Molecular architecture of a eukaryotic translational initiation complex

Abstract

The last step in eukaryotic translational initiation involves the joining of the large and small subunits of the ribosome, with initiator transfer RNA (Met-tRNA(i)(Met)) positioned over the start codon of messenger RNA in the P site. This step is catalyzed by initiation factor eIF5B. We used recent advances in cryo-electron microscopy (cryo-EM) to determine a structure of the eIF5B initiation complex to 6.6 angstrom resolution from <3% of the population, comprising just 5143 particles. The structure reveals conformational changes in eIF5B, initiator tRNA, and the ribosome that provide insights into the role of eIF5B in translational initiation. The relatively high resolution obtained from such a small fraction of a heterogeneous sample suggests a general approach for characterizing the structure of other dynamic or transient biological complexes.

Fig.1. Sample optimization strategy

Our sample preparation procedure was based on structural feedback provided by cryo-EM reconstructions, and consisted of five steps that are described in detail in the main text. Cryo-EM reconstructed density for 60S subunits is indicated in cyan; density for the 40S in yellow; density for eIF5B/tRNA in red and Stm1 protein is depicted as blue ribbon. A similar colour scheme is used throughout all figures.

Fig. 2. Micrograph example, maximum-likelihood classification scheme and final maps

(A) Example of a micrograph obtained after the protocol optimization (B) Maximum-likelihood classification scheme used to localize the subpopulation of particles where density for all four domains of eIF5B could be identified. In (C), gold-standard Fourier shell correlation (FSC) curves for the small class (P/I-tRNA, red) and the larger class (P/E-tRNA, cyan) after refinement and statistical movie processing in RELION (34). Representative densities for the two maps are shown in (D).

Fig.3. The eukaryotic 80S/ Met-tRNA_i^Met /eIF5B initiation complex

(A) Ratcheted overall conformation of the eukaryotic initiation complex. The 40S in the initiation complex (yellow) is rotated anticlockwise approximately 3.4 degrees from its position in the canonical 80S (gray (39)). (B) eIF5B (red), Met-tRNA_i^Met (green) and mRNA start codon (purple) bound to the 80S ribosome (60S in cyan, 40S in yellow). (C) Cryo-EM density showing the interaction between Met-tRNA_i^Met (green) and eIF5B (red) in two orientations. Domain IV of eIF5B contacts the 3′ CCA aminoacyl end of Met-tRNA_i^Met. (D) Conformational changes in eIF5B induced by during formation of the initiation complex on the ribosome. A coordinated displacement of domains III (circled) and IV, together with the linker α-helix 12 of eIF5B, is observed compared to the crystal structure of eIF5B in isolation (19).

Fig.4. Met-tRNA_i^Met conformation on the initiation complex

(A) Cryo-EM density for the Met-tRNA_i^Met allowed unambiguous docking of the tRNA (green) and the start codon of mRNA (purple). (B) Met-tRNA_i^Met (green) in the initiation complex compared to canonical P-site tRNA conformation (orange, PDB ID 2WDK/2WDL)(17). A displacement in the anticodon stem loop (ASL) is indicated. The 3′ CCA of the Met-tRNA_i^Met is stabilized in a conformation that prevents its entering the peptidyl transferase center (PTC), circled.

Fig.5. Role of domains III and the connecting helix 12 of eIF5B in GTPase activation

(A) Details showing the interaction of domain III of eIF5B with ribosomal protein S23 from the 40S (yellow) and the base of helix 12 with the sarcin ricin loop (SRL) of the 28S rRNA (B) Zoomed-in view of the interaction between helix 12 and the SRL around residue Y838. (C) A sequence alignment of IF2 from the bacteria Escherichia coli (EC), aIF5B from the archaea Methanothermobacter thermautotrophicus (MT) and eIF5B from the eukaryotes Saccharomyces cerevisiae (SC) and humans (HM) highlights the conservation of the Y838 and surrounding residues suggesting the importance of this interaction.

Fig.6. Two views of the eukaryotic ribosomal GTPase centre

The G-domain and domain II of eIF5B (red) contact the GTPase center of the 80S formed by the ribosomal proteins L9 (blue), L40 (purple) and regions of the 28S rRNA (cyan) and the 18S rRNA (yellow). Helix 6 of eIF5B packs tightly against the C-terminal domain of L9 and the N-terminal domain of the eukaryote-specific protein L40 (inset, top left). Density for the GTP analog GDPCP was clearly seen in the G-domain of eIF5B facing the 28S-rRNA SRL (inset, bottom left).