Fig. 4.

Vms1p exhibits tRNA hydrolase activity towards RQC substrates. a Time courses of S. cerevisiae in vitro translation (ScIVT) reactions prepared with a truncated mRNA (lacking a stop codon). Extract genotypes are indicated above. Peptides that have been CAT-tailed and released are denoted with a cat tail icon. b Quantification of peptidyl-tRNA species in a. Mean ± s.e.m.; n = 6. ^****P < 0.0001. The p value was calculated using a two-way ANOVA. c Time courses of ScIVT reactions prepared as in a. d Quantification of peptidyl-tRNA species in c. Mean ± s.e.m., n = 8. ^****P < 0.0001. The p value was calculated using a two-way ANOVA. e ScIVT reactions prepared as in a with a vms1Δ extract. At t = 15, buffer (−) or pure protein was added. Slopes indicate a titration series of decreasing protein concentrations (see Methods). FL full length Vms1, 1–417 N terminus through eRFL domain. f ScIVT reactions prepared as in a with a vms1Δ extract. At t = 15, buffer, WT (1–417) protein, or mutant (1–417) protein was added