title: Role of domains 4 and 5 in elongation factor G functions on the ribosome.
abstract: Elongation factor G (EF-G) is a large, five domain GTPase that catalyses the translocation of the tRNAs on the bacterial ribosome at the expense of GTP. In the crystal structure of GDP-bound EF-G, domain 1 (G domain) makes direct contacts with domains 2 and 5, whereas domain 4 protrudes from the body of the molecule. Here, we show that the presence of both domains 4 and 5 is essential for tRNA translocation and for the turnover of the factor on the ribosome, but not for rapid single-round GTP hydrolysis by EF-G. Replacement of a highly conserved histidine residue at the tip of domain 4, His583, with lysine or arginine decreases the rate of tRNA translocation at least 100-fold, whereas the binding of the factor to the ribosome, GTP hydrolysis and P(i) release are not affected by the mutations. Various small deletions in the tip region of domain 4 decrease the translocation activity of EF-G even further, but do not block the turnover of the factor. Unlike native EF-G, the mutants of EF-G lacking domains 4/5 do not interact with the alpha-sarcin stem-loop of 23 S rRNA. These mutants are not released from the ribosome after GTP hydrolysis or translocation, indicating that the contact with, or a conformational change of, the alpha-sarcin stem-loop is required for EF-G release from the ribosome.
authors.affiliation: Institute of Molecular Biology, University of Witten/Herdecke, Witten, 58448, Germany.
authors.forename: A
authors.initials: A
authors.lastname: Savelsbergh
authors.forename: N B
authors.initials: NB
authors.lastname: Matassova
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 / Q000235:genetics; D001665:Binding Sites; D002384:Catalysis; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D004722:Endoribonucleases / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry*; D005656:Fungal Proteins; D006160:Guanosine Triphosphate / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D020653:Peptide Elongation Factor G / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D012338:RNA, Ribosomal, 23S / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012343:RNA, Transfer / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion / Q000235:genetics; D013463:Sulfuric Acid Esters / Q000378:metabolism
chemical_list: D005656:Fungal Proteins; D020653:Peptide Elongation Factor G; D012338:RNA, Ribosomal, 23S; D016601:RNA-Binding Proteins; D013463:Sulfuric Acid Esters; C014801:alpha-sarcin; D006160:Guanosine Triphosphate; D012343:RNA, Transfer; D004722:Endoribonucleases; C007482:dimethyl sulfate