Letter | Published:

Structural basis for stop codon recognition in eukaryotes

Nature volume 524, pages 493496 (27 August 2015) | Download Citation


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.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.


Primary accessions

Electron Microscopy Data Bank

Data deposits

Maps have been deposited with the EMDB under accession codes 3038, 3039, and 3040. Atomic coordinates have been deposited with the Protein Data Bank under accession codes 3JAG, 3JAH and 3JAI.


  1. 1.

    , , & Release factors differing in specificity for terminator codons. Proc. Natl Acad. Sci. USA 61, 768–774 (1968)

  2. 2.

    et al. A highly conserved eukaryotic protein family possessing properties of polypeptide chain release factor. Nature 372, 701–703 (1994)

  3. 3.

    & The elongation, termination, and recycling phases of translation in eukaryotes. Cold Spring Harb. Perspect. Biol. 4, a013706 (2012)

  4. 4.

    , & Termination and post-termination events in eukaryotic translation. Adv. Protein Chem. Struct. Biol. 86, 45–93 (2012)

  5. 5.

    et al. Cryo-EM of ribosomal 80S complexes with termination factors reveals the translocated cricket paralysis virus IRES. Mol. Cell 57, 422–432 (2015)

  6. 6.

    et al. Cryo-EM structure of the mammalian eukaryotic release factor eRF1–eRF3-associated termination complex. Proc. Natl Acad. Sci. USA 109, 18413–18418 (2012)

  7. 7.

    et al. The role of ABCE1 in eukaryotic posttermination ribosomal recycling. Mol. Cell 37, 196–210 (2010)

  8. 8.

    & Kinetic analysis reveals the ordered coupling of translation termination and ribosome recycling in yeast. Proc. Natl Acad. Sci. USA 108, E1392–E1398 (2011)

  9. 9.

    et al. Mutations in the highly conserved GGQ motif of class 1 polypeptide release factors abolish ability of human eRF1 to trigger peptidyl-tRNA hydrolysis. RNA 5, 1014–1020 (1999)

  10. 10.

    et al. Cryoelectron microscopic structures of eukaryotic translation termination complexes containing eRF1-eRF3 or eRF1-ABCE1. Cell Rep. 8, 59–65 (2014)

  11. 11.

    et al. The crystal structure of human eukaryotic release factor eRF1—mechanism of stop codon recognition and peptidyl-tRNA hydrolysis. Cell 100, 311–321 (2000)

  12. 12.

    et al. Structural basis for translation termination on the 70S ribosome. Nature 454, 852–857 (2008)

  13. 13.

    et al. Insights into translational termination from the structure of RF2 bound to the ribosome. Science 322, 953–956 (2008)

  14. 14.

    et al. Crystal structure of a translation termination complex formed with release factor RF2. Proc. Natl Acad. Sci. USA 105, 19684–19689 (2008)

  15. 15.

    , , & Sequence analysis suggests that tetra-nucleotides signal the termination of protein synthesis in eukaryotes. Nucleic Acids Res. 18, 6339–6345 (1990)

  16. 16.

    et al. Quantitative analysis of ribosome–mRNA complexes at different translation stages. Nucleic Acids Res. 38, e15 (2010)

  17. 17.

    et al. Two-step model of stop codon recognition by eukaryotic release factor eRF1. Nucleic Acids Res. 41, 4573–4586 (2013)

  18. 18.

    , & The identity of the base following the stop codon determines the efficiency of in vivo translational termination in Escherichia coli. EMBO J. 14, 151–158 (1995)

  19. 19.

    , , & The invariant uridine of stop codons contacts the conserved NIKSR loop of human eRF1 in the ribosome. EMBO J. 21, 5302–5311 (2002)

  20. 20.

    et al. Three distinct peptides from the N domain of translation termination factor eRF1 surround stop codon in the ribosome. RNA 16, 1902–1914 (2010)

  21. 21.

    , & Highly conserved NIKS tetrapeptide is functionally essential in eukaryotic translation termination factor eRF1. RNA 8, 129–136 (2002)

  22. 22.

    et al. Optimal translational termination requires C4 lysyl hydroxylation of eRF1. Mol. Cell 53, 645–654 (2014)

  23. 23.

    et al. Invariant amino acids essential for decoding function of polypeptide release factor eRF1. Nucleic Acids Res. 33, 6418–6425 (2005)

  24. 24.

