title: Conformationally restricted elongation factor G retains GTPase activity but is inactive in translocation on the ribosome.
abstract: Elongation factor G (EF-G) from Escherichia coli is a large, five-domain GTPase that promotes tRNA translocation on the ribosome. Full activity requires GTP hydrolysis, suggesting that a conformational change of the factor is important for function. To restrict the intramolecular mobility, two cysteine residues were engineered into domains 1 and 5 of EF-G that spontaneously formed a disulfide cross-link. Cross-linked EF-G retained GTPase activity on the ribosome, whereas it was inactive in translocation as well as in turnover. Both activities were restored when the cross-link was reversed by reduction. These results strongly argue against a GTPase switch-type model of EF-G function and demonstrate that conformational mobility is an absolute requirement for EF-G function on the ribosome.
authors.affiliation: Institute of Molecular Biology, University of Witten/Herdecke, Witten, Germany.
authors.forename: F
authors.initials: F
authors.lastname: Peske
authors.forename: N B
authors.initials: NB
authors.lastname: Matassova
authors.forename: A
authors.initials: A
authors.lastname: Savelsbergh
authors.forename: M V
authors.initials: MV
authors.lastname: Rodnina
authors.forename: W
authors.initials: W
authors.lastname: Wintermeyer
mesh_terms: D019943:Amino Acid Substitution; D003432:Cross-Linking Reagents; D003545:Cysteine; D004926:Escherichia coli / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D020653:Peptide Elongation Factor G / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation; D012343:RNA, Transfer / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*; D016963:Thermus thermophilus / Q000378:metabolism
chemical_list: D003432:Cross-Linking Reagents; D020653:Peptide Elongation Factor G; D011994:Recombinant Proteins; D006153:Guanosine Diphosphate; D012343:RNA, Transfer; D020558:GTP Phosphohydrolases; D003545:Cysteine