tRNA Translocation by the Eukaryotic 80S Ribosome and the Impact of GTP Hydrolysis

Summary: Translocation moves the tRNA2⋅mRNA module directionally through the ribosome during the elongation phase of protein synthesis. Although translocation is known to entail large conformational changes within both the ribosome and tRNA substrates, the orchestrated events that ensure the speed and fidelity of this critical aspect of the protein synthesis mechanism have not been fully elucidated. Here, we present three high-resolution structures of intermediates of translocation on the mammalian ribosome where, in contrast to bacteria, ribosomal complexes containing the translocase eEF2 and the complete tRNA2⋅mRNA module are trapped by the non-hydrolyzable GTP analog GMPPNP. Consistent with the observed structures, single-molecule imaging revealed that GTP hydrolysis principally facilitates rate-limiting, final steps of translocation, which are required for factor dissociation and which are differentially regulated in bacterial and mammalian systems by the rates of deacyl-tRNA dissociation from the E site. : Translocation, the process by which tRNA and mRNA are moved relative to the ribosome during protein synthesis, is facilitated in eukaryotic cells by the conserved GTPase elongation factor 2. Here Flis et al. combine cryo-EM and single-molecule FRET to elucidate features and intermediate states of translocation on mammalian ribosomes. Keywords: mammalian ribosome, translation elongation, translocation, elongation factor eEF2, macromolecular machine, large-scale conformational changes, smFRET, cryo-EM, EF-

Transient disome complex formation in native polysomes during ongoing protein synthesis captured by cryo-EM

Abstract Structural studies of translating ribosomes traditionally rely on in vitro assembly and stalling of ribosomes in defined states. To comprehensively visualize bacterial translation, we reactivated ex vivo-derived E. coli polysomes in the PURE in vitro translation system and analyzed the actively elongating polysomes by cryo-EM. We find that 31% of 70S ribosomes assemble into disome complexes that represent eight distinct functional states including decoding and termination intermediates, and a pre-nucleophilic attack state. The functional diversity of disome complexes together with RNase digest experiments suggests that paused disome complexes transiently form during ongoing elongation. Structural analysis revealed five disome interfaces between leading and queueing ribosomes that undergo rearrangements as the leading ribosome traverses through the elongation cycle. Our findings reveal at the molecular level how bL9’s CTD obstructs the factor binding site of queueing ribosomes to thwart harmful collisions and illustrate how translation dynamics reshape inter-ribosomal contacts