Mechanism of Translation Termination: RF1 Dissociation Follows Dissociation of RF3 from the Ribosome

Release factors 1 and 2 (RF1 and RF2, respectively) bind to ribosomes that have a stop codon in the A site and catalyze the release of the newly synthesized protein. Following peptide release, the dissociation of RF1 and RF2 from the ribosome is accelerated by release factor 3 (RF3). The mechanism for RF3-promoted dissociation of RF1 and RF2 is unclear. It was previously proposed that RF3 hydrolyzes GTP and dissociates from the ribosome after RF1 dissociation. Here we monitored directly the dissociation kinetics of RF1 and RF3 using Forster resonance energy transfer-based assays. In contrast to the previous model, our data show that RF3 hydrolyzes GTP and dissociates from the ribosome before RF1 dissociation. We propose that RF3 stabilizes the ratcheted state of the ribosome, which consequently accelerates the dissociation of RF1 and RF2.