Radioactive tRNA-Protein Binding Assay

tRNA^Gln was uniformly ³²P-labeled with α-³²P UTP (PerkinElmer, Japan, 3,000 Ci/mmol) using an in vitro transcription kit (Promega, Japan) and then gel-purified. The ³²P-labeled tRNA^Gln (10 000 cpm) was incubated in 10 μl of a solution containing 50 mM Tris–Cl, pH 7.4, 10 mM MgCl₂, 50 mM NaCl, 10 mM β-mercaptoethanol, 10% (v/v) glycerol and various amounts of CdiA–CT^EC869_H281A, Tu, or single-chain Tu-Ts (sg–Tu–Ts), at 37°C for 10 min. The solutions were then cooled on ice. The solutions were separated by 6% (w/v) native acrylamide gel electrophoresis (1 × TBE) at room temperature (∼25°C), as described (39 (link)). ³²P-labeled bands were quantified using a BAS-5000 imager (FujiFilm, Japan).