Dual use of GTP hydrolysis by elongation factor G on the ribosome

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...

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Main Authors:	Cunha, Carlos E., Belardinelli, Riccardo, Peske, Frank, Holtkamp, Wolf, Wintermeyer, Wolfgang, Rodnina, Marina V.
Format:	Online
Language:	English
Published:	Taylor & Francis 2013
Online Access:	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4718068/

id	pubmed-4718068
recordtype	oai_dc
spelling	pubmed-47180682016-01-28 Dual use of GTP hydrolysis by elongation factor G on the ribosome Cunha, Carlos E. Belardinelli, Riccardo Peske, Frank Holtkamp, Wolf Wintermeyer, Wolfgang Rodnina, Marina V. Research Paper 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. Taylor & Francis 2013-04-01 /pmc/articles/PMC4718068/ /pubmed/26824016 http://dx.doi.org/10.4161/trla.24315 Text en Copyright © 2013 Landes Bioscience http://creativecommons.org/licenses/by-nc/3.0/ This is an open-access article licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported License. The article may be redistributed, reproduced, and reused for non-commercial purposes, provided the original source is properly cited.
repository_type	Open Access Journal
institution_category	Foreign Institution
institution	US National Center for Biotechnology Information
building	NCBI PubMed
collection	Online Access
language	English
format	Online
author	Cunha, Carlos E. Belardinelli, Riccardo Peske, Frank Holtkamp, Wolf Wintermeyer, Wolfgang Rodnina, Marina V.
spellingShingle	Cunha, Carlos E. Belardinelli, Riccardo Peske, Frank Holtkamp, Wolf Wintermeyer, Wolfgang Rodnina, Marina V. Dual use of GTP hydrolysis by elongation factor G on the ribosome
author_facet	Cunha, Carlos E. Belardinelli, Riccardo Peske, Frank Holtkamp, Wolf Wintermeyer, Wolfgang Rodnina, Marina V.
author_sort	Cunha, Carlos E.
title	Dual use of GTP hydrolysis by elongation factor G on the ribosome
title_short	Dual use of GTP hydrolysis by elongation factor G on the ribosome
title_full	Dual use of GTP hydrolysis by elongation factor G on the ribosome
title_fullStr	Dual use of GTP hydrolysis by elongation factor G on the ribosome
title_full_unstemmed	Dual use of GTP hydrolysis by elongation factor G on the ribosome
title_sort	dual use of gtp hydrolysis by elongation factor g on the ribosome
description	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.
publisher	Taylor & Francis
publishDate	2013
url	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4718068/
_version_	1613525563831484416

Dual use of GTP hydrolysis by elongation factor G on the ribosome

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