Inverse toeprinting locates ribosomes on the mRNA with codon resolution. Inverse toeprinting locates ribosomes on the mRNA with codon resolution. (A) Comparison between inverse toeprinting, ribosome profiling, and classical toeprinting. (B) Schematic overview of the inverse toeprinting workflow. Restriction enzymes used in odd (EcoRV) and even (ApoI) cycles are shown in red and blue, respectively. Stop codons are indicated as asterisks. (C) Removal of inverse toeprints featuring ribosomes that have reached the stop codon on the ermBL template (white triangle) using the EcoRV restriction enzyme. The black triangle corresponds to arrested ribosomes and the gray triangle to full-length mRNA. (D) Inverse toeprints for various Erm peptides in the absence or presence of Ery, SecM150–166, and TnaC12–24UAA25. The wild-type UGA25 stop codon for TnaC was replaced with a UAA25 stop codon, allowing its release by RF-1. (E) Size distribution of inverse toeprints from two biological replicates (NoAb1 and NoAb2) with a minimum Q-score of 30 obtained from an NNS15 library translated in the absence of any added antibiotic. The fragment size range shaded in gray corresponds to the band that was cut from a 12% TBE-acrylamide gel, whereas the range in yellow indicates fragments that were used in the subsequent analysis. (F) Analysis of inverse toeprints containing stop codons that were obtained in the absence of antibiotic reveals that RNase R cleaves +17 nucleotides downstream from the P-site. Britta Seip et al. LSA 2018;1:e201800148 © 2018 Seip et al.