Fig. 5: Read-through of stop codon serves as an arrest inhibition mechanism for PepNL.

a The wild-type (UGA) or mutated (UAG) pepNL mRNA was translated by PUREfrex in the presence or absence of tryptophan (Trp), and the PepNL-arrested ribosome was visualized by toeprint analysis as shown in Fig. 3c. (#) b Schematics of pepNL mRNAs analyzed. c The pepNL mRNAs indicated in Fig. 5b were translated by PUREfrex in the absence (lanes 3 and 4) or presence of tryptophan (lanes 1, 2, 5, 6, 7, and 8). The ³⁵S-methionine-labeled translation products were separated by neutral pH SDS-PAGE with optional RNase A (RN) pretreatment. The truncated pepNL NS−1 (14 aa) and NS−2 (27 aa) mRNA were also analyzed to serve as size markers (lanes 5 to 8). The PepNL (14 aa) or PepNL (read-through: RT) peptidyl-tRNA and PepNL (14 aa) or PepNL (RT) peptide are schematically indicated. The asterisk denotes the fMet-tRNA. (#) d The pepNL-stop (UGA/UAA/UAG)-lacZ mRNA was expressed in E. coli cells, and the frequency of stop codon read-through was calculated as described in the Methods section. The mean values ± SE estimated from three independent biological replicates (n = 3) are shown. e The pepNL mRNA was translated by PUREfrex without tryptophan and release factors　for 30 min at 37°C. Afterward, a final 25 µM of tryptophan was added and further incubated for the indicated duration. The ³⁵S-methionine-labeled translation products were analyzed as shown in Fig. 5c. The asterisk denotes the fMet-tRNA. (#) f Schematic illustration of the Trp-tRNA^Trp−dependent inhibition of the PepNL-induced translation arrest. RF2 inefficiently terminates the translation of pepNL due to the steric clash shown in Fig. 4. However, in the presence of sufficient tryptophan, the Trp-tRNA^Trp decodes the UGA of pepNL, leading to the stop codon read-through. Two potential scenarios could explain this event: 1) Trp-tRNA^Trp initiates read-through before the hairpin folds, which could otherwise inhibit Trp-tRNA^Trp accommodation (Supplementary Fig. 7f); or 2) Trp-tRNA^Trp releases the ribosome stalled by the hairpin-shaped PepNL, alleviating the translation arrest at a moderate rate. (#) A representative of three independent experiments (n = 3) is shown.