title: Elongation factor G stabilizes the hybrid-state conformation of the 70S ribosome.
abstract: Following peptide bond formation, transfer RNAs (tRNAs) and messenger RNA (mRNA) are translocated through the ribosome, a process catalyzed by elongation factor EF-G. Here, we have used a combination of chemical footprinting, peptidyl transferase activity assays, and mRNA toeprinting to monitor the effects of EF-G on the positions of tRNA and mRNA relative to the A, P, and E sites of the ribosome in the presence of GTP, GDP, GDPNP, and fusidic acid. Chemical footprinting experiments show that binding of EF-G in the presence of the non-hydrolyzable GTP analog GDPNP or GDP.fusidic acid induces movement of a deacylated tRNA from the classical P/P state to the hybrid P/E state. Furthermore, stabilization of the hybrid P/E state by EF-G compromises P-site codon-anticodon interaction, causing frame-shifting. A deacylated tRNA bound to the P site and a peptidyl-tRNA in the A site are completely translocated to the E and P sites, respectively, in the presence of EF-G with GTP or GDPNP but not with EF-G.GDP. Unexpectedly, translocation with EF-G.GTP leads to dissociation of deacylated tRNA from the E site, while tRNA remains bound in the presence of EF-G.GDPNP, suggesting that dissociation of tRNA from the E site is promoted by GTP hydrolysis and/or EF-G release. Our results show that binding of EF-G in the presence of GDPNP or GDP.fusidic acid stabilizes the ribosomal intermediate hybrid state, but that complete translocation is supported only by EF-G.GTP or EF-G.GDPNP.
authors.affiliation: Center for Molecular Biology of RNA, Department of Molecular, Cell and Developmental Biology, University of California-Santa Cruz 95064, USA.
authors.forename: P Clint
authors.initials: PC
authors.lastname: Spiegel
authors.forename: Dmitri N
authors.initials: DN
authors.lastname: Ermolenko
authors.forename: Harry F
authors.initials: HF
authors.lastname: Noller
mesh_terms: D004926:Escherichia coli; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D006868:Hydrolysis; D020653:Peptide Elongation Factor G / Q000737:chemistry / Q000502:physiology*; D014176:Protein Biosynthesis / Q000502:physiology*; D021381:Protein Transport; D012329:RNA, Bacterial / Q000737:chemistry / Q000378:metabolism*; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism; D012270:Ribosomes / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D020653:Peptide Elongation Factor G; D012329:RNA, Bacterial; D012333:RNA, Messenger; D006160:Guanosine Triphosphate