RELEASE FACTOR RF-3 GTPASE ACTIVITY ACTS IN DISASSEMBLY OF THE RIBOSOME TERMINATION COMPLEX

RF3 was initially characterized as a factor that stimulates translatio nal termination in an in vitro assay. The factor has a GTP binding sit e and shows sequence similarity to elongation factors EF-Tu and EF-G, Paradoxically, addition of GTP abolishes RF3 stimulation in the classi cal termination assay, using stop triplets. We here show GTP hydrolysi s, which is only dependent on the simultaneous presence of RF3 and rib osomes. Applying a new termination assay, which uses a minimessenger R NA instead of separate triplets, we show that GTP in the presence of R F3 stimulates termination at rate-limiting concentrations of RF1, We s how that RF3 can substitute for EF-G in RRF-dependent ribosome recycli ng reactions in vitro, This activity is GTP-dependent, In addition, ex cess RF3 and RRF in the presence of GTP caused release of nonhydrolyze d fmet-tRNA, This supports previous genetic experiments, showing that RF3 might be involved in ribosomal drop off of peptidyl-tRNA. In contr ast to GTP involvement of the above reactions, stimulation of terminat ion with RF2 by RF3 was independent of the presence of GTP. This is co nsistent with previous studies, indicating that RF3 enhances the affin ity of RF2 for the termination complex without GTP hydrolysis, Based o n our results, we propose a model of how RF3 might function in transla tional termination and ribosome recycling.