title: Complete kinetic mechanism for recycling of the bacterial ribosome.
abstract: How EF-G and RRF act together to split a post-termination ribosomal complex into its subunits has remained obscure. Here, using stopped-flow experiments with Rayleigh light scattering detection and quench-flow experiments with radio-detection of GTP hydrolysis, we have clarified the kinetic mechanism of ribosome recycling and obtained precise estimates of its kinetic parameters. Ribosome splitting requires that EF-G binds to an already RRF-containing ribosome. EF-G binding to RRF-free ribosomes induces futile rounds of GTP hydrolysis and inhibits ribosome splitting, implying that while RRF is purely an activator of recycling, EF-G acts as both activator and competitive inhibitor of RRF in recycling of the post-termination ribosome. The ribosome splitting rate and the number of GTPs consumed per splitting event depend strongly on the free concentrations of EF-G and RRF. The maximal recycling rate, here estimated as 25 sec(-1), is approached at very high concentrations of EF-G and RRF with RRF in high excess over EF-G. The present in vitro results, suggesting an in vivo ribosome recycling rate of ∼5 sec(-1), are discussed in the perspective of rapidly growing bacterial cells.
authors.affiliation: Department of Cell and Molecular Biology, Biomedical Center, Uppsala University, 751 24 Uppsala, Sweden.
authors.forename: Anneli
authors.initials: A
authors.lastname: Borg
authors.affiliation: Department of Cell and Molecular Biology, Biomedical Center, Uppsala University, 751 24 Uppsala, Sweden.
authors.forename: Michael
authors.initials: M
authors.lastname: Pavlov
authors.affiliation: Department of Cell and Molecular Biology, Biomedical Center, Uppsala University, 751 24 Uppsala, Sweden ehrenberg@xray.bmc.uu.se.
authors.forename: Måns
authors.initials: M
authors.lastname: Ehrenberg
mesh_terms: D018407:Bacterial Physiological Phenomena; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D012270:Ribosomes / Q000378:metabolism / Q000502:physiology*
chemical_list: D006160:Guanosine Triphosphate
keywords: bacterial ribosome recycling; elongation factor G; protein synthesis; ribosome recycling factor; translation rate optimization