Structural basis of early translocation events on the ribosome

Emily J Rundlet,Mikael Holm,Scott C Blanchard,Magdalena Schacherl,Christian M T Spahn,Roger B Altman,Alexander G Myasnikov,S Kundhavai Natchiar

doi:10.1038/s41586-021-03713-x

Emily J Rundlet, Mikael Holm + Show 6 more

Open Access

https://doi.org/10.1038/s41586-021-03713-x

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Abstract

Peptide-chain elongation during protein synthesis entails sequential aminoacyl-tRNA selection and translocation reactions that proceed rapidly (2–20 per second) and with a low error rate (around 10−3 to 10−5 at each step) over thousands of cycles1. The cadence and fidelity of ribosome transit through mRNA templates in discrete codon increments is a paradigm for movement in biological systems that must hold for diverse mRNA and tRNA substrates across domains of life. Here we use single-molecule fluorescence methods to guide the capture of structures of early translocation events on the bacterial ribosome. Our findings reveal that the bacterial GTPase elongation factor G specifically engages spontaneously achieved ribosome conformations while in an active, GTP-bound conformation to unlock and initiate peptidyl-tRNA translocation. These findings suggest that processes intrinsic to the pre-translocation ribosome complex can regulate the rate of protein synthesis, and that energy expenditure is used later in the translocation mechanism than previously proposed.

Highlights

Within the PRE complex, deacyl- and peptidyl-tRNAs can rapidly and spontaneously unlock from their ‘classical’ positions (PRE-C) after peptide-bond formation to achieve multiple ‘hybrid’ states (PRE-H)[18,19]
To gain insight into how tRNA2–mRNA movement is initiated by EF-G, and the role of GTP hydrolysis in translocation, we used smFRET to guide the capture of six cryo-electron microscopy structures of the ribosome in both early and late stages of translocation
This approach yielded six high-resolution (2.3–2.8 Å) ribosome structures programmed with deacyl-tRNAPhe and fMet-Phe-Lys-tRNALys at sequential stages of translocation (Extended Data Fig. 2, Supplementary Table 1), including the first—to our knowledge—structure of EF-G bound to a ribosome in an active conformation before inorganic phosphate

Summary

E PA Reverse swivel

These post-hydrolysis changes correlated with an upward shift and an approximately 15° rotation of the G domain of EF-G relative to the SRL (Extended Data Fig. 8e), together with inward displacement of the entire GTPase-activating centre towards the LSU central protuberance (Supplementary Video 2) Despite such extensive remodelling, EF-G DIV loops I and III remained in direct contact with the peptidyl-tRNA anticodon–mRNA codon pair, while losing contact with the tRNA body[6,9,10] (Extended Data Fig. 8b). Such changes are likely to contribute to reading frame maintenance while opening pathways through which deacyl-tRNA can shift position and/or dissociate[13,17,42]

Discussion

Methods