[35S]-Methionine incorporation assay

Wild type and nahA mutant cells were grown in defined S7 medium with glucose and 20 amino acids (concentrations listed in the “Plasmid and Strain Construction and Growth Conditions” section) to OD_600nm ~0.3. For amino acid starvation, cells were pelleted and resuspended in S7 glucose medium without amino acid. Alternatively, cells were treated with 0.5 mg/ml arginine hydroxamate. Aliquots of 200 μL culture were taken at indicated time points after treatment to label with 0.025 μCi/μL [³⁵S]-Methionine for 5 min, before adding 200 μL ice-chilled 20% (w/v) trichloroacetic acid. Samples were chilled before filtration. Glass fiber filters (24 mm, GF6, Whatman) were prewetted with 3 mL ice-chilled 5%(w/v) trichloroacetic acid and applied with samples. Filters were then washed three times by 3 × 10 mL ice-chilled 5%(w/v) trichloroacetic acid and dried by ethanol. Dried filters were put in scintillation vials, mixed with 5 mL scintillation fluid and then sent for scintillation count in the range of 2.0-18.6 eV. Counts per minutes (CPM) measured by scintillation counter, divided by the period of labeling and the OD_600nm of the corresponding culture, were used as the representative of translation rate.