Regulation of the mammalian elongation cycle by subunit rolling: a eukaryotic-specific ribosome rearrangement

Abstract

The extent to which bacterial ribosomes and the significantly larger eukaryotic ribosomes share the same mechanisms of ribosomal elongation is unknown. Here, we present subnanometer resolution cryoelectron microscopy maps of the mammalian 80S ribosome in the posttranslocational state and in complex with the eukaryotic eEF1A⋅Val-tRNA⋅GMPPNP ternary complex, revealing significant differences in the elongation mechanism between bacteria and mammals. Surprisingly, and in contrast to bacterial ribosomes, a rotation of the small subunit around its long axis and orthogonal to the well-known intersubunit rotation distinguishes the posttranslocational state from the classical pretranslocational state ribosome. We term this motion "subunit rolling." Correspondingly, a mammalian decoding complex visualized in substates before and after codon recognition reveals structural distinctions from the bacterial system. These findings suggest how codon recognition leads to GTPase activation in the mammalian system and demonstrate that in mammalia subunit rolling occurs during tRNA selection.

Figure 1. Reconstruction of eukaryotic 80S POST (A), PRE (classical-1) (B), PRE (classical-2) (C), codon sampling (D) and codon recognition / GTPase activation (E) complexes from rabbit liver

Left panels: Overall view of the cryo-EM reconstructions, displaying the 60S subunit (blue), 40S subunit (yellow), A-tRNA (pink), P-tRNA (green), E-site tRNA (orange), A/T-tRNA (dark violet), eEF1A (red) and mRNA (blue). Middle and right panels: Individual mesh representation of the subunit maps with docked models, showing the ribosomal ligands relative to the 40S subunit (ribosomal RNA yellow and S proteins gray) and the 60S subunit (rRNA blue and L proteins orange), respectively. Landmarks are denoted for 40S: beak (bk), left foot (lf), right foot (rf), head (h), shoulder (sh) and 60S: central protuberance (CP), L1 stalk (L1), stalk base (SB) and stalk (St). See also Figure S1. Heterogeneity in the 80S ribosome POST complexes from rabbit liverand resolution curves. Table S1. Summary of the modelled proteins in the rabbit ribosome.

Figure 2. Eukaryotic-specific features of the mRNA path visible from intersubunit space (A) and solvent side (B) of 40S subunit

(A) Interaction of the P- and E-site tRNAs of the 80S POST complex (transparent gray) with mRNA. (B) Comparison of eukaryotic (transparent gray) and prokaryotic (red ribbon, Jenner et al., 2010, PDB ID 3I8G) mRNA paths. The model for the additional four nucleotides on the 3′-end of the eukaryotic mRNA is shown in cyan. Densities for the ligands were segmented from the cryo-EM map presented in Figure 1A. The models for tRNAs, mRNA and ribosomal proteins from the homology model of the human 80S ribosome presented in this paper. Ribosome orientations are indicated by orientation aids. See also Figure S2. Eukaryotic-specific contacts between P-tRNA and large ribosomal proteins.

Figure 3. 40S subunit rolling

Comparison of the 40S subunit positions in classical-1 state (orange) to the POST state (yellow) represented by (A) cryo-EM maps and (B) ribbons. Comparisons are based on a common 60S alignment. Arrows indicate the direction of movement during transition between the two different states. The distance changes in the 40S subunit positions resulting from the rigid body transformation are color-coded in Å units. Landmarks for 40S aredenoted: head (h), body (b) and beak (bk). See also Figure S3. 40S rolling and its effect on integrity of ribosomal intersubunit bridges and tRNA positions. Movie S1. Effects of 40S rolling on the intersubunit space.

Figure 4. Conformation of the mammalian ternary complex and differences from the bacterial counterpart

Overall fitting of crystallographic models of Aeropyrum pernix aEF1A (Kobayashi et al., 2012) (red ribbon, PDB ID 3VMF) and T.thermophilus A/T-tRNA (pink ribbon, modified from PDB IDs 2XQD and 1TTT for ASL and tRNA body, respectively) to the ternary complex cryo-EM maps (transparent gray) of (A) codon sampling state and (B) codon recognition / GTPase activation state. Densities for the ternary complex are extracted from the cryo-EM maps presented in Figure 1D and E. (C) Superposition of the codon sampling state (transparent gray) and codon recognition / GTPase activation state ternary complex molecular models on the 40S subunit surface. The alignment was based on the 40S subunit densities. (D–E) Comparisons of the mammalian (D) codon sampling state and (E) codon recognition / GTPase recognition state (E) ternary complexes with the bacterial ternary complex stalled by GTP analog (Voorhees et al, Science 2010) (PDB ID 2XQD; transparent gray). The alignment was based on conserved parts of the 18S/16S rRNA. Ribosome orientations are indicated by orientation aids. (C–E) The distances between positions of the ternary complexes are color coded (capped at 6 Å). We note that these distances essentially reflect rigid body transformations of eEF1A/EF-Tu and ASL or body of tRNA. See also Figure S4. Protein sequence alignment of EF1A homologs from different species. Movie S2. Codon recognition induces conformational changes in the A/T-tRNA, leading to a seesaw-like motion of the eEF1A on the 40S surface.

Fig. 5. Interactions of the ternary complexes with the 80S ribosome

(A) Contacts of eEF1A with the shoulder of the 40S ribosomal subunit. Contacts of (B) eEF1A and (C) A/T-tRNA inside of the ternary complexes with SRL of 28S rRNA. (D) Contact of the eEF1A domain III with the C-terminus of uS12. Left column represents the codon sampling state, right column represents the codon recognition / GTPase activation state. Ribosome orientations are indicated by orientation aids. The UniProt numbering which includes the leading methionine, was used, resulting in a +1sequence shift, such that e.g. eukaryotic His94 is His95 according to our numbering. See also Figure S5. Re-organisation of the mammalian ternary complex to adapt to a new ribosomal environment.

Figure 6. Codon recognition and 40S domain closure at the decoding center

(A) Models for the codon recognition / GTPase activation state complex. The decoding center is shown from the 60S side, with h44 in light blue, h18 containing the 626 (530, E.colinumbering ) loop in dark blue, A/T-tRNA in pink, mRNA in green and uS12 in gold. Bases 1824 and 1825 (1492 and 1493, E.coli numbering) of h44 are highlighted in red and are shown in the flipped out positions modelled to the codon recognition state. Panels B–F show density maps for the (B) POST state, (C) the codon sampling state, (D) the codon recognition / GTPase activation state, (E) the PRE classical-2 and (F) the PRE classical-1 state. The models are kept fixed in the recognition state in all panels for better comparison. Electron density maps are aligned to h44 and 40S platform. For clarity, the density around h44 and uS12 has been segmented to show only the surface-most layer. See also Movie S3. 40S domain closure in the decoding center and shoulder domain.

Figure 7. The mammalian elongation circle features unique motions of the 40S subunit

Cartoon representation of the eukaryotic elongation circle highlighting the individual subunit motions necessary to convert one functional intermediate state to the next. Movements of the small ribosomal subunit shared with the prokaryotic system are noted in black, while eukaryotic specific intersubunit movements, i.e. rolling and back-rolling, are noted in red.