    , , , & Selectivity of stop codon recognition in translation termination is modulated by multiple conformations of GTS loop in eRF1. Nucleic Acids Res. 40, 5751–5765 (2012)

  25. 25.

    et al. Structural insights into eRF3 and stop codon recognition by eRF1. Genes Dev. 23, 1106–1118 (2009)

  26. 26.

    , & Conversion of omnipotent translation termination factor eRF1 into ciliate-like UGA-only unipotent eRF1. EMBO Rep. 3, 881–886 (2002)

  27. 27.

    et al. The surveillance complex interacts with the translation release factors to enhance termination and degrade aberrant mRNAs. Genes Dev. 12, 1665–1677 (1998)

  28. 28.

    , , & Therapeutics based on stop codon readthrough. Annu. Rev. Genomics Hum. Genet. 15, 371–394 (2014)

  29. 29.

    , , & A meta-analysis of nonsense mutations causing human genetic disease. Hum. Mutat. 29, 1037–1047 (2008)

  30. 30.

    , & Listerin-dependent nascent protein ubiquitination relies on ribosome subunit dissociation. Mol. Cell 50, 637–648 (2013)

  31. 31.

    , , & in Protein Secretion 619, 339–363 (Humana Press, 2010)

  32. 32.

    & Reconstitution of a minimal ribosome-associated ubiquitination pathway with purified factors. Mol. Cell 55, 880–890 (2014)

  33. 33.

    , , & Ribosome structures to near-atomic resolution from thirty thousand cryo-EM particles. eLife 2, e00461 (2013)

  34. 34.

    et al. Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM. Nature Methods 10, 584–590 (2013)

  35. 35.

    Semi-automated selection of cryo-EM particles in RELION-1.3. J. Struct. Biol. 189, 114–122 (2015)

  36. 36.

    RELION: Implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180, 519–530 (2012)

  37. 37.

    Beam-induced motion correction for sub-megadalton cryo-EM particles. eLife 3, e03665 (2014)

  38. 38.

    et al. High-resolution noise substitution to measure overfitting and validate resolution in 3D structure determination by single particle electron cryomicroscopy. Ultramicroscopy 135, 24–35 (2013)

  39. 39.

    & Optimal determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy. J. Mol. Biol. 333, 721–745 (2003)

  40. 40.

    , & Quantifying the local resolution of cryo-EM density maps. Nature Methods 11, 63–65 (2014)

  41. 41.

    , , & Structure of the mammalian ribosome-Sec61 complex to 3.4 Å resolution. Cell 157, 1632–1643 (2014)

  42. 42.

    , , & Structure and assembly pathway of the ribosome quality control complex. Mol. Cell 57, 433–444 (2015)

  43. 43.

    et al. UCSF Chimera–a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004)

  44. 44.

    , & X-ray structure of the complete ABC enzyme ABCE1 from Pyrococcus abyssi. J. Biol. Chem. 283, 7962–7971 (2008)

  45. 45.

    et al. Tools for macromolecular model building and refinement into electron cryo-microscopy reconstructions. Acta Crystallogr. D 71, 136–153 (2015)

  46. 46.

    Structure of the 70S ribosome complexed with mRNA and tRNA. Science 313, 1935–1942 (2006)

  47. 47.

    & GtRNAdb: a database of transfer RNA genes detected in genomic sequence. Nucleic Acids Res. 37, D93–D97 (2009)

  48. 48.

    et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D 66, 12–21 (2010)

  49. 49.

    et al. Structure of the yeast mitochondrial large ribosomal subunit. Science 343, 1485–1489 (2014)

Download references


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 (kzhang@mrc-lmb.cam.ac.uk). 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.

Author information

Author notes

    • Alan Brown
    •  & Sichen Shao

    These authors contributed equally to this work


  1. MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK

    • Alan Brown
    • , Sichen Shao
    • , Jason Murray
    • , Ramanujan S. Hegde
    •  & V. Ramakrishnan


  1. Search for Alan Brown in:

  2. Search for Sichen Shao in:

  3. Search for Jason Murray in:

  4. Search for Ramanujan S. Hegde in:

  5. Search for V. Ramakrishnan in:


A.B., S.S., R.S.H. and V.R. designed the study. S.S. purified complexes and prepared samples. A.B., S.S. and J.M. collected data. A.B. calculated the cryo-EM reconstructions, built the atomic models and interpreted the structure. A.B., S.S., R.S.H and V.R. wrote the manuscript. All authors discussed and commented on the final manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Ramanujan S. Hegde or V. Ramakrishnan.

Extended data

About this article

Publication history






Further reading


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.