Visualization of translation termination intermediates trapped by the Apidaecin\xa0137 peptide during RF3-mediated recycling of RF1

Michael Graf,Daniel N Wilson,Cristina Maracci,Paul Huter,Miroslav Peterek,Marina V Rodnina

doi:10.1038/s41467-018-05465-1

Abstract

During translation termination in bacteria, the release factors RF1 and RF2 are recycled from the ribosome by RF3. While high-resolution structures of the individual termination factors on the ribosome exist, direct structural insight into how RF3 mediates dissociation of the decoding RFs has been lacking. Here we have used the Apidaecin 137 peptide to trap RF1 together with RF3 on the ribosome and visualize an ensemble of termination intermediates using cryo-electron microscopy. Binding of RF3 to the ribosome induces small subunit (SSU) rotation and swivelling of the head, yielding intermediate states with shifted P-site tRNAs and RF1 conformations. RF3 does not directly eject RF1 from the ribosome, but rather induces full rotation of the SSU that indirectly dislodges RF1 from its binding site. SSU rotation is coupled to the accommodation of the GTPase domain of RF3 on the large subunit (LSU), thereby promoting GTP hydrolysis and dissociation of RF3 from the ribosome.

Highlights

During translation termination in bacteria, the release factors RF1 and RF2 are recycled from the ribosome by RF3
Based on our ensemble of termination intermediates, as well as the available literature, we suggest a revised model for RF3-mediated dissociation of RF1 during translation termination (Fig. 7a–e)
A stop codon in the A site is recognized by the decoding factors RF1, which catalyse hydrolysis of the peptidyl-tRNA (Fig. 7a)

Summary

Introduction

During translation termination in bacteria, the release factors RF1 and RF2 are recycled from the ribosome by RF3. SSU rotation is coupled to the accommodation of the GTPase domain of RF3 on the large subunit (LSU), thereby promoting GTP hydrolysis and dissociation of RF3 from the ribosome. Structures of RF1 and RF2 in complex with termination state ribosomes have revealed how conserved residues within the superdomain 2/ 4 recognize the stop codon on the small subunit (SSU) 10–14. Cryo-EM and X-ray structures exist of RF3-GDP(C/N)P (nonhydrolysable GTP analogues) bound to rotated ribosomes with P/ E-hybrid state tRNAs but without the decoding release factors (Fig. 1b)[17,27,28,29]. Superimposition of the RF3 and RF1/RF2 ribosome structures revealed no overlap in the factor binding sites, suggesting that RF3 indirectly promotes RF1/RF2 dissociation indirectly via inducing ribosomal subunit rotation[14,17,27,28,29,30]. RF3mediated subunit rotation plays a dual role during termination, namely, to dislodge the decoding release factors from the ribosome, and to facilitate dissociation of RF3 itself

Methods

Results

Conclusion