Gel Retardation Assay for tRNA-Protein Binding

The gel retardation assay was performed essentially as described (43 (link),45 (link)). Recombinant TrmO (15–45 pmol) and in vitro transcribed tRNA (15 pmol) were incubated at 37°C for 30 min in a 10 μl mixture containing 50 mM Tris-HCl (pH 8.0), 5 mM MgCl₂, 100 mM KCl, and 2 mM spermine. The tRNA–protein complex was resolved by 4% native polyacrylamide gel electrophoresis with a running buffer consisting of 50 mM Tris and 5 mM Mg(OAc)₂ (pH 8.0, adjusted with acetic acid). After electrophoresis, the gel was stained with SYBR gold (Invitrogen) to visualize tRNA, and then stained with Coomassie Brilliant Blue (Nacalai Tesque) to visualize the protein.