Mechanism of HflX-mediated recycling of E. coli ribosome studied by time-resolved cryo-EM

Abstract

Recycling of stalled, translationally inactive ribosomes is one of the response mechanisms underlying resistance to environmental stress. HflX, a bacterial GTPase overexpressed upon heat shock and exposure to antibiotics, catalyzes the splitting of stalled ribosomes. Due to its rapid rate, an attempt to follow this process in real time at room temperature requires time-resolved techniques on the scale of tens of milliseconds. Here we show the time course of the splitting of E. coli 70S ribosome by HflX in the presence of GTP using time-resolved cryo-EM with a microfluidic device. We report the structures of three short-lived intermediate states within 140 ms of mixing 70S ribosomes with HflX and GTP. Binding of HflX and GTP hydrolysis induces breakage of multiple intersubunit bridges and opening of the 70S ribosome in a clamshell-like manner as the 30S subunit rotates around an axis with hinges formed by the remaining intersubunit bridges B3 and B7a. At 900 ms virtually all ribosomes are dissociated, while HflX remains bound to the 50S subunit. Our analysis of the structural intermediates not only allows the mechanism of HflX-catalyzed ribosome splitting to be followed in molecular detail but also to shed light on related processes: recycling of the post-termination complex by RRF/EF-G.

Competing Interest Statement

The authors have declared no competing interest.