To prepare ICs, 70S ribosomes were incubated with a 2-fold excess of mRNA, 1.7-fold excess of initiation factors, 3-fold excess of f[3H]Met-tRNAfMet, and 1 mM GTP in TAKM7 buffer [50 mM tris-HCl (pH 7.5) at 37°C, 70 mM NH4Cl, 30 mM KCl, and 7 mM MgCl2] for 30 min at 37°C (38). Ternary complex (TC) was prepared by incubating EF-Tu (threefold excess over tRNA) with 1 mM GTP, 3 mM phosphoenolpyruvate, and 0.5% pyruvate kinase in TAKM7 buffer for 15 min at 37°C and subsequent addition of aminoacyl-tRNAs cognate to the mRNA coding sequence. Pretranslocation complex (PRE) was formed by mixing IC and TC (two- to fivefold excess over IC). Purification of IC and PRE was performed by centrifugation through a 1.1 M sucrose cushion in TAKM21 buffer [50 mM tris-HCl (pH 7.5) at 37°C, 70 mM NH4Cl, 30 mM KCl, and 21 mM MgCl2]. Pellets were dissolved in TAKM21 buffer, and the concentration of purified complex was determined by nitrocellulose filtration (21). Posttranslocation complex (POST) was prepared by mixing purified PRE and EF-G (4 μM) and incubated for a minute at 37°C before it was used. The magnesium concentration of purified complexes was adjusted to working concentration before use. All experiments were carried out in TAKM7, unless stated otherwise.

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