Physical evidence for distinct mechanisms of translational control by upstream open reading frames.
The Saccharomyces cerevisiae GCN4 mRNA 5'-leader contains four upstream open reading frames (uORFs) and the CPA1 leader contains a single uORF. To determine how these uORFs control translation, we examined mRNAs containing these leaders in cell-free translation extracts to determine where ribosomes were loaded first and where they were loaded during steady-state translation. Ribosomes predominantly loaded first at GCN4 uORF1. Following its translation, but not the translation of uORF4, they efficiently reinitiated protein synthesis at Gcn4p. Adding purified eIF2 increased reinitiation at uORFs 3 or 4 and reduced reinitiation at Gcn4p. This indicates that eIF2 affects the site of reinitiation following translation of GCN4 uORF1 in vitro. In contrast, for mRNA containing the CPA1 uORF, ribosomes reached the downstream start codon by scanning past the uORF. Addition of arginine caused ribosomes that had synthesized the uORF polypeptide to stall at its termination codon, reducing loading at the downstream start codon, apparently by blocking scanning ribosomes, and not by affecting reinitiation. The GCN4 and CPA1 uORFs thus control translation in fundamentally different ways.
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Department of Biochemistry and Molecular Biology, OGI School of Science and Engineering, Oregon Health and Science University, 20 000 NW Walker Road, Beaverton, OR 97006-8921, USA.
Wang
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Krishnamoorthy
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Hinnebusch
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D020121:5' Untranslated Regions; D001483:Base Sequence; D001665:Binding Sites; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing); D002474:Cell-Free System; D003062:Codon; D017931:DNA Primers / Q000378:metabolism; D004268:DNA-Binding Proteins; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism; D005656:Fungal Proteins / Q000378:metabolism; D005786:Gene Expression Regulation; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009492:Neurospora crassa / Q000235:genetics; D016366:Open Reading Frames; D010455:Peptides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D014176:Protein Biosynthesis; D011494:Protein Kinases / Q000378:metabolism; D012313:RNA / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D013997:Time Factors
D020121:5' Untranslated Regions; D003062:Codon; D017931:DNA Primers; D004268:DNA-Binding Proteins; D015852:Eukaryotic Initiation Factor-2; D005656:Fungal Proteins; D010455:Peptides; D029701:Saccharomyces cerevisiae Proteins; D012313:RNA; D011494:Protein Kinases; C403521:CPA1 protein, S cerevisiae; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)