Structure
Volume 24, Issue 12, 6 December 2016, Pages 2092-2101
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Article
Key Intermediates in Ribosome Recycling Visualized by Time-Resolved Cryoelectron Microscopy

https://doi.org/10.1016/j.str.2016.09.014Get rights and content
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Highlights

  • The joint action of RRF and EF-G attacking bridge B2a during ribosome recycling has been visualized

  • Observation of IF3- and tRNA- bound 30S subunits helps elucidate possible alternative recycling pathways

  • This study shows cryo-EM is able to depict short-lived states in molecular interactions

Summary

Upon encountering a stop codon on mRNA, polypeptide synthesis on the ribosome is terminated by release factors, and the ribosome complex, still bound with mRNA and P-site-bound tRNA (post-termination complex, PostTC), is split into ribosomal subunits, ready for a new round of translational initiation. Separation of post-termination ribosomes into subunits, or “ribosome recycling,” is promoted by the joint action of ribosome-recycling factor (RRF) and elongation factor G (EF-G) in a guanosine triphosphate (GTP) hydrolysis-dependent manner. Here we used a mixing-spraying-based method of time-resolved cryo-electron microscopy (cryo-EM) to visualize the short-lived intermediates of the recycling process. The two complexes that contain (1) both RRF and EF-G bound to the PostTC or (2) deacylated tRNA bound to the 30S subunit are of particular interest. Our observations of the native form of these complexes demonstrate the strong potential of time-resolved cryo-EM for visualizing previously unobservable transient structures.

Keywords

time-resolved
cryo-EM
mixing-spraying
ribosome
recycling

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