Termi-Luc: a versatile assay to monitor full-protein release from ribosomes

Competitive inhibition of eRF1-induced release of nanoluciferase from purified 80S pretermination complexes. (A) Luminescence in the presence of wild-type eRF1 or equimolar amounts of wild-type eRF1 and catalytically inactive eRF1^AGQ (0.6 µM). (B) Luminescence in the presence of wild-type eRF1 and different concentrations of G418. (C) Dependence of catalytic rates (k_obs) for eRF1-mediated release of nanoluciferase on G418 concentration. IC₅₀ was determined by hyperbola fitting (red) of k_obs values obtained at different concentrations of G418 and 0.6 µM human eRF1. Error bars represent standard deviation of the mean (n = 3). (D) Alignment of G418-bound 80S decoding center (red; Prokhorova et al. 2017) with that of eRF1-bound 80S termination complex carrying a UAA stop codon (gray; Brown et al. 2015) shows steric clash of G418 with A1824.