Removal of EF-G•GDPNP from the transition state with GDP. (a) Time-dependent cleavage of mRNA by 166 nM RelE in transT* complex in the presence of 2 μM EF-G and 0.32 mM GDPNP (GDPNP case) or after further addition of GDP to a concentration of 1 mM to remove EF-G from the ribosome (GDPNP + GDP case). In each case, GTP was added to a final concentration of 1 mM at 29 min to show the fraction of ribosomes that was active in translocation (lanes 3 and 6). (b,c) Time-dependent release of fMet-Ile by (b) 0.4 mM puromycin or (c) 0.5 μM RF2; 2 μM EF-G was pre-incubated with 46 nM preT complex and 40 μM GDPNP or polymix buffer for 3 min at 37°C. Then, buffer or 2 mM GDP was added and the incubation was continued for 1 min. Finally, (b) 0.4 mM puromycin or (c) 0.5 μM RF2 was added and the extent of peptide release was observed over time. (d) Exchange of [33P]-tRNAfMet on 88 nM preT complex, pre-incubated with 2 μM EF-G and 100 μM GDPNP or with buffer, with 2 μM tRNAfMet in the presence or absence of 2 mM GDP.