A small uORF with three start codons in perfect context is able to sequester all scanning initiation complexes in vitro. (A) Design of reinitiation-specific nanoLuciferase (nLuc) reporters used in this study. A 16 nt spacer between the variable-sized uORF and nLuc ORF allows specific detection of reinitiation. (B) Schematic of ribosome toeprinting with FAM-labeled primers to detect sites of initiation with lactimidomycin preincubation. (C,D) Ribosome toeprinting of 80S ribosomes after start codon recognition on the (C) small uORF nLuc reporter mRNA and (D) mutated uORF nLuc reporter mRNA from in vitro translation. Signal from unused primer is seen at 20 nt. Signal from duplicate samples is shown in black and red.
