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. 2022 Jan 25;13(1):476.
doi: 10.1038/s41467-022-27967-9.

High-resolution structures of a thermophilic eukaryotic 80S ribosome reveal atomistic details of translocation

Miglė Kišonaitė  1 , Klemens Wild  1 , Karine Lapouge  1 , Thomas Ruppert  2 , Irmgard Sinning  3
Affiliations

High-resolution structures of a thermophilic eukaryotic 80S ribosome reveal atomistic details of translocation

Miglė Kišonaitė et al. Nat Commun. .

Abstract

Ribosomes are complex and highly conserved ribonucleoprotein assemblies catalyzing protein biosynthesis in every organism. Here we present high-resolution cryo-EM structures of the 80S ribosome from a thermophilic fungus in two rotational states, which due to increased 80S stability provide a number of mechanistic details of eukaryotic translation. We identify a universally conserved 'nested base-triple knot' in the 26S rRNA at the polypeptide tunnel exit with a bulged-out nucleotide that likely serves as an adaptable element for nascent chain containment and handover. We visualize the structure and dynamics of the ribosome protective factor Stm1 upon ribosomal 40S head swiveling. We describe the structural impact of a unique and essential m1acp3 Ψ 18S rRNA hyper-modification embracing the anticodon wobble-position for eukaryotic tRNA and mRNA translocation. We complete the eEF2-GTPase switch cycle describing the GDP-bound post-hydrolysis state. Taken together, our data and their integration into the structural landscape of 80S ribosomes furthers our understanding of protein biogenesis.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Overall architecture of the Ct80S ribosome.
a The 40S subunit viewed from the intersubunit interface (left) and in the crown view (right) with ribosomal proteins indicated. b Same presentation for the 60S subunit from the backside (left) and in the crown view (right). The L1-stalk is missing and the P-stalk only partially built.
Fig. 2
Fig. 2. The second copy of eL41 at the periphery of the 60S subunit.
Cryo-EM maps of 80S ribosomes from C. thermophilum and S. cerevisiae. The second copy of eL41 (red) in C. thermophilum is tightly surrounded by the 26S rRNA, while the absence of eL41 in Sc80S ribosome leaves a deep protrusion in the ribosomal surface (void with red spots).
Fig. 3
Fig. 3. Ct80S ribosomes retain nascent chains.
a Cryo-EM map for the (TI)-POST state of Ct80S with NC density shown in magenta. The narrowing at the ribosomal tunnel exit is highlighted by a green circle. b Zoom into the C. thermophilum 80S tunnel exit region covered by cryo-EM-density (3.5 σ, 1.5 σ for NC) with highlighted nucleotides of 28S rRNA H50. The tunnel diameter and relevant hydrogen-bonds (colored dashed lines) are indicated. c Comparison of the tunnel exit to S. cerevisiae. Helix H50 forms a unique ‘nested base-triple (pseudo-)knot’. With the flipped-in C1502 (corresponding to G1485) two base-triples are formed. Same color code as in panel b. d, e Schemes of the nested base-triple knot with two intertwined triples and an exposed base stack as platform for the bulged-out nucleotide, as shown for C. thermophilum (d) and S. cerevisiae (e). Base-flipping is indicated for the respective O. cuniculus G2416 nucleotide (light blue, dashed outline) as revisited here for the mammalian SRP-ribosome complex. f Mammalian SRP-ribosome complex. The close-by SRP54M domain recognizes the N-terminal signal (upper left corner) of the NC in the tunnel (parts connected by dotted line). The guanine base is built flipped-in, but according EM-density is at least partially bulged-out (indicated by double-arrow, see also Supplementary Fig. 20).
Fig. 4
Fig. 4. Ribosome-associated factor Stm1 and the m1acp3Ψ hypermodification.
