UC Santa Cruz

During protein synthesis, the ribosome must move the tRNAs and mRNA together in single codon steps after the addition of each amino acid to the polypeptide. This process of translocation is catalyzed by the GTPase elongation factor G in prokaryotes. EF-G hydrolyzes GTP during each round of translocation, yet the purpose of this energy expenditure is unclear. Here, we first ask how inhibiting GTP hydrolysis using GTP analogs and a mutant form of EF-G impacts the structural rearrangements of the ribosome that take place during translocation, monitored with Förster resonance energy transfer (FRET). We find that hydrolysis is required only for reverse rotation of the 30S head domain, an event that occurs late in translocation after the tRNAs and mRNA have completed the bulk of their movement. We then investigate the specific role and timing of phosphate (Pi) release from EF-G, which is delayed relative to hydrolysis and is the step responsible for the bulk of the energy derived from hydrolyzing GTP. We determine the timing of Pi release relative to the structural rearrangements of the ribosome by monitoring structural dynamics with FRET and in parallel observing the kinetics of Pi release with a fluorescence-based reporter. We find that Pi release occurs after forward head rotation and, surprisingly, is coupled to reverse intersubunit rotation. Further, we show that both Pi release and EF-G dissociation are required for reverse head rotation. To account for these findings, we propose that delayed Pi release prevents premature dissociation of EF-G; this ensures that the codon-anticodon duplex is stabilized by EF-G throughout its movement to prevent a frameshift. We conclude that the GTPase function of EF-G, rather than driving tRNA movement, is crucial for enforcing accuracy during translocation. This function may well extend to other translational GTPases such as IF2 and EF-Tu, which also exhibit delayed Pi release and have critical roles in enforcing accuracy during different steps of protein synthesis.