Preparation of 70S translocation complexes with or without EF-G•GDPCP

70S•mRNA•fMet-tRNA^fMet•Pro-tRNA^Pro(UGG)•EF-G•GDPCP complexes were prepared as follows, separately for the slippery and non-slippery mRNAs. In each, 0.33 µM 30S subunits (all concentrations specified for the final solution) were pre-activated at 42 °C for 5 min in the ribosome-reconstitution buffer (20 mM HEPES-KOH pH 7.5, 120 mM NH₄Cl, 20 mM MgCl₂, 2 mM spermidine, 0.05 mM spermine, 6 mM βME). These activated 30S subunits were added with 0.33 µM 50S subunits with 1.33 µM mRNA and incubated for 10 min at 37 °C. Subsequently, 0.33 µM fMet-tRNA^fMet was added and the solution was incubated for 3 min at 37 °C, to form the 70S complex with the P-site tRNA.

Pro-tRNA^Pro (UGG) (0.33 µM), EF-Tu (0.5 µM), and GTP (8.3 µM) were added to the solution and incubated for 10 min at 37 °C to form the A-site bound 70S complex. Next, EF-G (5.3 µM) and GDPCP (0.66 mM) were added and incubated for 5 min at 37 °C, then cooled down to room temperature, resulting in 70S translocation complexes with EF-G•GDPCP.

Pre-translocation 70S•mRNA•fMet-tRNA^fMet•Pro-tRNA^Pro(UGG) complex that yielded Structure I^rot-FS was prepared with the slippery mRNA as above excluding the addition of EF-G and GDPCP.