The structure of a human translation initiation complex reveals two independent roles for the helicase eIF4A

Figures

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  Fig. 1. Cryo-EM 3D reconstruction and the structure of a human 48S complex. a–c, Overview of the cryo-EM density map shown in different orientations. d–f, Overview of the molecular model of human 48S shown in different orientations. The map contains densities for 40S small ribosomal subunit, eIF3, eIF1A, eIF5-NTD, eIF2, tRNAiMet, mRNA, eIF4F (eIF4G and eIF4A) and a second molecule of eIF4A bound to eIF3 at the mRNA channel entry site. The map also contains a possible density for eIF4B in contact with the second molecule of eIF4A at the entry site.

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  Fig. 2. Structure of human 48S reveals a second molecule of eIF4A bound at the mRNA channel entry site. a, Atomic model of human eIF4A fitted (correlation 0.9024) into the cryo-EM map filtered to local resolution (6 to 10 Å). b, Atomic model of eIF4A, eIF4F, mRNA and tRNAiMet fitted into a low-pass filter cryo-EM map. c, Interactions of eIF4A at the entry site. d, The interface between eIF4A-CTD and eIF3a-CTD.

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  Fig. 3. Conformational differences between the two molecules of eIF4A and a possible location of eIF4B in the 48S. a,b, Superposition of eIF4F with entry site eIF4A to highlight conformational differences shown in in different orientations. eIF4G-HEAT-1 would clash with eIF3i and eIF3b. c,d, Atomic model of human eIF4B-RRM (PDB: 2J76)31 fit into the unassigned cryo-EM density map at the entry site shown in in different orientations. The possible location of eIF4B places it in close proximity to eIF4A-NTD, eIF3g-RRM, uS3, eS10 and uS10.

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  Fig. 4. Structure of eIF4F and its interactions with eIF3 and ribosomal protein eS7. a–c, Cryo-EM map to highlight eIF4F and its interaction network shown in in different orientations. d–f, Atomic model of human eIF4A and eIF4G fitted into the cryo-EM map shown in in different orientations. g–i, The insets highlight the interaction network of eIF4F with eIF3. j, Close-up to highlight the interaction between eIF4F and ribosomal protein eS7. k, Superposition of ubiquitin (PDB:8C83)53 with the structure of human 48S to highlight the possible clash between eIF4A and ubiquitin at its predicted binding site (Lys85).

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  Fig. 5. Codon–anticodon base pairing in the P site. a, Close-up of the P site to highlight the codon–anticodon base pairing. b, eIF5-NTD replaces eIF1 after AUG start codon recognition. c, Close-up view illustrating the interaction between eIF5-NTD, tRNA, and mRNA in the P site. d, Superposition of eIF5-NTD with the structure of human eIF1 during scanning (PDB: 6ZMW)4. e, The conformation of eIF1 during scanning would clash with the anticodon stem loop of the tRNAiMet after start codon recognition.

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  Fig. 6. Model for translational regulation by eIF4F and eIF4A. a–c, The cap-binding complex eIF4F (a) binds at the 5′ end of mRNA and recruits the 43S (b) to form the 48S complex (c). The fuzzy line represents the mRNA into the channel in the 40S. eIF4F binds to eIF3 and ribosomal protein eS7 located upstream of the 43S. The position of eIF4F is compatible with a model whereby the mRNA is slotted into the mRNA channel in the 40S small ribosomal subunit. In addition to eIF4F, a second molecule of eIF4A at the mRNA entry site is likely to play a role in this process by facilitating the accommodation of the mRNA into the channel. The ATPase activity of eIF4A is stimulated by eIF4B and eIF3bgi module 15 located at the entry site. d, Thus, eIF4A at the entry site is likely to use the energy from ATP hydrolysis to unwind the RNA secondary structure downstream of the 43S. e,f, It remains unclear whether eIF4E remains attached to the rest of eIF4F (e) or is released (f) during the scanning process, but the structure suggests the latter.

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  Extended Data Fig. 1. Analysis of capped mRNA after Poly(A) tailing. The polyadenylated mRNA has additional ~90 nucleotides at the 3’ end. The poly(A) tailing was performed according to the protocol provided by the manufacturer (NEB; M0276). The final mRNA, containing a poly(A) stretch, was stored and used to assemble the complex for cryo-EM.

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  Extended Data Fig. 2. Cryo-EM data analysis of human 48S. We performed two independent data collections, and each dataset (datasets 1 and 2) was analyzed independently. After polishing and CTF refinement, the two datasets were merged, which yielded the 2.9 Å 3D reconstruction of a human 48S.

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  Extended Data Fig. 3. Overall and local resolution. (a and b) Fourier shell correlation (FSC) curve and local resolution of the 48S complex after focus 3D focus classification at the entry site, eIF4F, and ternary complex. (c and d) Overall and local resolution of eIF4F-eIF3 after multi-body refinement.

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  Extended Data Fig. 4. Cryo-EM 3D reconstruction of a human 48S complex assembled without Rocaglamide (RocA). (a to c) Overview of the cryo-EM density map. The complex contains both eIF4F and the second molecule of eIF4A bound at the mRNA channel entry site. To assembly this complex, we used an mRNA with highly stable RNA stem-loop structure (DLP) located 83 nucleotides downstream of the cap. This mRNA also has an AUG start codon.

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  Extended Data Fig. 5. Conformation of molecule of eIF4A bound at the mRNA entry site. (a) Superposition of eIF4A with the structure of a yeast eIF4A in complex with eIF4G (PDB:2VSO)23. (b) Superposition of eIF4A with the structures of eIF4A in complex with Pdcd4 (PDB:3eiq)24.

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  Extended Data Fig. 6. Cryo-EM 3D reconstruction of a human 48S assembled with a truncated eIF4G (residues 557–1137) or without eIF4B. (a to c) Overview of the cryo-EM density map of a 48S complex assembled with a truncated eIF4G (residues 557–1137). The eIF4A density is visible even without 3D focus classification. The eIF4F density is visible, but is faint without focus classification. (d to f) Overview of the cryo-EM density map of a 48S complex assembled without eIF4B. The red oval highlights the absence of the density in the complex without eIF4B (g) that can be seen in the structure of 48S assembled eIF4B (h).