Radioactive tRNA Binding Assay

Lyophilized E. coli MRE 600 tRNA (Roche, Germany) was dissolved in ultrapure MilliQ water treated with diethylpyrocarbonate (DEPC). The 5′-ends of the tRNA molecules were labeled with [γ-³²P]-ATP using T4 polynucleotide kinase (Thermo Scientific, Lithuania). The 5′-labeled RNAs were incubated with target proteins for 30 min at 37 °C in a binding mixture, containing 20 mM Tris-HCl (pH 7.5), 300 mM KCl, 10 mM MgCl₂ or 10 mM CoCl₂, and 100 ng/µL bovine serum albumin, similarly as described by Kim et al. [5 (link)]. The final ratio of tRNA to protein was 1:100. After the incubation, samples were loaded onto 8% (v/v) native polyacrylamide gel. Electrophoresis was conducted at 150 V for 150 min at 1× TBE buffer. The visualization was performed by phosphorimaging (Fuji, Japan).