A single amino acid change of translation termination factor eRF1 switches between bipotent and omnipotent stop-codon specificity†

In eukaryotes a single class-1 translation termination factor eRF1 decodes the three stop codons: UAA, UAG and UGA. Some ciliates, like Euplotes, have a variant code, and here eRF1s exhibit UAR-only specificity, whereas UGA is reassigned as a sense codon. Since eukaryote eRF1 stop-codon recognition is associated with its N-terminal domain, structural features should exist in the N domain of ciliate eRF1s that restrict their stop-codon specificity. Using an in vitro reconstituted eukaryotic translation system we demonstrate here that a chimeric eRF1 composed of the N domain of Euplotes aediculatus eRF1 fused to the MC domains of human eRF1 exhibits UAR-only specificity. Functional analysis of eRF1 chimeras constructed by swapping Euplotes N domain sequences with the cognate regions from human eRF1 as well as site-directed mutagenesis of human eRF1 highlighted the crucial role of the alanine residue in position 70 of E. aediculatus eRF1 in restricting UGA decoding. Switching the UAR-only specificity of E. aediculatus eRF1 to omnipotent mode is due to a single point mutation. Furthermore, we examined the influence of eRF3 on the ability of chimeric and mutant eRF1s to induce peptide release in response to different stop codons.

[1]  Y. Inagaki,et al.  Convergence and constraint in eukaryotic release factor 1 (eRF1) domain 1: the evolution of stop codon specificity. , 2002, Nucleic acids research.

[2]  L. Frolova.  THE CRYSTAL STRUCTURE OF HUMAN EUKARYOTIC RELEASE FACTOR ERF1-MECHANISM OF STOP CODON RECOGNITION AND PEPTIDYL-TRNA HYDROLYSIS , 2000 .

[3]  T. Merkulova,et al.  C‐terminal domains of human translation termination factors eRF1 and eRF3 mediate their in vivo interaction , 1999, FEBS letters.

[4]  L. Klobutcher,et al.  Distinct Paths To Stop Codon Reassignment by the Variant-Code Organisms Tetrahymena and Euplotes , 2006, Molecular and Cellular Biology.

[5]  S. Thompson,et al.  Distinct eRF3 requirements suggest alternate eRF1 conformations mediate peptide release during eukaryotic translation termination. , 2008, Molecular cell.

[6]  I. Stansfield,et al.  Terminating eukaryote translation: domain 1 of release factor eRF1 functions in stop codon recognition. , 2000, RNA.

[7]  Yan Wang,et al.  Functional characterization of polypeptide release factor 1b in the ciliate Euplotes. , 2010, Bioscience reports.

[8]  A. Beaudet,et al.  Mammalian release factor; in vitro assay and purification. , 1974, Methods in Enzymology.

[9]  Andre R. O. Cavalcanti,et al.  Decoding the Decoding Region: Analysis of Eukaryotic Release Factor (eRF1) Stop Codon-Binding Residues , 2005, Journal of Molecular Evolution.

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

[11]  N. Go,et al.  Newly sequenced eRF1s from ciliates: the diversity of stop codon usage and the molecular surfaces that are important for stop codon interactions. , 2005, Gene.

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

[13]  M. Ehrenberg,et al.  Release factor RF3 in E.coli accelerates the dissociation of release factors RF1 and RF2 from the ribosome in a GTP‐dependent manner , 1997, The EMBO journal.

[14]  L. Kisselev,et al.  Termination of translation in eukaryotes is governed by two interacting polypeptide chain release factors, eRF1 and eRF3. , 1995, The EMBO journal.

[15]  P. Vlasov,et al.  Translation termination in pyrrolysine‐utilizing archaea , 2009, FEBS letters.

[16]  L. Frolova,et al.  Omnipotent decoding potential resides in eukaryotic translation termination factor eRF1 of variant-code organisms and is modulated by the interactions of amino acid sequences within domain 1 , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[17]  J. Rousset,et al.  Different modes of stop codon restriction by the Stylonychia and Paramecium eRF1 translation termination factors , 2007, Proceedings of the National Academy of Sciences.

[18]  H. Pelham.  Leaky UAG termination codon in tobacco mosaic virus RNA , 1978, Nature.

[19]  Lev L. Kisselev,et al.  In Vitro Reconstitution of Eukaryotic Translation Reveals Cooperativity between Release Factors eRF1 and eRF3 , 2006, Cell.

[20]  M. Gorovsky,et al.  An unusual genetic code in nuclear genes of Tetrahymena. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[21]  A. Hasilik,et al.  UGA is translated as cysteine in pheromone 3 of Euplotes octocarinatus. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[22]  J. Rousset,et al.  How does Euplotes translation termination factor eRF1 fail to recognize the UGA stop codon? , 2007, Molecular Biology.

[23]  I. Stansfield,et al.  The C‐terminus of eRF1 defines a functionally important domain for translation termination in Saccharomyces cerevisiae , 1999, Molecular microbiology.

[24]  D. Bedwell,et al.  GTP Hydrolysis by eRF3 Facilitates Stop Codon Decoding during Eukaryotic Translation Termination , 2004, Molecular and Cellular Biology.

[25]  L. Frolova,et al.  Class-1 translation termination factors: invariant GGQ minidomain is essential for release activity and ribosome binding but not for stop codon recognition. , 2001, Nucleic acids research.

[26]  T. Merkulova,et al.  Translation termination in eukaryotes: polypeptide release factor eRF1 is composed of functionally and structurally distinct domains. , 2000, RNA.

[27]  Yoshikazu Nakamura,et al.  Making sense of mimic in translation termination. , 2003, Trends in biochemical sciences.

[28]  Y. Nakamura,et al.  The stretch of C-terminal acidic amino acids of translational release factor eRF1 is a primary binding site for eRF3 of fission yeast. , 1998, RNA.

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

[30]  M. Ehrenberg,et al.  Termination of translation: interplay of mRNA, rRNAs and release factors? , 2003, The EMBO journal.

[31]  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.

[32]  C. Kitanaka,et al.  Molecular mechanism of stop codon recognition by eRF1: a wobble hypothesis for peptide anticodons , 2001, Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme.

[33]  I. Stansfield,et al.  The products of the SUP45 (eRF1) and SUP35 genes interact to mediate translation termination in Saccharomyces cerevisiae. , 1995, The EMBO journal.

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

[35]  Daniel N. Wilson,et al.  Molecular mimicry in the decoding of translational stop signals. , 2003, Progress in nucleic acid research and molecular biology.

[36]  O. Jean-Jean,et al.  Stop codon selection in eukaryotic translation termination: comparison of the discriminating potential between human and ciliate eRF1s , 2003, The EMBO journal.

[37]  G. Puca,et al.  Detection of a nonsense mutation in the dystrophin gene by multiple SSCP. , 1992, Human molecular genetics.

[38]  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.