Dynamics of ribosomes and release factors during translation termination in E. coli

Release factors RF1 and RF2 promote hydrolysis of peptidyl-tRNA during translation termination. The GTPase RF3 promotes recycling of RF1 and RF2. Using single molecule FRET together with ensemble kinetics, we show that ribosome termination complexes that carry two factors, RF1–RF3 or RF2–RF3, are dynamic and fluctuate between non-rotated and rotated states, while each factor alone has its distinct signature on the ribosome dynamics and conformation. Dissociation of RF1 depends on peptide release and the presence of RF3, whereas RF2 can dissociate spontaneously. RF3 binds in the GTP-bound state and can rapidly dissociate without GTP hydrolysis from termination complex carrying RF1. GTP cleavage helps RF3 release from ribosomes stalled in the rotated state in the absence of RF1. Our data suggest how the stochastic assembly of the ribosome–RF1–RF3–GTP complex, peptide release, and ribosome fluctuations promote termination of protein synthesis and recycling of the release factors.