Properties of the transition state. (a) Time-dependent exchange of [33P]-tRNAfMet bound to the P site of 70 nM preT complex with 1 μM unlabeled tRNAfMet or tRNAPhe after the addition of 2 μM EF-G and 1 mM nucleotide. (b) The fraction of [33P]-tRNAfMet exchanged with tRNAfMet or tRNAPhe after 9 min incubation of 70 nM preT with 2 μM EF-G, 1 mM nucleotide and 0–2 μM tRNAfMet or tRNAPhe. (c) Fraction of [33P]-tRNAfMet on 88 nM preT ribosomes exchanged after 7 min incubation with 2 μM unlabeled tRNAfMet, 2 μM EF-G and 0–240 μM GDPNP to estimate the fraction of ribosomes containing EF-G•GDPNP. (d) Exchange of [33P]-tRNAfMet with 2 μM tRNAfMet or tRNAPhe added to 78 nM preT incubated with 2 μM EF-G, 0.4 nM GDPNP with or without 80 nM RelE. At 27.5 min, 1 mM GTP was added to translocate [33P]-tRNAfMet to the E site.