title: Identification of compounds that decrease the fidelity of start codon recognition by the eukaryotic translational machinery.
abstract: Translation initiation in eukaryotes involves more than a dozen protein factors. Alterations in six factors have been found to reduce the fidelity of start codon recognition by the ribosomal preinitiation complex in yeast, a phenotype referred to as Sui(-). No small molecules are known that affect the fidelity of start codon recognition. Such compounds would be useful tools for probing the molecular mechanics of translation initiation and its regulation. To find compounds with this effect, we set up a high-throughput screen using a dual luciferase assay in S. cerevisiae. Screening of over 55,000 compounds revealed two structurally related molecules that decrease the fidelity of start codon selection by approximately twofold in the dual luciferase assay. This effect was confirmed using additional in vivo assays that monitor translation from non-AUG start codons. Both compounds increase translation of a natural upstream open reading frame previously shown to initiate translation at a UUG. The compounds were also found to exacerbate increased use of UUG as a start codon (Sui(-) phenotype) conferred by haploinsufficiency of wild-type eukaryotic initiation factor (eIF) 1, or by mutation in eIF1. Furthermore, the effects of the compounds are suppressed by overexpressing eIF1, which is known to restore the fidelity of start codon selection in strains harboring Sui(-) mutations in various other initiation factors. Together, these data strongly suggest that the compounds affect the translational machinery itself to reduce the accuracy of selecting AUG as the start codon.
authors.affiliation: Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
authors.forename: Julie E
authors.initials: JE
authors.lastname: Takacs
authors.forename: Timothy B
authors.initials: TB
authors.lastname: Neary
authors.forename: Nicholas T
authors.initials: NT
authors.lastname: Ingolia
authors.forename: Adesh K
authors.initials: AK
authors.lastname: Saini
authors.forename: Pilar
authors.initials: P
authors.lastname: Martin-Marcos
authors.forename: Jerry
authors.initials: J
authors.lastname: Pelletier
authors.forename: Alan G
authors.initials: AG
authors.lastname: Hinnebusch
authors.forename: Jon R
authors.initials: JR
authors.lastname: Lorsch
mesh_terms: D018387:Codon, Initiator / Q000502:physiology*; D015977:Eukaryotic Initiation Factor-1 / Q000235:genetics / Q000378:metabolism*; D057895:Haploinsufficiency; D006571:Heterocyclic Compounds / Q000494:pharmacology*; D008156:Luciferases / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D010442:Peptide Chain Initiation, Translational / Q000187:drug effects* / Q000502:physiology*; D010641:Phenotype; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D054682:Ribosome Subunits, Small, Eukaryotic / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D054852:Small Molecule Libraries
chemical_list: D018387:Codon, Initiator; D015977:Eukaryotic Initiation Factor-1; D006571:Heterocyclic Compounds; D012333:RNA, Messenger; C072076:SUI1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D054852:Small Molecule Libraries; D008156:Luciferases