TRNA Dissociation from EF-Tu after GTP Hydrolysis: Primary Steps and Antibiotic Inhibition

Malte Warias,Helmut Grubmüller,Lars V Bock

doi:10.1016/j.bpj.2019.10.028

Abstract

In each round of ribosomal translation, the translational GTPase elongation factor Tu (EF-Tu) delivers a transfer RNA (tRNA) to the ribosome. After successful decoding, EF-Tu hydrolyzes GTP, which triggers a conformational change that ultimately results in the release of the tRNA from EF-Tu. To identify the primary steps of these conformational changes and how they are prevented by the antibiotic kirromycin, we employed all-atom explicit-solvent molecular dynamics simulations of the full ribosome-EF-Tu complex. Our results suggest that after GTP hydrolysis and Pi release, the loss of interactions between the nucleotide and the switch 1 loop of EF-Tu allows domain D1 of EF-Tu to rotate relative to domains D2 and D3 and leads to an increased flexibility of the switch 1 loop. This rotation induces a closing of the D1-D3 interface and an opening of the D1-D2 interface. We propose that the opening of the D1-D2 interface, which binds the CCA tail of the tRNA, weakens the crucial EF-Tu-tRNA interactions, which lowers tRNA binding affinity, representing the first step of tRNA release. Kirromycin binds within the D1-D3 interface, sterically blocking its closure, but does not prevent hydrolysis. The resulting increased flexibility of switch 1 explains why it is not resolved in kirromycin-bound structures.

Highlights

Elongation factor Tu (EF-Tu) is a central part of the bacterial translation machinery
After GTP hydrolysis and the release of the inorganic phosphate Pi, the ribosome-bound EF-Tu undergoes a conformational change that precedes the release of EF-Tu from the ribosome [6,21]
This conformational change results in the release of the transfer RNA (tRNA) from EF-Tu, allowing the tRNA to fully accommodate into the A site and EF-Tu to dissociate from the ribosome

Summary

Introduction

Elongation factor Tu (EF-Tu) is a central part of the bacterial translation machinery. The transition of EF-Tu into a reorganized catalytic configuration in the GTPase-activated state catalyzes GTP hydrolysis to GDP [4,5], followed by the release of inorganic phosphate (Pi) and a conformational change of EF-Tu [6]. Because the conformational change of EF-Tu occurs rapidly and its rate is limited by Pi release, EF-Tu was suggested to behave like a ‘‘loaded spring’’ whose tension is relaxed after Pi dissociation [6]. This conformational change eventually leads to the transfer RNA (tRNA) being released from EF-Tu, followed by the accommodation of the tRNA into the A site and the dissociation of EF-Tu from the ribosome [7]. Ensemble and singlemolecule kinetic experiments indicate that EF-Tu dissociates first from the 30 end of the tRNA and from the GTPase-associated center of the ribosome and subsequently from the rest of the tRNA [8]. tRNA accommodation proceeds in a stepwise manner [9,10,11,12] via an intermediate elbow-accommodated conformation, and EF-Tu has been suggested to help the tRNA to assume an intermediate step before full accommodation [13]

Methods

Results

Conclusion