Structural basis for stop codon recognition in eukaryotes

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 bases. By contrast, eukaryotes rely on an evolutionarily unrelated omnipotent release factor (eRF1) to recognize all three stop codons. 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 termination.

[1]  Alan Brown,et al.  Structure and Assembly Pathway of the Ribosome Quality Control Complex , 2015, Molecular cell.

[2]  R. Henderson,et al.  Optimal determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy. , 2003, Journal of molecular biology.

[3]  R. Hegde,et al.  Listerin-Dependent Nascent Protein Ubiquitination Relies on Ribosome Subunit Dissociation , 2013, Molecular cell.

[4]  L. Frolova,et al.  Highly conserved NIKS tetrapeptide is functionally essential in eukaryotic translation termination factor eRF1. , 2002, RNA.

[5]  Chris M. Brown,et al.  The identity of the base following the stop codon determines the efficiency of in vivo translational termination in Escherichia coli. , 1995, The EMBO journal.

[6]  D. Bedwell,et al.  Therapeutics based on stop codon readthrough. , 2014, Annual review of genomics and human genetics.

[7]  A. Haenni,et al.  A highly conserved eukaryotic protein family possessing properties of polypeptide chain release factor , 1994, Nature.

[8]  R. Hegde,et al.  In vitro dissection of protein translocation into the mammalian endoplasmic reticulum. , 2010, Methods in molecular biology.

[9]  Matthew Mort,et al.  A meta‐analysis of nonsense mutations causing human genetic disease , 2008, Human mutation.

[10]  Conrad C. Huang,et al.  UCSF Chimera—A visualization system for exploratory research and analysis , 2004, J. Comput. Chem..

[11]  A. Spirin,et al.  Quantitative analysis of ribosome–mRNA complexes at different translation stages , 2009, Nucleic acids research.

[12]  Hemant D. Tagare,et al.  The Local Resolution of Cryo-EM Density Maps , 2013, Nature Methods.

[13]  M. Ruiz-Echevarría,et al.  The surveillance complex interacts with the translation release factors to enhance termination and degrade aberrant mRNAs. , 1998, Genes & development.

[14]  D. Barford,et al.  The Crystal Structure of Human Eukaryotic Release Factor eRF1—Mechanism of Stop Codon Recognition and Peptidyl-tRNA Hydrolysis , 2000, Cell.

[15]  Israel S. Fernández,et al.  Structure of the Mammalian Ribosome-Sec61 Complex to 3.4 Å Resolution , 2014, Cell.

[16]  M. Hentze,et al.  The role of ABCE1 in eukaryotic posttermination ribosomal recycling. , 2010, Molecular cell.

[17]  Sjors H.W. Scheres,et al.  Semi-automated selection of cryo-EM particles in RELION-1.3 , 2015, Journal of structural biology.

[18]  Vincent B. Chen,et al.  Correspondence e-mail: , 2000 .

[19]  R. Green,et al.  Cryoelectron Microscopic Structures of Eukaryotic Translation Termination Complexes Containing eRF1-eRF3 or eRF1-ABCE1 , 2014, Cell reports.

[20]  Sabine Petry,et al.  Insights into Translational Termination from the Structure of RF2 Bound to the Ribosome , 2008, Science.

[21]  K. Hopfner,et al.  X-ray Structure of the Complete ABC Enzyme ABCE1 from Pyrococcus abyssi* , 2008, Journal of Biological Chemistry.

[22]  T. Mielke,et al.  Cryo-EM of ribosomal 80S complexes with termination factors reveals the translocated cricket paralysis virus IRES. , 2015, Molecular cell.

[23]  L. Frolova,et al.  Three distinct peptides from the N domain of translation termination factor eRF1 surround stop codon in the ribosome. , 2010, RNA.

[24]  Patricia P. Chan,et al.  GtRNAdb: a database of transfer RNA genes detected in genomic sequence , 2008, Nucleic Acids Res..

