Structural mechanism of GTPase-powered ribosome-tRNA movement

GTPases are regulators of cell signaling acting as molecular switches. The translational GTPase EF-G stands out, as it uses GTP hydrolysis to generate force and promote the movement of the ribosome along the mRNA. The key unresolved question is how GTP hydrolysis drives molecular movement. Here, we visualize the GTPase-powered step of ongoing translocation by time-resolved cryo-EM. EF-G in the active GDP–Pi form stabilizes the rotated conformation of ribosomal subunits and induces twisting of the sarcin-ricin loop of the 23 S rRNA. Refolding of the GTPase switch regions upon Pi release initiates a large-scale rigid-body rotation of EF-G pivoting around the sarcin-ricin loop that facilitates back rotation of the ribosomal subunits and forward swiveling of the head domain of the small subunit, ultimately driving tRNA forward movement. The findings demonstrate how a GTPase orchestrates spontaneous thermal fluctuations of a large RNA-protein complex into force-generating molecular movement.


Supplementary Fig. 1 | Cryo-EM analysis of early translocation intermediates. a)
Sorting of cryo-EM data and masks used for focused classification on EF-G domains (dashed box). See Methods for details. b) Ribosome population distribution depending on the nucleotide-binding state of EF-G derived by cryo-EM particle sorting. EF-G-GDP-Pi binding alters the ribosome population from predominantly C to exclusively H, but does not significantly shift the equilibrium between the H1 and H2 states. c) Fourier-Shell-Correlation (FSC) curves for final cryo-EM reconstructions. d) Cryo-EM density for the GTP binding pocket in the H2-EF-G-GDP-Pi structure at 4 Å modelled with GDP-Pi (cyan) and, alternatively, GTP (grey). See main text for details.

Supplementary Fig. 2 | Biochemical characterization at low temperature and Apr binding to the ribosome. a)
Effect of polyamines (P) and Apr (A) on mRNA translocation. Translocation was monitored using a fluorescence-labeled mRNA 34 . The rate of translocation at 4°C is 0.13 s -1 (black), in the presence of polyamines 0.016 s -1 (cyan; 0.6 mM spermine and 0.4 mM spermidine). Addition of Apr (50 µM) abolishes translocation (gray without and red with polyamines). Time courses are averages of 6 technical replicates (N=6). b) Translocation monitored by tripeptide formation. Note the split Y-axis used to visualize the low translocation activity in the presence of Apr. Error bars represent standard deviation of 3 independent experiments (N=3). c) GTP hydrolysis by EF-G at multiple-turnover conditions. The close to 1:1 stoichiometry of single round GTP hydrolysis was validated by addition of the antibiotic fusidic acid, which blocks EF-G on the ribosome after one round of translocation 60 . Error bars represent standard deviation of 3 independent experiments (N=3). d) Pi release. Pi release was measured in the absence and presence of Apr under the same conditions as GTP hydrolysis. Time courses are averages of 4 technical replicates (N=4). e) Apr binding sites. Apr binding sites are shown in the free SSU subunit 22 (SSU, PDB 4AQY), state C, H1 state with EF-G-GDP-Pi, and CHI1 state with EF-G-GDP. Apr binds to the secondary binding site on h44 of 16S rRNA only at low SSU rotation degrees. f) Primary Apr binding site in the SSU decoding center.
Top: The decoding center and Apr are similar in the C and H1 states, despite the large difference in global ribosome conformation. Bottom: Rearrangement of Apr between the C and CHI states. The glucopyranose ring of Apr rotates to accommodate the changes in A1492 and 1493 upon release of the mRNA-tRNA complex from the SSU body.

