G-domain dimerization orchestrates the tRNA wobble modification reaction in the MnmE/GidA complex.

TitleG-domain dimerization orchestrates the tRNA wobble modification reaction in the MnmE/GidA complex.
Publication TypeJournal Article
Year of Publication2009
AuthorsMeyer, S., A. Wittinghofer, and W. Versées
JournalJ Mol Biol
Volume392
Issue4
Pagination910-22
Date Published2009 Oct 2
ISSN1089-8638
KeywordsBacterial Proteins, Escherichia coli Proteins, GTP Phosphohydrolases, Guanosine Triphosphate, Humans, Hydrolysis, Models, Biological, Models, Molecular, Multiprotein Complexes, Protein Binding, Protein Multimerization, Protein Structure, Tertiary, RNA Processing, Post-Transcriptional, RNA, Transfer, Yeasts
Abstract

MnmE and GidA are involved in the modification of wobble uridine to carboxymethylaminomethyl uridine in certain tRNAs. Malfunctioning of the human orthologs has been implicated in mitochondrial diseases. MnmE is a conserved G protein activated by dimerization. Here, we show that complex formation between MnmE and GidA involves large conformational changes that induce G-domain dimerization of MmnE and that GidA co-stimulates GTP hydrolysis on MnmE. Starting from a structural model of the complex, we identify interface mutations disrupting complex formation or communication. Although GidA does not directly contact the G-domains, conformational changes in MnmE, induced by G-domain dimerization in the triphosphate state, regulate the affinity for GidA. We developed a tRNA modification assay and demonstrate for the first time in vitro that the MnmE/GidA complex catalyzes incorporation of glycine into tRNA. An intact MnmE/GidA complex rather than their sequential action is crucial for in vitro modification. Since only GTP, but not GDP or non-hydrolyzable GTP analogs, drives the MnmE/GidA-catalyzed modification reaction, we conclude that GTP hydrolysis is essential for activity. We finally show that an active GTPase, an intact MnmE/GidA communication, and dimerization of G-domains are necessary for in vivo functioning since mutations disrupting either result in a respiratory deficient phenotype in yeast.

DOI10.1016/j.jmb.2009.07.004
Alternate JournalJ. Mol. Biol.
PubMed ID19591841
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