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The mammalian mitochondrial genome contains two noncanonical stop codons, AGA and AGG. Here, the authors show that mtRF1, a mitochondrial protein of unknown function, triggers translation termination at AGA or AGG, placing mtRF1 among mitochondrial translation factors.

Ataluren is the only nonsense suppressor drug currently approved for clinical use. Here, the authors determine where ataluren binds to the ribosome and how it inhibits termination at nonsense codons.

Premature termination codons (PTCs) account for ~12% of all human disease mutations. Translation readthrough-inducing drugs (TRIDs) are prominent among the several therapeutic approaches being used to overcome PTCs. Ataluren is the only TRID that has been approved for treating patients suffering from a PTC disease, Duchenne muscular dystrophy, but it gives variable readthrough results in cells isolated from patients suffering from other PTC diseases. We recently elucidated ataluren’s mechanism of action as a competitive inhibitor of release factor complex (RFC) catalysis of premature termination and identified ataluren’s binding sites on the ribosome responsible for such an inhibition. These results suggest the possibility of discovering new TRIDs, which would retain ataluren’s low toxicity while displaying greater potency and generality in stimulating readthrough via the inhibition of termination. Here we present a detailed description of a new in vitro plate reader assay that we are using both to screen small compound libraries for the inhibition of RFC-dependent peptide release and to better understand the influence of termination codon identity and sequence context on RFC activity.