title: GCD10, a translational repressor of GCN4, is the RNA-binding subunit of eukaryotic translation initiation factor-3.
abstract: GCN4 mRNA is translated by a reinitiation mechanism involving four short upstream open reading frames (uORFs) in its leader sequence. Decreasing the activity of eukaryotic initiation factor-2 (eIF-2) by phosphorylation inhibits general translation in yeast but stimulates GCN4 expression by allowing ribosomes to scan past the uORFs and reinitiate at GCN4 instead. GCD10 was first identified genetically as a translational repressor of GCN4. We show here that GCD10 is an essential protein of 54.6 kD that is required in vivo for the initiation of total protein synthesis. GCD10 binds RNA in vitro and we present strong biochemical evidence that it is identical to the RNA-binding subunit of yeast initiation factor-3 (eIF-3). eIF-3 is a multisubunit complex that stimulates translation initiation in vitro at several different steps. We suggest that gcd10 mutations decrease the ability of eIF-3 to stimulate binding of eIF-2/GTP/Met-tRNA(iMet) ternary complexes to small ribosomal subunits in vivo. This would explain why mutations in eIF-3 mimic eIF-2 alpha phosphorylation in allowing ribosomes to bypass the uORFs and reinitiate at GCN4. Our results indicate that GCN4 expression provides a sensitive in vivo assay for the function of eIF-3 in initiation complex formation.
authors.affiliation: Instituto de Microbiologia-Bioquimica, Consejo Superior de Investigaciones Cientificas/Universidad de Salamanca, Facultad de Biologia, Spain.
authors.forename: M T
authors.initials: MT
authors.lastname: Garcia-Barrio
authors.forename: T
authors.initials: T
authors.lastname: Naranda
authors.forename: C R
authors.initials: CR
authors.lastname: Vazquez de Aldana
authors.forename: R
authors.initials: R
authors.lastname: Cuesta
authors.forename: A G
authors.initials: AG
authors.lastname: Hinnebusch
authors.forename: J W
authors.initials: JW
authors.lastname: Hershey
authors.forename: M
authors.initials: M
authors.lastname: Tamame
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins; D039621:Eukaryotic Initiation Factor-3; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005800:Genes, Fungal; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D016366:Open Reading Frames; D010448:Peptide Initiation Factors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D014176:Protein Biosynthesis; D011487:Protein Conformation; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D012313:RNA / Q000378:metabolism*; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins
chemical_list: D004268:DNA-Binding Proteins; D039621:Eukaryotic Initiation Factor-3; D005656:Fungal Proteins; D010448:Peptide Initiation Factors; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D012313:RNA; D011494:Protein Kinases