title: Control of phosphate release from elongation factor G by ribosomal protein L7/12.
abstract: Ribosomal protein L7/12 is crucial for the function of elongation factor G (EF-G) on the ribosome. Here, we report the localization of a site in the C-terminal domain (CTD) of L7/12 that is critical for the interaction with EF-G. Single conserved surface amino acids were replaced in the CTD of L7/12. Whereas mutations in helices 5 and 6 had no effect, replacements of V66, I69, K70, and R73 in helix 4 increased the Michaelis constant (KM) of EF-G.GTP for the ribosome, suggesting an involvement of these residues in EF-G binding. The mutations did not appreciably affect rapid single-round GTP hydrolysis and had no effect on tRNA translocation on the ribosome. In contrast, the release of inorganic phosphate (Pi) from ribosome-bound EF-G.GDP.Pi was strongly inhibited and became rate-limiting for the turnover of EF-G. The control of Pi release by interactions between EF-G and L7/12 appears to be important for maintaining the conformational coupling between EF-G and the ribosome for translocation and for timing the dissociation of the factor from the ribosome.
authors.affiliation: Institute of Molecular Biology, University of Witten/Herdecke, Witten, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Savelsbergh
authors.forename: Dagmar
authors.initials: D
authors.lastname: Mohr
authors.forename: Ute
authors.initials: U
authors.lastname: Kothe
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Wintermeyer
authors.forename: Marina V
authors.initials: MV
authors.lastname: Rodnina
mesh_terms: D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins; D006160:Guanosine Triphosphate / Q000737:chemistry; D006868:Hydrolysis; D007700:Kinetics; D008954:Models, Biological; D008958:Models, Molecular; D009154:Mutation; D020653:Peptide Elongation Factor G / Q000378:metabolism / Q000502:physiology*; D010710:Phosphates / Q000737:chemistry / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D021381:Protein Transport; D012333:RNA, Messenger / Q000378:metabolism; D012343:RNA, Transfer / Q000737:chemistry / Q000378:metabolism; D012269:Ribosomal Proteins / Q000737:chemistry* / Q000502:physiology*; D012270:Ribosomes / Q000737:chemistry / Q000378:metabolism; D013997:Time Factors
chemical_list: D029968:Escherichia coli Proteins; D020653:Peptide Elongation Factor G; D010710:Phosphates; D012333:RNA, Messenger; D012269:Ribosomal Proteins; C512844:rplL protein, E coli; C016700:ribosomal protein L7-L12; D006160:Guanosine Triphosphate; D012343:RNA, Transfer