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. 2021 Dec 13;12(1):7236.
doi: 10.1038/s41467-021-27415-0.

Time-resolved cryo-EM visualizes ribosomal translocation with EF-G and GTP

Christine E Carbone  1 , Anna B Loveland  1 , Howard B Gamper Jr  2 , Ya-Ming Hou  2 , Gabriel Demo  3   4 , Andrei A Korostelev  5
Affiliations

Time-resolved cryo-EM visualizes ribosomal translocation with EF-G and GTP

Christine E Carbone et al. Nat Commun. .

Abstract

During translation, a conserved GTPase elongation factor-EF-G in bacteria or eEF2 in eukaryotes-translocates tRNA and mRNA through the ribosome. EF-G has been proposed to act as a flexible motor that propels tRNA and mRNA movement, as a rigid pawl that biases unidirectional translocation resulting from ribosome rearrangements, or by various combinations of motor- and pawl-like mechanisms. Using time-resolved cryo-EM, we visualized GTP-catalyzed translocation without inhibitors, capturing elusive structures of ribosome•EF-G intermediates at near-atomic resolution. Prior to translocation, EF-G binds near peptidyl-tRNA, while the rotated 30S subunit stabilizes the EF-G GTPase center. Reverse 30S rotation releases Pi and translocates peptidyl-tRNA and EF-G by ~20 Å. An additional 4-Å translocation initiates EF-G dissociation from a transient ribosome state with highly swiveled 30S head. The structures visualize how nearly rigid EF-G rectifies inherent and spontaneous ribosomal dynamics into tRNA-mRNA translocation, whereas GTP hydrolysis and Pi release drive EF-G dissociation.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Time-resolved cryo-EM of translocation with EF-G and GTP.
a Scheme of the translocation reaction of the 70S•mRNA•fMet-tRNAfMet•Pro-tRNAPro complex with EF-G•GTP. b Segmented cryo-EM maps of 8 states of the translocation reaction, and their assignment as substrates, EF-G-bound intermediates, or products of the reaction. The maps are colored to show the 50S ribosomal subunit (light blue), 30S ribosomal subunit body (yellow) and head (tan), tRNAfMet (dark blue), tRNAPro (green), mRNA (magenta) and EF-G (red). c Relative abundance of substrates (blue), EF-G intermediates (red), and translocation products (green) over time, obtained from particle distributions in cryo-EM datasets. d Domain organization of EF-G; Arabic numerals denote the five conserved domains of the elongation factor. e Cryo-EM density of the EF-G GTPase center in the transient pre-translocation and pre-Pi-release state (III). For additional density views, see Fig. 4 and Supplementary Figs. 3, 4, and 5.
Fig. 2
Fig. 2. Structures of translocation intermediates with EF-G.
a–c Structures III, IV and V with EF-G. 16S nucleotides at the A, P, and E sites (G530, C1400 and G693, respectively) are shown as black surfaces for reference. Unresolved part of EF-G in Structure V is shown in transparent red in panel c. d–e high-resolution density identifying pre-translocation (d) and post-translocation (e) tRNA anticodon and mRNA codon in the P sites of Structures I and VII. f–g Transitions of tRNA and EF-G between Structures III and IV (f) and Structures IV and V (g). h Degrees of 30S head swivel and body rotation in Structures I through VII.
Fig. 3
Fig. 3. Positions and interactions of EF-G in translocation intermediates.
a–b Positions of EF-G and tRNAs relative to the decoding center (yellow) in Structures III and IV. c Superposition of EF-G in Structures III and IV demonstrates an overall similar extended conformation. d Movement of EF-G relative to the 30S subunit from Structure III (gray) to IV (colored). Structures are aligned on 16S rRNA and colored ribosomal elements are from Structure III. e Movement of EF-G relative to the 30S subunit from Structure IV (colored) to V (blue-gray). Structures are aligned on 16S rRNA and colored ribosomal elements are from Structure IV. f Buried surface area (contact area) showing the extent of interactions of EF-G domains with the ribosome, mRNA, and/or tRNA in Structures III to V.
Fig. 4
Fig. 4. The GTPase center of EF-G in translocation Structures III, III-vio and IV.
a–c Cryo-EM densities are consistent with GDP•Pi in Structures III and III-vio, and with GDP in Structure IV. Grey model shows GTP for reference (1WDT). d Positions of sw-I in Structure III (ordered) and IV (gray, disordered) between the SRL and h14 of the 30S subunit. e The catalytic conformation of His92 in Structure III (colored) differs from position of this side chain in the off-ribosome EF-G homolog (archaeal EF-2, gray) bound with GTP analog. f Pretranslocation 70S•EF-G•GDP•Pi structure captured with viomycin (III-vio; gray) is nearly identical to Structure III. Box shows density for EF-G GTPase center in Structure III-vio (His92 density is shown in Supplementary Fig. 2c).
Fig. 5
Fig. 5. Schematic of ribosomal translocation catalyzed by EF-G and GTP.
a–i Progression of translocation and rearrangements of ribosomal subunits, EF-G and tRNAs. j–l Rearrangement of EF-G GTPase switch loop I, showing the coupling of Pi release with 30S rotation and translocation.
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