Ribosome-dependent exchange of GDP to GTP on EF-G. (a) Scatchard plot from a nitrocellulose-filtration experiment to obtain the dissociation constant for the binding of [3H]-GDP to free EF-G. (b) Chase of [3H]-GDP from free EF-G by unlabeled GTP or, as a control, GDP. The dissociation constant for GTP binding to free EF-G was obtained from the corresponding constant for GDP binding in (a) and from the inhibition of [3H]-GDP binding to EF-G by GTP addition. The figure shows the results of two independent experiments (1 and 2). (c) Time-dependent release of fMet-Ile by 0.5 μM RF2 after translocation of fMet-Ile-tRNAIle from the A to the P site by a catalytic amount of EF-G (10 nM) added to 23 nM preT ribosomes together with 0.5 mM GTP and 0–0.8 mM GDP. CM(GTP) is the GTP concentration and I50 is the GDP concentration at which the rate of translocation is reduced to half-maximal value. (d) Inhibition of EF-G•GDPNP binding to post-termination (PostTerm) complexes or naked 70S ribosomes (Nakedribo) in the presence of 1 μM [3H]-GDPNP and 0–2 mM unlabeled GDP. (e) Fraction of [3H]-GDPNP (total concentration 1 μM) bound to EF-G• [3H]-GDPNP in postTerm complexes or in naked ribosomes as a function of time after addition of unlabeled GDP to a concentration of 2 mM. (f) Time-dependence of EF-G• [3H]-GDPNP binding to postTerm ribosomes in the presence of 1 μM [3H]-GDPNP: in the absence of RF2 (control), after addition of [3H]-GDPNP to EF-G pre-incubated with RF2 and postTerm ribosomes, or after addition of RF2 to EF-G pre-incubated with [3H]-GDPNP and postTerm ribosomes.