[25]  A. Favre,et al.  The invariant uridine of stop codons contacts the conserved NIKSR loop of human eRF1 in the ribosome , 2002, The EMBO journal.

[26]  Alan Brown,et al.  Structure of the Yeast Mitochondrial Large Ribosomal Subunit , 2014, Science.

[27]  R. Green,et al.  The elongation, termination, and recycling phases of translation in eukaryotes. , 2012, Cold Spring Harbor perspectives in biology.

[28]  Jianyu Zhu,et al.  Crystal structure of a translation termination complex formed with release factor RF2 , 2008, Proceedings of the National Academy of Sciences.

[29]  L. Frolova,et al.  Selectivity of stop codon recognition in translation termination is modulated by multiple conformations of GTS loop in eRF1 , 2012, Nucleic acids research.

[30]  P. Kryuchkova,et al.  Two-step model of stop codon recognition by eukaryotic release factor eRF1 , 2013, Nucleic acids research.

[31]  Sjors H.W. Scheres,et al.  RELION: Implementation of a Bayesian approach to cryo-EM structure determination , 2012, Journal of structural biology.

[32]  H. Noller,et al.  Structural basis for translation termination on the 70S ribosome , 2008, Nature.

[33]  M. Selmer,et al.  Structure of the 70S Ribosome Complexed with mRNA and tRNA , 2006, Science.

[34]  L. Frolova,et al.  Conversion of omnipotent translation termination factor eRF1 into ciliate‐like UGA‐only unipotent eRF1 , 2002, EMBO reports.

[35]  D. Agard,et al.  Electron counting and beam-induced motion correction enable near atomic resolution single particle cryoEM , 2013, Nature Methods.

[36]  S. Scheres.  Beam-induced motion correction for sub-megadalton cryo-EM particles , 2014, eLife.

[37]  L. Frolova,et al.  Invariant amino acids essential for decoding function of polypeptide release factor eRF1 , 2005, Nucleic acids research.

[38]  S. Scheres,et al.  Ribosome structures to near-atomic resolution from thirty thousand cryo-EM particles , 2013, eLife.

[39]  R. Green,et al.  Kinetic analysis reveals the ordered coupling of translation termination and ribosome recycling in yeast , 2011, Proceedings of the National Academy of Sciences.

[40]  M. Nirenberg,et al.  Release factors differing in specificity for terminator codons. , 1968, Proceedings of the National Academy of Sciences of the United States of America.

[41]  R. Henderson,et al.  High-resolution noise substitution to measure overfitting and validate resolution in 3D structure determination by single particle electron cryomicroscopy☆ , 2013, Ultramicroscopy.

[42]  Chris M. Brown,et al.  Sequence analysis suggests that tetra-nucleotides signal the termination of protein synthesis in eukaryotes. , 1990, Nucleic acids research.

[43]  Richard J Jackson,et al.  Termination and post-termination events in eukaryotic translation. , 2012, Advances in protein chemistry and structural biology.

[44]  Alan Brown,et al.  Tools for macromolecular model building and refinement into electron cryo-microscopy reconstructions , 2015, Acta crystallographica. Section D, Biological crystallography.

[45]  V. Blinov,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. , 1999, RNA.

[46]  Kazuki Saito,et al.  Structural insights into eRF3 and stop codon recognition by eRF1. , 2009, Genes & development.

[47]  R. Hegde,et al.  Reconstitution of a Minimal Ribosome-Associated Ubiquitination Pathway with Purified Factors , 2014, Molecular cell.

[48]  L. Frolova,et al.  Optimal Translational Termination Requires C4 Lysyl Hydroxylation of eRF1 , 2014, Molecular cell.

[49]  J. Frank,et al.  Cryo-EM structure of the mammalian eukaryotic release factor eRF1–eRF3-associated termination complex , 2012, Proceedings of the National Academy of Sciences.