title: Ribosome-induced tuning of GTP hydrolysis by a translational GTPase.
abstract: GTP hydrolysis by elongation factor Tu (EF-Tu), a translational GTPase that delivers aminoacyl-tRNAs to the ribosome, plays a crucial role in decoding and translational fidelity. The basic reaction mechanism and the way the ribosome contributes to catalysis are a matter of debate. Here we use mutational analysis in combination with measurements of rate/pH profiles, kinetic solvent isotope effects, and ion dependence of GTP hydrolysis by EF-Tu off and on the ribosome to dissect the reaction mechanism. Our data suggest that--contrary to current models--the reaction in free EF-Tu follows a pathway that does not involve the critical residue H84 in the switch II region. Binding to the ribosome without a cognate codon in the A site has little effect on the GTPase mechanism. In contrast, upon cognate codon recognition, the ribosome induces a rearrangement of EF-Tu that renders GTP hydrolysis sensitive to mutations of Asp21 and His84 and insensitive to K(+) ions. We suggest that Asp21 and His84 provide a network of interactions that stabilize the positions of the γ-phosphate and the nucleophilic water, respectively, and thus play an indirect catalytic role in the GTPase mechanism on the ribosome.
authors.affiliation: Department of Physical Biochemistry, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany.
authors.forename: Cristina
authors.initials: C
authors.lastname: Maracci
authors.affiliation: Department of Physical Biochemistry, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany.
authors.forename: Frank
authors.initials: F
authors.lastname: Peske
authors.affiliation: Department of Physical Biochemistry, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany.
authors.forename: Ev
authors.initials: E
authors.lastname: Dannies
authors.affiliation: Department of Physical Biochemistry, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany.
authors.forename: Corinna
authors.initials: C
authors.lastname: Pohl
authors.affiliation: Department of Physical Biochemistry, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany rodnina@mpibpc.mpg.de.
authors.forename: Marina V
authors.initials: MV
authors.lastname: Rodnina
mesh_terms: D001224:Aspartic Acid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D020134:Catalytic Domain; D003062:Codon / Q000235:genetics / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000737:chemistry / Q000378:metabolism*; D006639:Histidine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D008968:Molecular Conformation; D015394:Molecular Structure; D009154:Mutation; D010444:Peptide Elongation Factor Tu / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D014176:Protein Biosynthesis; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism; D012270:Ribosomes / Q000737:chemistry / Q000378:metabolism*
chemical_list: D003062:Codon; D012346:RNA, Transfer, Amino Acyl; D001224:Aspartic Acid; D006639:Histidine; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; D010444:Peptide Elongation Factor Tu