title: Dual use of GTP hydrolysis by elongation factor G on the ribosome.
abstract: Elongation factor G (EF-G) is a GTPase that catalyzes tRNA and mRNA translocation during the elongation cycle of protein synthesis. The GTP-bound state of the factor on the ribosome has been studied mainly with non-hydrolyzable analogs of GTP, which led to controversial conclusions about the role of GTP hydrolysis in translocation. Here we describe a mutant of EF-G in which the catalytic His91 is replaced with Ala. The mutant EF-G does not hydrolyze GTP, but binds GTP with unchanged affinity, allowing us to study the function of the authentic GTP-bound form of EF-G in translocation. Utilizing fluorescent reporter groups attached to the tRNAs, mRNA, and the ribosome we compile the velocity map of translocation seen from different perspectives. The data suggest that GTP hydrolysis accelerates translocation up to 30-fold and facilitates conformational rearrangements of both 30S subunit (presumably the backward rotation of the 30S head) and EF-G that lead to the dissociation of the factor. Thus, EF-G combines the energy regime characteristic for motor proteins, accelerating movement by a conformational change induced by GTP hydrolysis, with that of a switch GTPase, which upon Pi release switches the conformations of EF-G and the ribosome to low affinity, allowing the dissociation of the factor.
authors.affiliation: Max Planck Institute for Biophysical Chemistry; Department of Physical Biochemistry; Göttingen, Germany.
authors.forename: Carlos E
authors.initials: CE
authors.lastname: Cunha
authors.affiliation: Max Planck Institute for Biophysical Chemistry; Department of Physical Biochemistry; Göttingen, Germany.
authors.forename: Riccardo
authors.initials: R
authors.lastname: Belardinelli
authors.affiliation: Max Planck Institute for Biophysical Chemistry; Department of Physical Biochemistry; Göttingen, Germany.
authors.forename: Frank
authors.initials: F
authors.lastname: Peske
authors.affiliation: Max Planck Institute for Biophysical Chemistry; Department of Physical Biochemistry; Göttingen, Germany.
authors.forename: Wolf
authors.initials: W
authors.lastname: Holtkamp
authors.affiliation: Max Planck Institute for Biophysical Chemistry; Department of Physical Biochemistry; Göttingen, Germany.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Wintermeyer
authors.affiliation: Max Planck Institute for Biophysical Chemistry; Department of Physical Biochemistry; Göttingen, Germany.
authors.forename: Marina V
authors.initials: MV
authors.lastname: Rodnina
keywords: Ribosomes; molecular motors; protein synthesis; tRNA; translational GTPases
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