Phenylalanine specific e coli trna

Thermus thermophilus ribosomes were a kind gift from V. Ramakrishnan. His-tagged EF-G from T. thermophilus and His-tagged phenylalanine-tRNA synthetase (PheRS) from Escherichia coli were overexpressed in E. coli BL21 (DE3) using a T7 expression system and then isolated using Ni²⁺ pre-charged HiTrap chelating columns according to the manufacturer's instructions (GE Healthcare) (17 (link)). All protein concentrations were measured on a SimpliNano spectrophotometer (GE Healthcare). Phenylalanine-specific E. coli tRNA was purchased from Sigma. Aminoacylation was performed by incubating 1 μM tRNA at 37°C for 30 min in 50 mM HEPES-KOH (pH 7.5), 60 mM NH₄Cl, 7 mM MgCl₂, 0.1 mM ethylenediaminetetraacetic acid (EDTA), 2 mM ATP, 30 μM phenylalanine and 1 μM PheRS. Stalled ribosomes were obtained by incubating 1 μM 70S ribosomes with 2 μM E. coli aminoacylated tRNAPhe and 2 μM mRNA with the AGGAGGUGAGGUUUU sequence containing a Shine-Dalgarno sequence and a phenylalanine codon (underlined), for 30 min at 37°C in buffer III made of 5 mM HEPES-KOH (pH 7.5), 10 mM NH₄Cl, 10 mM MgOAc, 50 mM KCl, 0.1 mM EDTA and 6 mM β-mercaptoethanol. The final complexes were obtained by putting 1 μM of stalled ribosomes and 20 μM of EF-G with 2 mM GTP and mixing for 15 min at 42°C.