Extensive ribosome and RF2 rearrangements during translation termination

Abstract

Protein synthesis ends when a ribosome reaches an mRNA stop codon. Release factors (RFs) decode the stop codon, hydrolyze peptidyl-tRNA to release the nascent protein, and then dissociate to allow ribosome recycling. To visualize termination by RF2, we resolved a cryo-EM ensemble of E. coli 70S•RF2 structures at up to 3.3 Å in a single sample. Five structures suggest a highly dynamic termination pathway. Upon peptidyl-tRNA hydrolysis, the CCA end of deacyl-tRNA departs from the peptidyl transferase center. The catalytic GGQ loop of RF2 is rearranged into a long β-hairpin that plugs the peptide tunnel, biasing a nascent protein toward the ribosome exit. Ribosomal intersubunit rotation destabilizes the catalytic RF2 domain on the 50S subunit and disassembles the central intersubunit bridge B2a, resulting in RF2 departure. Our structures visualize how local rearrangements and spontaneous inter-subunit rotation poise the newly-made protein and RF2 to dissociate in preparation for ribosome recycling.

ArfB can displace mRNA to rescue stalled ribosomes

“…An~10°rotation of the 30S subunit relative to the large subunit shifts the tRNA's CCA end into the E site of the 50S subunit, so the tRNA adopts a hybrid P/E state. Intersubunit rotation prepares the ribosome for release-factor dissociation 27,28 and recycling [29][30][31] .…”

“…A 3.8-Å structure (+2-I) reports a non-rotated ribosome with the P-site tRNA and a vacant A site ( Fig. 2a), resembling a pre-release or post-release state without a release factor 28 . Two 3.7 Å maps (+2-II and +2-IV) and a 3.8 Å map (+2-III) report non-rotated ribosomes with ArfB in different conformations ( Fig.…”

“…+2 and +9 complexes differ in the occupancies of ArfB on the rotated 70S ribosome, suggesting that ArfB dissociation during intersubunit rotation occurs differently. Similarly to the post-termination 70S complexes with RF2 28 , the A site of the rotated Structure +2-V (~7°) is vacant ( Fig. 2e and Supplementary Fig.…”

Ribosome inhibition byC9ORF72-ALS/FTD-associated poly-PR and poly-GR proteins revealed by cryo-EM

“…Data for the S. cerevisae 80S•PR 20 or 80S•GR 20 were collected on a Titan Krios electron microscope (ThermoFisher Scientific) operating at 300 kV and equipped with a Gatan Image Filter (Gatan Inc.) and a K2 Summit direct electron (Gatan Inc.) targeting 0.5 to 2.0-μm underfocus. For 80S•PR 20 , a dataset of 203,089 particles from 3033 movies was collected automatically using SerialEM (Mastronarde, 2005) using beam tilt to collected 5 movies per hole at 4 holes between stage movements (Svidritskiy et al, 2019). The movies had a total of 30 frames with 1 e -/Å 2 per frame for a total dose of 30 e -/Å 2 on the sample.…”

“…Data collection was automated using SerialEM (Mastronarde, 2005) using beam tilt to collect multiple movies (e.g. 4 movies per hole at 4 holes) at each stage position (Svidritskiy et al, 2019). The E. coli dataset had a total of 20 frames per movie, with 1.1 e -/Å 2 per frame for a total dose of 36 e -/Å 2 on the sample, comprising 1024 movies yielding 172,278 particles for the 70S•PR 20 complex.…”