a Surface representation of the Ct80S in both rotational states with bound Stm1 (red), eEF2 (orange), and pe/E tRNA (blue). b Zoom on the pathway of Stm1 following the P- and A-sites, and the mRNA entry tunnel. A-, P-, and E-sites are marked with circles. c In the idle POST state Stm1 occupies the mRNA position in the P-site. The strictly conserved m1acp3Ψ hypermodification in eukaryotic 18S rRNA (CtU1188) lines the P-site (cyan line). Cryo-EM map is shown for central features (2 σ). d In the rotated (TI)-POST state of Ct80S, Stm1 conformation is changed and m1acp3Ψ1188 forms an interaction with the pe/E-site tRNA-ASL in the E-site. e In presence of the codon-anticodon, the modified nucleotide (not built originally) forms a lid described here as wobble-seal together with a conserved cytosine stacking on the wobble.
Fig. 5
Fig. 5. Ct80S modifications and ligands.
a N-terminal acetylation of Ser2 of uL13 (SAC2) is shown in its cryo-EM-density and with close interactors (highlighted by color and with dashed connector lines). b Conserved 2’-O methylation in the PTC. The high resolution allows for detailed modelling of sugar methylations and conformations (OMG2578), and of magnesium coordination. The peptidyl-tRNA is modeled from a superposed high-resolution Sc80S structure. c Typical example for a magnesium ion (magenta) with its hydration shell tethering two rRNA helices of 5.8S and 26S rRNA, respectively. Interactions are indicated by dashed lines. All cryo-EM maps are contoured at a 2.5 σ level.
Fig. 6
Fig. 6. eEF2 Dph modification and GTPase switch cycle.
a Top: (TI)-POST state of Ct80S with eEF2-GDP shows the position of the conserved diphthamide (Dph) histidine modification at the apex of eEF2 domain IV. The monitoring adenines of the DC are marked in magenta. Bottom: The (TI)-POST state with tRNA2•mRNA module (although still with non-hydrolyzable eEF2-‘GTP’). Dph is in same post-conformation as in our (TI)-POST state. Codons and associated tRNAs are color-coded. b The rotated ribosome acts as GAP for GTP hydrolysis in eEF2. Top: The full-rotated ribosome (40S body helix H5) pushes on the ordered switch 1 region (sw1) of eEF2 and places an intrinsic arginine finger. Bottom: Upon back-rotation (indicated by arrows), as observed in the (TI)-POST state of Ct80S with eEF2-GDP-Mg2+, sw1 becomes disordered. c, d Switch 2 (sw2, green) conformations during the eEF2 switch cycle. In context of the PRE ribosome, the SRL is in tight contact with sw2 and the P-loop (light blue) of eEF2-‘GTP’. Upon GTP hydrolysis and as detailed for (TI)-POST Ct80S, the P-loop and sw2 relax and contacts are loose. Magnesium ions (magenta spheres) mediate SRL-eEF2 contacts. GDP is shown with its cryo-EM map (3 σ). e, f Switch 1 conformations during the eEF2 switch cycle. In the full-rotated state, the arginine finger within sw1 is placed on top of the scissile bond (finger-in) and the ribosome acts as GAP (not described there). Upon back-rotation of the 40S body (indicated by gray arrow), as seen in the (TI)-POST state of Ct80S, sw1 becomes disordered and eEF2 relaxes in a relay system (distant domains II and III).
Fig. 7
Fig. 7. Scheme of eEF2-catalyzed ribosome translocation.
From left to right: Schematic for eukaryotic ribosomal states along a translocation cycle starting with the PRE state after peptide-bond formation. The m1acp3Ψ hypermodification (magenta bracket) within the 40S head, on top of position one of the anticodon in the wobble, helps as part of the wobble-seal in coupling ribosomal rotation to tRNA2•mRNA module movements between the classical A/A P/P and the A/P P/E hybrid states. Dph (highlighted in blue) within domain IV of eEF2-GTP acts as a doorstop (pawl) in the A-site and the full-rotated state is stabilized. The full-rotated ribosome acts as GAP inducing GTP hydrolysis in eEF2, marking the transition to the translocated POST state. Back-rotation of the 40S head is uncoupled from the tRNA2•mRNA module and Dph acts as ‘post-pawl’ in contact with the P-site. eEF2-GDP binding is weakened due to the internal relay and SRL-contact loosening and upon back-swiveling of the 40S head, eEF2-GDP is released. Translocation is complete in the P/P E/E state.
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