The structural basis for release-factor activation during translation termination revealed by time-resolved cryogenic electron microscopy

Abstract

When the ribosome encounters a stop codon, it recruits a release factor (RF) to hydrolyze the ester bond between the peptide chain and tRNA. RFs have structural motifs that recognize stop codons in the decoding center and a GGQ motif for induction of hydrolysis in the peptidyl transfer center 70 Å away. Surprisingly, free RF2 is compact, with only 20 Å between its codon-reading and GGQ motifs. Cryo-EM showed that ribosome-bound RFs have extended structures, suggesting that RFs are compact when entering the ribosome and then extend their structures upon stop codon recognition. Here we use time-resolved cryo-EM to visualize transient compact forms of RF1 and RF2 at 3.5 and 4 Å resolution, respectively, in the codon-recognizing ribosome complex on the native pathway. About 25% of complexes have RFs in the compact state at 24 ms reaction time, and within 60 ms virtually all ribosome-bound RFs are transformed to their extended forms.

Solvent quality and chromosome folding inEscherichia coli

“…Sub-volumes of the detected ribosomes were aligned and classified (STAR Methods, Figure S4C-D) (Winkler, 2007;Winkler et al, 2009). The average ribosome structure calculated using the detected ribosomes was similar to that of the low-pass filtered reference structure ( Figure 6C; Video S4) (Fu et al, 2019), suggesting a high degree of accuracy in our detection procedure. In total, we detected 5,028 ribosomes in the tomogram represented in Figure 6D.…”

“…C. Comparison between the reference ribosome structure (gray) and the averaged structure (yellow) derived from ribosomes localized in the lamella tomogram shown in panel B. An E. coli 70S ribosome structure (EMD-20173) was used as the reference (Fu et al, 2019). See also Video S4 for a 3D Bar graph showing the mean volume of 1,000 simulated chromosomes in each type of solvent.…”

“…The software package emClarity (Himes and Zhang, 2018) was used to perform the 3D template search to identify ribosomes from the tomograms. A low-pass filtered (~ 4-nm resolution) E. coli 70S ribosome structure (Fu et al, 2019) was used as a reference for the template search ( Figure S4C).…”

“…The left panel shows the original GFP fluorescence signal from the particle, and the right panel shows the additional annotation based on the single-particle tracking results: the particle center was denoted by the red point, and the yellow tail represents a short trajectory indicating the particle positions within the last 5 frames. This video shows a 3D comparison between the low-pass filtered (~3 nm resolution) reference structure (grey) obtained from (Fu et al, 2019) and the average structure (yellow) based on all ribosomes localized in the tomogram shown in Figure 6B and Video S3. and good ( = 0.58).…”

Integrative approaches in cryogenic electron microscopy: Recent advances in structural biology and future perspectives

“…For instance, compact form of release factor crystal structure as compared to the “open” structures of its ribosome-bound state raised serious questions long back ( Kjeldgaard, 2003 ). A recent trCryo-EM study resolved this long-standing issue by capturing a transient state of ribosome-bound release factor in the compact form ( Fu et al., 2019 ). Evidently, resolving structures of the short-lasting intermediates using the time-resolved technique in combination with cryo-EM improves and fine-tunes our understanding of the mechanisms of fundamental cellular processes ( Figure 2 ).…”