Release factor RF-3 GTPase activity acts in disassembly of the ribosome termination complex

Abstract

RF3 was initially characterized as a factor that stimulates translational termination in an in vitro assay. The factor has a GTP binding site and shows sequence similarity to elongation factors EF-Tu and EF-G. Paradoxically, addition of GTP abolishes RF3 stimulation in the classical termination assay, using stop triplets.We here show GTP hydrolysis, which is only dependent on the simultaneous presence of RF3 and ribosomes. Applying a new termination assay, which uses a minimessenger RNA instead of separate triplets, we show that GTP in the presence of RF3 stimulates termination at rate-limiting concentrations of RF1. We show that RF3 can substitute for EF-G in RRF-dependent ribosome recycling reactions in vitro. This activity is GTP-dependent. In addition, excess RF3 and RRF in the presence of GTP caused release of nonhydrolyzed fmet-tRNA. This supports previous genetic experiments, showing that RF3 might be involved in ribosomal drop off of peptidyl-tRNA. In contrast to GTP involvement of the above reactions, stimulation of termination with RF2 by RF3 was independent of the presence of GTP. This is consistent with previous studies, indicating that RF3 enhances the affinity of RF2 for the termination complex without GTP hydrolysis. Based on our results, we propose a model of how RF3 might function in translational termination and ribosome recycling.

Amber mutations in ribosome recycling factors of Escherichia coli and Thermus thermophilus: evidence for C‐terminal modulator element

“…Grentzmann et al [8] have observed similar results and found that RF3 can substitute for EF-G in RRFdependent ribosome recycling reactions in vitro. One contradiction that remains to be resolved is whether RRF is able to dissociate the mRNA or not.…”

Expression Profiling of Translation-associated Genes in SporulatingBacillus subtilisand Consequence of Sporulation by Gene Inactivation

“…[34][35][36] In addition, RRF maintains translational accuracy during the elongation process. 37) In B. subtilis, the frr gene was expressed at a moderately high level during the early stages of sporulation (Fig.…”

Interaction of RRF and EF-G from E. coli and T. thermophilus with ribosomes from both origins—insight into the mechanism of the ribosome recycling step

“…On the other hand, our finding that ec-RRF can work with EF-G from other species is consistent with the report that Mycobacterium tuberculosis EF-G (mt-EF-G) can complement an ec-EF-G temperature-sensitive mutant, indicating that mt-EF-G can function with ec-RRF for disassembly in vivo (Rao and Varshney 2001). This is not due to RF3 doing the role of EF-G in the RRF reaction because RF3 does not function in place of EF-G for disassembly of model post-termination complexes (data not shown) despite the fact that RF3 substituted EF-G for disassembly of complexes with artificial short nucleotides (Grentzmann et al 1998). Therefore, tt-EF-G must function with ec-RRF in vivo.…”