Supplementary Fig. 3 | Details of the H-EF-G-GDP-Pi structures. a)
Comparison of the H1-EF-G-GDP-Pi structure with the structure of EF-G-GDPCP bound to the POST ribosome 15 (PDB 4V9H). The global EF-G position on the ribosome, including the SRL, is different (left), but the overall EF-G conformation is similar (RMSD 2.0 Å with substate 3 of H1-EF-G-GDP-Pi excluding sw1). Also, the nucleotide binding pocket appears remarkably similar in the two structures (right). The sw1 region is mostly disordered in EF-G-GDPCP, except for the short helix (residues 52-64) that is structured. b) Cryo-EM density for the sw1 region in the H1-EF-G-GDP-Pi structure at 3.1 Å. c) Structural comparison of H1-EF-G-GDP-Pi and POST-EF-G-GDPNP 13 (PDB 4V9K). The overall conformation of EF-G is similar (left), despite the different global position on the ribosome (not shown; similar in refs. 13 and 15 ). The sw1 region (right) adopts an extended conformation in the POST complex and points away from the SSU. d) Unique conformational change of the SRL in EF-G-GDP-Pi.
Left: Structural dynamics of the SRL. The two major conformational modes of the SRL were determined by principal component analysis of ribosome structures with and without different translational GTPases (Methods). The first "common" mode describes the prevalent dynamics of the SRL, the second "twisting" mode reflects the unique change observed in the H-EF-G-GDP-Pi state. Right: Conformational dynamics of SRL residues (solid bars) projected onto the SRL backbone structure in the H1-EF-G-GDP-Pi (dark red) and C state (white). The common mode (grey bars) uniformly affects the entire SRL structure, whereas the twisting mode (light red bars) mostly affects the crucial bases A2660-A2662. In the minor population of H1-EF-G-GDP complexes, the sw1 region is unstructured and EF-G moves in a similar way as in the CHI1 state, indicating that Pi has been released. However, the ribosome remains in the rotated state with tRNAs in hybrid states (H1). There is no density for D4, most likely due to high flexibility, which corroborates the importance of D4 progression into the decoding center to promote mRNA-tRNA movement.

Supplementary Fig. 5 | EF-G interactions and domain dynamics in H1. a)
Interactions of EF-G D3 with protein uS12. Upon Pi release EF-G slides on uS12, switching the contact with His76 of uS12 from Lys424 in the H1 state to Asp427 in the CHI1 state. b) Conformations of EF-G-GDP-Pi D4 in H1. Left: The dynamics of D4 in the H1 state were resolved by sorting the corresponding cryo-EM data into three sub-states and computing separate cryo-EM maps. c) Superposition showing the three distinct sub-states of D4. Notably, the broad conformational range sampled by D4 has no effect on the ribosome conformation or tRNA positions. D4 moves largely independent of the other EF-G domains, facilitated by flexible hinge regions (black dots).
Supplementary Fig. 6 | EF-G reorientation and dynamics upon Pi release. a) Rotation of EF-G on the ribosome upon transition from H2 (grey ribbons) to CHI1 (colored ribbons), shown as cut-away view from SSU side. b) Cα-distance plot demonstrating local changes of sw1 and the β-hairpin in D2 (β-hp), but no global conformational changes within EF-G upon Pi release. CTT, flexible C-terminal tail. c) Pi coordination and SRL contacts of EF-G in H1-EF-G-GDP-Pi vs. CHI1-EF-G-GDP. In contrast to sw1, the conformation of sw2 (containing the catalytic His91) changes only slightly upon Pi release, whereas the contacts with the SRL change substantially. d) Change in EF-G dynamics upon Pi release. The model to map real-space-cross-correlation (RSSC) is plotted for each EF-G residue of the H1-EF-G-GDP-Pi (blue) and the CHI1-EF-G-GDP (red) structures; darker lines correspond to the smoothened curves using the Savitzky-Golay filter. The more dynamic sw1 region in CHI1 state was modelled at 5 Å resolution and its RSSC determined at the same resolution.

Supplementary Fig. 7 | Details of tRNA movement.
Movement of the tRNAs from the C (top) to H1 (middle) to CHI1 (bottom) state. Close-ups of the decoding center (left) and the peptidyl transferase center (right). On the SSU, the A-site fMetPhe-tRNA Phe moves from the A site in C and H1 states to the ap state in CHI1, while the P-site tRNA fMet moves from the P site to the pe state. On the LSU, fMetPhe-CCA is in the A site in C and in the P site in the H1 and CHI1 states. Note the refolding of residues 55-64 of the compact sw1 into a β-hairpin upon Pi release and the substantial changes in the interdomain arrangement, which relax interactions of EF-Tu with the aminoacyl-tRNA (aa-tRNA) and ribosome. For visual clarity, the SRL is not shown in the bottom panels.