Translocation with catalytic amounts of EF-G (turnover reaction) and spontaneous translocation in the absence of EF-G were measured using the puromycin assay. Pretranslocation complexes carrying tRNAfMet in the P site and f[3H]Met[14C]Phe-tRNAPhe in the A site were prepared as described (30). Pretranslocation complexes (wild type or mutant) (0.1 μM) were incubated with a catalytic amount of EF-G (1 nM) or without EF-G in TAKM7 buffer [50 mM Tris-HCl (pH 7.5) 70 mM NH4Cl, 30 mM KCl, and 7 mM MgCl2]. Samples were taken and reacted with puromycin (1 mM) for 10 s before being quenched with 1.5 M sodium acetate (pH 4.5) saturated with MgSO4. f[3H]Met[14C]Phe-puromycin was extracted with ethyl acetate and quantified by radioactivity counting. Single-round translocation experiments were carried out in TAKM7 buffer containing 1 mM GTP in a stopped-flow apparatus (SX-20MV, Applied Photophysics) at 37°C. Pretranslocation complexes programmed with an Alexa Fluor 405–labeled mRNA (mMF14Alx405) (0.05 μM) were rapidly mixed with EF-G (4 μM). The dye was excited at 400 nm, and fluorescence was measured after passing a KV418 cutoff filter (Schott).