Sec61β (human numbering) with the stop codon in the A-site (see also Extended Data Fig. 1a). A stacking interaction between an aromatic residue of the nascent chain and U4555 (blue) lining the ribosomal exit tunnel can also be observed. b, Densities for the interactions between the UAG stop codon (grey), a portion of h44 of 18S rRNA (yellow) and the YxCxxxF and NIKS motifs of eRF1(AAQ) (purple). The invariant isoleucine of the NIKS motif provides a hydrophobic base for the stacking of the +2 and +3 bases of the stop codon with A1825. Unlike the tyrosine and cysteine residues of the YxCxxxF motif, the phenylalanine does not contribute to stop codon recognition, but to the hydrophobic packing of the eRF1 N domain.
Chemical diagrams of uridine and cytidine with hydrogen bond donors (blue) and acceptors (magenta) indicated. Two of the three hydrogen bonds that uridine forms with Asn61 and Lys63 of the NIKS motif of eRF1(AAQ) (purple) are not possible with cytidine (see also Fig. 4a).
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Brown, A., Shao, S., Murray, J. et al. Structural basis for stop codon recognition in eukaryotes. Nature 524, 493–496 (2015). https://doi.org/10.1038/nature14896
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