EF-Tu dynamics during pre-translocation complex formation: EF-Tu·GDP exits the ribosome via two different pathways

Nucleic Acids Research
Wei LiuBarry S Cooperman

Abstract

The G-protein EF-Tu, which undergoes a major conformational change when EF-Tu·GTP is converted to EF-Tu·GDP, forms part of an aminoacyl(aa)-tRNA·EF-Tu·GTP ternary complex (TC) that accelerates the binding of aa-tRNA to the ribosome during peptide elongation. Such binding, placing a portion of EF-Tu in contact with the GTPase Associated Center (GAC), is followed by GTP hydrolysis and Pi release, and results in formation of a pretranslocation (PRE) complex. Although tRNA movement through the ribosome during PRE complex formation has been extensively studied, comparatively little is known about the dynamics of EF-Tu interaction with either the ribosome or aa-tRNA. Here we examine these dynamics, utilizing ensemble and single molecule assays employing fluorescent labeled derivatives of EF-Tu, tRNA, and the ribosome to measure changes in either FRET efficiency or fluorescence intensity during PRE complex formation. Our results indicate that ribosome-bound EF-Tu separates from the GAC prior to its full separation from aa-tRNA, and suggest that EF-Tu·GDP dissociates from the ribosome by two different pathways. These pathways correspond to either reversible EF-Tu·GDP dissociation from the ribosome prior to the major conformational chan...Continue Reading

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Citations

Mar 15, 2016·Biopolymers·Cristina Maracci, Marina V Rodnina
Nov 1, 2016·Nature Communications·Jeffrey K Noel, Paul C Whitford
Nov 12, 2016·Proceedings of the National Academy of Sciences of the United States of America·Ka-Weng IeongMåns Ehrenberg
Feb 1, 2017·Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences·Marina V RodninaHolger Stark
Aug 30, 2017·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Wei LiuBarry S Cooperman
Jul 24, 2018·Proteins·Huan YangPaul C Whitford
Feb 7, 2020·Proceedings of the National Academy of Sciences of the United States of America·Justin C MorseScott C Blanchard
Aug 15, 2018·Nucleic Acids Research·Darius KavaliauskasCharlotte R Knudsen
Aug 11, 2019·Nucleic Acids Research·Erica N ThomasHani S Zaher
Jan 9, 2021·Proceedings of the National Academy of Sciences of the United States of America·Martin Y NgBarry S Cooperman
Oct 19, 2017·Biochemistry·Jonathan LaiZaida Luthey-Schulten

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Methods Mentioned

BETA
GTPase
fluorescence
FRET
GTPases

Software Mentioned

OriginLab
Origin
smFRET

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