Key Intermediates in Ribosome Recycling Visualized by Time-Resolved Cryoelectron Microscopy

Abstract

Upon encountering a stop codon on messenger RNA (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 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.

Time-resolved cryo-electron microscopy: Recent progress

“…The 30S subunit, the other product of recycling, exhibits a bound tRNA (and mRNA, not shown) at 140 ms but after a longer time span, the tRNA is replaced by IF3. This observation is relevant in the long-drawn controversy on the fate of tRNA and the role of IF3 in recycling (see discussion in Fu et al, 2016). …”

“…If this is a slow process, e.g., in the range of minutes, then all we have to do is start the reaction and then take samples in some intervals for visualization of the molecules with negative-staining EM (Mulder et al, 2010) or standard cryo-EM (Fischer et al, 2010). However, if the process is fast compared to the time required for pipetting and blotting (several seconds), then a special apparatus is needed which uses a microfluidic chip for mixing and reacting two reactants, and rapid deposition of the reaction product on the grid (Lu et al, 2009; Sheikh et al, 2014; Chen et al, 2014; Chen and Frank, 2016; Fu et al, 2016). We refer to this method as “mixing/spraying,” in contradistinction to an earlier method of “spraying/mixing” developed by Berriman and Unwin (1994), in which one reactant is sprayed on a grid already covered with another reactant, and the mixing takes place on the grid itself.…”

“…An article describing the sample preparation, data collection and outcome of this experiment has recently been published (Fu et al, 2016), and here it will suffice to give a brief summary reflecting on the gain in biological knowledge. A total of 84,000 particle images were collected from droplet areas for different time points.…”

Single‐Particle Reconstruction of Biological Molecules—Story in a Sample (Nobel Lecture)

“…This potential for resolving dynamic changes of molecules in equilibrium is augmented by the development of timeresolved techniques that are able to trap short-lived states evolving in an on-equilibrium experiment. [84][85][86][87][88][89] Ultimately, even the recovery of acontinuum of structures reflecting the states of abiological molecule at work, and the mapping of its free-energy landscape are no longer distant goals. [90][91][92] This most recent development is made possible by our ability to collect large quantities of data, ensuring that even states encountered with low probability are represented in the ensemble.…”

Research in morphology and flow cytometry is at the heart of hematology

“…Yet, in parallel, other methods are developed to get a better grasp on the "physical" structure/morphology of organelles such as ribosomes, i.e., with cryo-electron microscopy. 16 Meanwhile, classical methods retain their intrinsic value to check on the morphology of manipulated cells or to use antibodies to assess their immunophenotypic or secretory profile.…”