Fig. 3: PepNL nascent peptide turns back toward the entrance of the ribosome tunnel.

a Serial dilution spot assay to assess the cytotoxicity upon overexpression of wild-type PepNL (WT) or its frameshift mutant (FS) in E. coli cells. b The PepNL-tRNA accumulated in E. coli cells expressing the indicated pepNL variants was detected by northern blotting using an anti-tRNA^Asp probe. An asterisk indicates the unprocessed rrnC or rrnH transcripts that include the unprocessed tRNA^Asp sequence. (#) c The wild-type or frameshifted pepNL mRNA was translated by PUREfrex in the absence of tryptophan, and the PepNL-arrested ribosome was visualized by toeprint analysis. Thiostrepton, which inhibits translation elongation, was pre-included where indicated. The pepNL mRNA was translated in the absence of release factors (RF⁻) to prepare the ribosomes stalled at the stop codon for the position marker (lane 5). (#) d Overall cross section of the cryo-EM density map at the peptide exit tunnel of the 70S ribosome (gray), showing P-tRNA (green), RF2 (cyan), and PepNL peptide (orange). e, f PepNL peptide and interacting 23S rRNA nucleotides in the ribosome exit tunnel. The N-terminus and C-terminus of PepNL were labeled as “N” and “C”, respectively. Close-up views of the intramolecular interactions within the PepNL peptide (g) and intermolecular interactions between the PepNL peptide and 23S rRNA nucleotides (h). g, panel 1: A hydrophobic interaction between Ile3 and Tyr9. g, panel 2: β-sheet-like interactions involving Lys2 with Ala10 and Leu4 with Ile8. h, panel 1: A hydrophobic interaction between Ile3 and U2609. h, panel 2: A hydrophobic interaction between Leu4 and A2062. h, panel 3: Hydrophobic interactions between Ile8 and A2058-A2059. h, panel 4: A hydrophobic interaction between Tyr9 and U2610. i Structural comparison of the PepNL nascent peptide (orange) with nascent peptides that lack RAP activity {PDB: 8CVJ (nascent peptide sequence: fMSEAC, pink) and 8CVL (fMTHSMRC, purple)}. j Serial dilution spot assay to assess the cytotoxicity upon overexpression of wild-type PepNL (WT) or its variants carrying the indicated amino acid substitution in E. coli cells. (#) A representative of three independent experiments (n = 3) is shown.