Translational readthrough-inducing drug (TRID) effects on eukaryotic termination investigated at the single-molecule level.

Abstract

Many genetic disorders are caused by premature stop codon (PSC) mutations, but only one TRID (Translational readthrough-inducing drug), ataluren, has been approved for clinical use. Recently, we used single-molecule TIRF with a cell-free in vitro assay and found that ataluren competitively inhibits productive release factor complex (RFC, eRF1.eRF3.GTP) binding to the pre-termination complex. We found that such inhibition occurs before or at the peptidyl-tRNA hydrolysis step (Huang et al., Nat. Comm., 2022, 13: 2413). Here we report new results using an sm-FRET assay with a Cy3-labeled peptidyl-tRNA bound in the ribosomal P-site adjacent to a UGA stop codon in the A-site and Cy5-labeled human eRF1. Upon RFC binding, we observe transient FRET efficiency, E = ∼0.25, between Cy5-eRF1 and Cy3-tRNA, consistent with the successful accommodation of eRF1 within the A-site. Following peptidyl-tRNA hydrolysis, we find a strong correlation between eRF1 and tRNA dissociation times, unlike the weak correlation between peptide release and tRNA dissociation times that we observed previously (Huang et al., ibid.). Additionally, we found that ataluren and added near-cognate suppressor tRNA each had a significant inhibitory effect on the arrival time of RFCs at the ribosome, and that ataluren in combination with the aminoglycoside G418 had an even stronger inhibitory effect compared with ataluren or G418 alone. These results support our earlier suggestion (Ng et al., PNAS, 2021, 118: e2020599118) that ataluren or ataluren-like TRIDs could potentiate the therapeutic effects of aminoglycosides on PSC diseases. We expect that our ongoing studies will aid in elucidating the readthrough mechanisms of other TRIDs leading to enhanced and safer treatments of PSC diseases.