Elongation factor G (EF-G) is a GTPase that is involved in the translocation of bacterial ribosomes along messenger RNA during protein biosynthesis. In contrast to current models, EF-G-dependent GTP hydrolysis is shown to precede, and greatly accelerate, the rearrangement of the ribosome that leads to translocation. Domain IV of the EF-G structure is crucial for both rapid translocation and subsequent release of the factor from the ribosome. By coupling the free energy of GTP hydrolysis to translocation, EF-G serves as a motor protein to drive the directional movement of transfer and messenger RNAs on the ribosome.

The translocation step of protein synthesis entails large-scale rearrangements of the ribosome-transfer RNA (tRNA) complex. Here we have followed tRNA movement through the ribosome during translocation by time-resolved single-particle electron cryomicroscopy (cryo-EM). Unbiased computational sorting of cryo-EM images yielded 50 distinct three-dimensional reconstructions, showing the tRNAs in classical, hybrid and various novel intermediate states that provide trajectories and kinetic information about tRNA movement through the ribosome. The structures indicate how tRNA movement is coupled with global and local conformational changes of the ribosome, in particular of the head and body of the small ribosomal subunit, and show that dynamic interactions between tRNAs and ribosomal residues confine the path of the tRNAs through the ribosome. The temperature dependence of ribosome dynamics reveals a surprisingly flat energy landscape of conformational variations at physiological temperature. The ribosome functions as a Brownian machine that couples spontaneous conformational changes driven by thermal energy to directed movement.

The kinetic mechanism of elongation factor Tu (EF-Tu