The major 5' determinant in stop codon read-through involves two adjacent adenines.

The aim of this approach was to identify the major determinants, located at the 5' end of the stop codon, that modulate translational read-through in Saccharomyces cerevisiae. We developed a library of oligonucleotides degenerate at the six positions immediately upstream of the termination codon, cloned in the ADE2 reporter gene. Variations at these positions modulated translational read-through efficiency approximately 16-fold. The major effect was imposed by the two nucleotides immediately upstream of the stop codon. We showed that this effect was neither mediated by the last amino acid residues present in the polypeptide chain nor by the tRNA present in the ribosomal P site. We propose that the mRNA structure, depending on the nucleotides in the P site, is the main 5' determinant of read-through efficiency.

[1]  K. Flanigan,et al.  Sequence specificity of aminoglycoside‐induced stop codon readthrough: Potential implications for treatment of Duchenne muscular dystrophy , 2000, Annals of neurology.

[2]  O. Namy,et al.  Nonsense-mediated decay mutants do not affect programmed -1 frameshifting. , 2000, RNA.

[3]  I. Stansfield,et al.  Endless possibilities: translation termination and stop codon recognition. , 2001, Microbiology.

[4]  S. Peltz,et al.  The role of Upf proteins in modulating the translation read‐through of nonsense‐containing transcripts , 2001, The EMBO journal.

[5]  J. Burke,et al.  Suppression of a nonsense mutation in mammalian cells in vivo by the aminoglycoside antibiotics G-418 and paromomycin. , 1985, Nucleic acids research.

[6]  A. Mesecar,et al.  Genetic interaction between yeast Saccharomyces cerevisiae release factors and the decoding region of 18 S rRNA. , 2001, Journal of molecular biology.

[7]  M. Philippe,et al.  Poly(A)-Binding Protein Acts in Translation Termination via Eukaryotic Release Factor 3 Interaction and Does Not Influence [PSI+] Propagation , 2002, Molecular and Cellular Biology.

[8]  Ulrich Melcher,et al.  Predominance of six different hexanucleotide recoding signals 3' of read-through stop codons. , 2002, Nucleic acids research.

[9]  M. Gautschi,et al.  Nascent-polypeptide-associated complex , 2002, Cellular and Molecular Life Sciences CMLS.

[10]  Isabelle Hatin,et al.  Impact of the six nucleotides downstream of the stop codon on translation termination , 2001, EMBO reports.

[11]  G. C. Johnston,et al.  A yeast glutamine tRNA signals nitrogen status for regulation of dimorphic growth and sporulation. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[12]  G. Fink,et al.  A positive selection for mutants lacking orotidine-5′-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance , 1984, Molecular and General Genetics MGG.

[13]  S. Paushkin,et al.  Itt1p, a novel protein inhibiting translation termination in Saccharomyces cerevisiae , 2001, BMC Molecular Biology.

[14]  J. Rousset,et al.  Versatile vectors to study recoding: conservation of rules between yeast and mammalian cells. , 1995, Nucleic acids research.

[15]  E. Friedberg,et al.  Normal yeast tRNA(CAGGln) can suppress amber codons and is encoded by an essential gene. , 1986, Journal of molecular biology.

[16]  L. Isaksson,et al.  Only the last amino acids in the nascent peptide influence translation termination in Escherichia coli genes , 1997, FEBS letters.

[17]  Pavel V Baranov,et al.  Recoding: translational bifurcations in gene expression. , 2002, Gene.

[18]  P. Noirot,et al.  A new mutation delivery system for genome‐scale approaches in Bacillus subtilis , 2002, Molecular microbiology.

[19]  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.

[20]  D. Söll,et al.  Transfer RNA‐dependent cognate amino acid recognition by an aminoacyl‐tRNA synthetase. , 1996, The EMBO journal.

[21]  M. Ehrenberg,et al.  Regulatory Nascent Peptides in the Ribosomal Tunnel , 2002, Cell.

[22]  M. Tuite,et al.  The influence of 5' codon context on translation termination in Saccharomyces cerevisiae. , 1998, European journal of biochemistry.

[23]  K. Murata,et al.  Transformation of intact yeast cells treated with alkali cations. , 1984, Journal of bacteriology.

[24]  N. Sonenberg,et al.  A Novel Role of the Mammalian GSPT/eRF3 Associating with Poly(A)-binding Protein in Cap/Poly(A)-dependent Translation* , 2002, The Journal of Biological Chemistry.

[25]  H. Beier,et al.  Misreading of termination codons in eukaryotes by natural nonsense suppressor tRNAs. , 2001, Nucleic acids research.

[26]  J. F. Atkins,et al.  The signal for a leaky UAG stop codon in several plant viruses includes the two downstream codons. , 1991, Journal of molecular biology.

[27]  R. Buckingham,et al.  Translational termination comes of age. , 2000, TIBS -Trends in Biochemical Sciences. Regular ed.

[28]  L. Kisselev,et al.  Eukaryotic polypeptide chain release factor eRF3 is an eRF1- and ribosome-dependent guanosine triphosphatase. , 1996, RNA.

[29]  Daniel N. Wilson,et al.  Ribosomal crystallography: peptide bond formation and its inhibition. , 2003, Biopolymers.

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

[31]  D. Bedwell,et al.  Aminoglycoside antibiotics restore CFTR function by overcoming premature stop mutations , 1996, Nature Medicine.

[32]  G. Piluso,et al.  Gentamicin administration in Duchenne patients with premature stop codon. Preliminary results. , 2003, Acta myologica : myopathies and cardiomyopathies : official journal of the Mediterranean Society of Myology.

[33]  V. Ramakrishnan,et al.  Ribosome Structure and the Mechanism of Translation , 2002, Cell.

[34]  S. Peltz,et al.  Mtt1 is a Upf1-like helicase that interacts with the translation termination factors and whose overexpression can modulate termination efficiency. , 2000, RNA.

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

[36]  Jean-Pierre Rousset,et al.  UAG readthrough in mammalian cells: Effect of upstream and downstream stop codon contexts reveal different signals , 2001, BMC Molecular Biology.

[37]  D. Bedwell,et al.  The efficiency of translation termination is determined by a synergistic interplay between upstream and downstream sequences in Saccharomyces cerevisiae. , 1995, Journal of molecular biology.

[38]  L. Bossi,et al.  Context effects: translation of UAG codon by suppressor tRNA is affected by the sequence following UAG in the message. , 1983, Journal of molecular biology.

[39]  M. Philippe,et al.  Poly(A)‐binding protein and eRF3 are associated in vivo in human and Xenopus cells , 2002, Biology of the cell.

[40]  O. Namy,et al.  Translational readthrough of the PDE2 stop codon modulates cAMP levels in Saccharomyces cerevisiae , 2002, Molecular microbiology.

[41]  H. Sweeney,et al.  Aminoglycoside antibiotics restore dystrophin function to skeletal muscles of mdx mice. , 1999, The Journal of clinical investigation.