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

• Published in: Cell reports (Cambridge) 2018-12, Vol.25 (10), p.2676-2688.e7
• Main Authors: Flis, Julia, Holm, Mikael, Rundlet, Emily J., Loerke, Justus, Hilal, Tarek, Dabrowski, Marylena, Bürger, Jörg, Mielke, Thorsten, Blanchard, Scott C., Spahn, Christian M.T., Budkevich, Tatyana V.
• Format: Article
• Language: English
• Subjects: Animals; Bacteria - metabolism; cryo-EM; EF-G; elongation factor eEF2; Guanosine Triphosphate - chemistry; Guanosine Triphosphate - metabolism; Humans; Hydrolysis; Internal Ribosome Entry Sites; large-scale conformational changes; macromolecular machine; mammalian ribosome; Mammals - metabolism; Models, Molecular; Peptide Elongation Factor 2 - chemistry; Peptide Elongation Factor 2 - metabolism; Protein Domains; Ribosomes - chemistry; Ribosomes - metabolism; RNA, Messenger - chemistry; RNA, Messenger - metabolism; RNA, Transfer - chemistry; RNA, Transfer - metabolism; smFRET; translation elongation; translocation
• ISSN: 2211-1247; 2211-1247
• DOI: 10.1016/j.celrep.2018.11.040

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. [Display omitted] •Translocation intermediates revealed on eEF2-bound mammalian 80S ribosomes•Inhibition of GTP hydrolysis specifically inhibits final steps of translocation•E-site tRNA release regulates the reversibility of bacterial and mammal translocation 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.