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

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.

Fig. 1. Time-resolved cryo-EM of translocation with EF-G and GTP.

a Scheme of the translocation reaction of the 70S•mRNA•fMet-tRNA^fMet•Pro-tRNA^Pro 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), tRNA^fMet (dark blue), tRNA^Pro (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. 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. 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. 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. 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.