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. 2011 Jul;9(7):e1001095.
doi: 10.1371/journal.pbio.1001095. Epub 2011 Jul 5.

The Cryo-EM structure of a complete 30S translation initiation complex from Escherichia coli

Patricia Julián  1 , Pohl Milon, Xabier Agirrezabala, Gorka Lasso, David Gil, Marina V Rodnina, Mikel Valle
Affiliations

The Cryo-EM structure of a complete 30S translation initiation complex from Escherichia coli

Patricia Julián et al. PLoS Biol. 2011 Jul.

Abstract

Formation of the 30S initiation complex (30S IC) is an important checkpoint in regulation of gene expression. The selection of mRNA, correct start codon, and the initiator fMet-tRNA(fMet) requires the presence of three initiation factors (IF1, IF2, IF3) of which IF3 and IF1 control the fidelity of the process, while IF2 recruits fMet-tRNA(fMet). Here we present a cryo-EM reconstruction of the complete 30S IC, containing mRNA, fMet-tRNA(fMet), IF1, IF2, and IF3. In the 30S IC, IF2 contacts IF1, the 30S subunit shoulder, and the CCA end of fMet-tRNA(fMet), which occupies a novel P/I position (P/I1). The N-terminal domain of IF3 contacts the tRNA, whereas the C-terminal domain is bound to the platform of the 30S subunit. Binding of initiation factors and fMet-tRNA(fMet) induces a rotation of the head relative to the body of the 30S subunit, which is likely to prevail through 50S subunit joining until GTP hydrolysis and dissociation of IF2 take place. The structure provides insights into the mechanism of mRNA selection during translation initiation.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Cryo-EM reconstruction and classification of the 30S IC.
(A) Initial reconstruction of the 30S IC with partial densities for tRNA and IF2 on the surface of the 30S subunit (arrow). (B) Cryo-EM map of class 1 particles after ML3D classification of the total set of images. No density for tRNA or IF2 can be seen. (C) The cryo-EM map of class 2 particles showing improved density for tRNA and IF2 on the 30S subunit (arrow). This map was assigned to the 30S IC complex. (D and E) Two orientations of the cryo-EM map for the 30S IC complex after segmentation and fitting of atomic coordinates attributed to the 30S subunit (gray), IF2 (green), IF1 (blue), fMet-tRNAfMet (red), and IF3 (orange). Smaller thumbnails depict segmented map with solid densities in different colors, while large renderings in semi-transparent representation show fitted atomic coordinates. Landmarks on the 30S subunit in all figures indicate: h, head; sp, spur; sh, shoulder; pt, platform; h44, helix 44 from the 16S rRNA.
Figure 2
Figure 2. Conformation of the 30S subunit within the 30S IC.
(A) 30S subunit conformations in reconstructions of particles from class 1 (red mesh) and class 2 (semitransparent yellow). The arrow indicates the clockwise rotation of the 30S subunit head. The inset shows the positions of protein S13 in the two maps. (B) The current 30S IC map (class 2 after ML3D classification) aligned with the cryoEM map for the 70S IC (EM database code 1249 [12]) in two distinct orientations. (C) Comparison of the 30S·mRNA map (class 1 after classification) with the 70S IC map . The alignment between density maps was performed by maximum overlap of the body of the 30S subunits.
Figure 3
Figure 3. Homology model for IF2 guided by the cryo-EM map.
(A) Close-up view of the fMet-tRNAfMet·IF2 complex on the 30S IC. Densities for fMet-tRNAfMet and IF2 were rendered semitransparent to show the fitted IF2 (ribbon representation). For fMet-tRNAfMet (pdb code 2FMT; [31]), a rigid-body fitting was performed. Domains G2 (orange), G3 (green), C1 (grey), and C2 (purple) of IF2 are indicated. The NTD was modeled as three sub-domains (yellow, blue, and pink). (B) Domain structure of IF2. Colors are as in (A). Below the segment for each domain, the homology T-Coffee score between IF2 from E. coli and the atomic coordinates used is shown.
Figure 4
Figure 4. IF2 on the 30S IC.
(A) Interactions of the NTD of IF2 with IF1 and S12. Thumbnail shows orientation. (B) Interactions of domains G2 and G3 of IF2 with 16S rRNA. Residues of helix h14 involved in formation of the intersubunit bridge B8 are highlighted in orange. (C) Position of IF2 relative to intersubunit bridges. The binding site for IF2 entails 16S rRNA helices h5 and h14 (green), and proteins IF1 (blue) and S12 (pink). The crystal structure of IF1 bound to the 30S subunit is taken from (pdb code: 1HRO; [7]). Intersubunit bridges are shown in orange. Bridges in the head of the 30S subunit are highlighted in red and the arrow indicates the direction of the 30S subunit head movement.
Figure 5
Figure 5. Orientation of fMet-tRNAfMet bound to the 30S IC.
(A) