Methods.

Pretranslocation complexes were formed from fluorescently labeled ribosomes, a 39-nt transcribed mRNA, tRNA^Met in the P/E state, and N-Ac-Met-Val-tRNA^Val in the A/P state. S6-L9 ribosomes were used to measure intersubunit rotation, and S12-S19 and S11-S13 ribosomes were used to measure 30S head rotation. Overall translocation rates were measured using a 24-nt mRNA containing a 3′-terminal pyrene. Fluorescence changes over a single round of translocation following rapid mixing of the pretranslocation complex with EF-G and GTP (or nonhydrolyzable analog) were monitored using an Applied Photophysics SX20 stopped-flow fluorometer at 22 °C. GTP hydrolysis was blocked using the nonhydrolyzable analogs GDPNP and GDPCP or the H91L mutant of EF-G or slowed using the GTP analog GTPγS. Fusidic acid was introduced together with EF-G and GTP when used. Fluorescence traces were fit to single- or double-exponential functions to obtain apparent rate constants. Inhibition of hydrolysis of α-[³²P]-GTP was measured by thin-layer chromatography. Full details of materials and methods are presented in the SI Appendix.