title: Codon-dependent conformational change of elongation factor Tu preceding GTP hydrolysis on the ribosome.
abstract: The mechanisms by which elongation factor Tu (EF-Tu) promotes the binding of aminoacyl-tRNA to the A site of the ribosome and, in particular, how GTP hydrolysis by EF-Tu is triggered on the ribosome, are not understood. We report steady-state and time-resolved fluorescence measurements, performed in the Escherichia coli system, in which the interaction of the complex EF-Tu.GTP.Phe-tRNAPhe with the ribosomal A site is monitored by the fluorescence changes of either mant-dGTP [3'-O-(N-methylanthraniloyl)-2-deoxyguanosine triphosphate], replacing GTP in the complex, or of wybutine in the anticodon loop of the tRNA. Additionally, GTP hydrolysis is measured by the quench-flow technique. We find that codon-anticodon interaction induces a rapid rearrangement within the G domain of EF-Tu around the bound nucleotide, which is followed by GTP hydrolysis at an approximately 1.5-fold lower rate. In the presence of kirromycin, the activated conformation of EF-Tu appears to be frozen. The steps following GTP hydrolysis--the switch of EF-Tu to the GDP-bound conformation, the release of aminoacyl-tRNA from EF-Tu to the A site, and the dissociation of EF-Tu-GDP from the ribosome--which are altogether suppressed by kirromycin, are not distinguished kinetically. The results suggest that codon recognition by the ternary complex on the ribosome initiates a series of structural rearrangements resulting in a conformational change of EF-Tu, possibly involving the effector region, which, in turn, triggers GTP hydrolysis.
authors.affiliation: Institute of Molecular Biology, University Witten/Herdecke, Germany.
authors.forename: M V
authors.initials: MV
authors.lastname: Rodnina
authors.forename: R
authors.initials: R
authors.lastname: Fricke
authors.forename: L
authors.initials: L
authors.lastname: Kuhn
authors.forename: W
authors.initials: W
authors.lastname: Wintermeyer
mesh_terms: D000926:Anticodon / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D003062:Codon / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006868:Hydrolysis; D009690:Nucleic Acid Conformation; D010444:Peptide Elongation Factor Tu / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D011728:Pyridones / Q000494:pharmacology; D012346:RNA, Transfer, Amino Acyl / Q000737:chemistry / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*; D062367:ortho-Aminobenzoates / Q000378:metabolism
chemical_list: D000926:Anticodon; D003062:Codon; D011728:Pyridones; D012346:RNA, Transfer, Amino Acyl; D062367:ortho-Aminobenzoates; C065197:3'-(methylanthraniloyl)-2'-deoxy-guanosine diphosphate; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D010444:Peptide Elongation Factor Tu; C008536:mocimycin