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.

Functional Elements in Initiation Factors 1, 1A, and 2β Discriminate against Poor AUG Context and Non-AUG Start Codons

“…Thus, SUI5 might stabilize the closed complex only in response to the UUGanticodon mismatch and be unable to compensate for the destabilizing effect of poor context. The SUI3-2 mutation in eIF2␤, by contrast, increases initiation from nearly all functional near-cognate triplets in vivo (21,46) in addition to its ability (shown here) to overcome poor context at SUI1.…”

How Ribosomes Translate Cancer

“…If fidelity is impaired, combining L-Leucine with a drug that corrects fidelity defects may be required. Indeed, drugs targeting ribosomal fidelity are an active field of research: drug screens have identified compounds that decrease the fidelity of start codon initiation (170). Moreover, ataluren (Translarna), a drug promoting premature stop-codon read-through (171), has received market authorization from the European Commission and is in clinical trials for treatment of diseases caused by nonsense mutations such as Duchenne muscular dystrophy and cystic fibrosis (172,173).…”

Conserved residues in yeast initiator tRNA calibrate initiation accuracy by regulating preinitiation complex stability at the start codon

“…We asked whether they also differ in their effects on utilization of different near-cognates by comparing expression of firefly luciferase reporters harboring different start codons normalized for expression of a renilla luciferase reporter bearing an AUG codon (Takacs et al 2011). Interestingly, the G31A:C39U ASL substitution elevates utilization of UUG, CUG, or GUG triplets, all first-base mismatches, but not AUA or ACG near-cognates with second-or third-base mismatches; whereas this bias does not exist for the G70A substitution in the acceptor stem (Supplemental Fig.…”