mRNA sequences influencing translation and the selection of AUG initiator codons in the yeast Saccharomyces cerevisiae

Authors

  • Ding-Fang Yun,

    1. Department of Biochemistry, University of Rochester, School of Medicine and Dentistry, Rochester, New York 14642, USA.
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    • These authors contributed equally to this work.

  • Thomas M. Laz,

    1. Department of Biochemistry, University of Rochester, School of Medicine and Dentistry, Rochester, New York 14642, USA.
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    • These authors contributed equally to this work.

    • Synaptic Pharmaceuticals, 21 5 College Road, Paramus, New Jersey, USA

  • John M. Clements,

    1. Department of Biochemistry, University of Rochester, School of Medicine and Dentistry, Rochester, New York 14642, USA.
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      British Bio-technology, Watlington Road, Cowley, Oxford OX4 5LY

  • Fred Sherman

    Corresponding author
    1. Department of Biochemistry, University of Rochester, School of Medicine and Dentistry, Rochester, New York 14642, USA.
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Summary

The secondary structure and sequences influencing the expression and selection of the AUG initiator codon in the yeast Saccharomyces cerevisiae were investigated with two fused genes, which were composed of either the CYC7 or CYC1 leader regions, respectively, linked to the lacZ coding region. In addition, the strains contained the upf1-Δ disruption, which stabilized mRNAs that had premature termination codons, resulting in wild-type levels. The following major conclusions were reached by measuring β-galactosidase activities in yeast strains having integrated single copies of the fused genes with various alterations in the 89 and 38 nucleotide-long untranslated CYC7 and CYC1 leader regions, respectively. The leader region adjacent to the AUG initiator codon was dispensable, but the nucleotide preceding the AUG initiator at position −3 modified the efficiency of translation by less than twofold, exhibiting an order of preference A>G>C>U. Upstream out-of-frame AUG triplets diminished initiation at the normal site, from essentially complete inhibition to approximately 50% inhibition, depending on the position of the upstream AUG triplet and on the context (−3 position nucleotides) of the two AUG triplets. In this regard, complete inhibition occurred when the upstream and downstream AUG triplets were closer together, and when the upstream and downstream AUG triplets had, respectively, optimal and suboptimal contexts. Thus, leaky scanning occurs in yeast, similar to its occurrence in higher eukaryotes. In contrast, termination codons between two AUG triplets causes reinitiation at the downstream AUG in higher eukaryotes, but not generally in yeast. Our results and the results of others with GCN4 mRNA and its derivatives indicate that reinitiation is not a general phenomenon in yeast, and that special sequences are required.

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