Structural mechanism of GTPase-powered ribosome-tRNA movement

Valentyn Petrychenko,Niels Fischer,Frank Peske,Marina V Rodnina,Bee-Zen Peng,Ana C De A P Schwarzer

doi:10.1038/s41467-021-26133-x

Valentyn Petrychenko, Niels Fischer + Show 4 more

Open Access

https://doi.org/10.1038/s41467-021-26133-x

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Abstract

GTPases are regulators of cell signaling acting as molecular switches. The translational GTPase EF-G stands out, as it uses GTP hydrolysis to generate force and promote the movement of the ribosome along the mRNA. The key unresolved question is how GTP hydrolysis drives molecular movement. Here, we visualize the GTPase-powered step of ongoing translocation by time-resolved cryo-EM. EF-G in the active GDP–Pi form stabilizes the rotated conformation of ribosomal subunits and induces twisting of the sarcin-ricin loop of the 23 S rRNA. Refolding of the GTPase switch regions upon Pi release initiates a large-scale rigid-body rotation of EF-G pivoting around the sarcin-ricin loop that facilitates back rotation of the ribosomal subunits and forward swiveling of the head domain of the small subunit, ultimately driving tRNA forward movement. The findings demonstrate how a GTPase orchestrates spontaneous thermal fluctuations of a large RNA-protein complex into force-generating molecular movement.

Highlights

GTPases are regulators of cell signaling acting as molecular switches
We visualized the early steps of translocation by time-resolved cryo-electron microscopy (EM) (Fig. 1 and Supplementary Fig. 1) using native E. coli pretranslocation complexes (PRE) and elongation factor G (EF-G)–guanosine triphosphate (GTP), but slowed down translocation by lowering the reaction temperature, by adding polyamines and the antibiotic apramycin (Apr)[21]
The major population of PRE ribosomes does not contain EF-G and is found in the non-rotated state with transfer RNAs (tRNAs) in their classical A/A and P/P positions, which we denote as classical (C) state (Fig. 1a, b); the preference for the C state is likely due to the presence of polyamines and Apr in the buffer

Summary

Introduction

GTPases are regulators of cell signaling acting as molecular switches. The translational GTPase EF-G stands out, as it uses GTP hydrolysis to generate force and promote the movement of the ribosome along the mRNA. Refolding of the GTPase switch regions upon Pi release initiates a largescale rigid-body rotation of EF-G pivoting around the sarcin-ricin loop that facilitates back rotation of the ribosomal subunits and forward swiveling of the head domain of the small subunit, driving tRNA forward movement. Small and large ribosomal subunits (SSU and LSU) rotate relative to each other and the SSU head domain swivels relative to the SSU body[2,3]. These principal motions of the ribosome components are spontaneous and rapid even in the absence of EFG7–10. How EF-G powered by GTP hydrolysis synchronizes the spontaneous fluctuations of the ribosome and the tRNAs into a rapid, directed motion is the key unresolved question

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