In vitro translation

Translation was carried out at 37°C in buffer A with 25 nM purified initiation complex, 15 μM EF‐Tu, 2 μM EF‐G, 0.1 μM EF‐Ts, total aminoacyl‐tRNA containing 2.7 μM [¹⁴C]Leu‐tRNA^Leu (estimated 15 μM total aa‐tRNA), 1 mM GTP, 3 mM phosphoenolpyruvate, and 10 μg/ml pyruvate kinase, with or without 0.5 μM nanodisc‐embedded SecYEG. To obtain translation time courses, translation was quenched by the addition of 2% ammonia (final concentration), and peptidyl tRNA was hydrolyzed for 30 min at 37°C. Samples were then dried in a SpeedVac and dissolved in SDS‐loading buffer. Translation products were separated on 16% Tris‐Tricine SDS–PAGE (Schagger, 2006) and imaged on an FLA‐9000 fluorescence scanner (Fujifilm). Gels were analyzed using ImageJ software.

To determine translational efficiency, translation with Atto655‐labeled initiation complexes was carried out in the presence of 0.5 μM SecYEG as above. The samples were overlaid on 1.1 M sucrose solution, and RNCs were then purified from free aminoacyl‐tRNA by centrifugation at 180,000 g in a TLA‐100 rotor (Beckman) at 4°C for one hour. Pelleted RNCs were dissolved, the concentration of ribosomes was determined by UV absorbance (ɛ₂₆₀ = 43.5 μM⁻¹cm⁻¹), and the nascent chain concentration was determined by counting ³H‐Met and ¹⁴C‐Leu. Translation efficiency, calculated as the ratio of nascent chain per ribosome, was determined to be 79%, 76%, and 96% for EmrD135, EmrD(–)135, and LepB94, respectively.