Figure 2

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-tRNA^Ile 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.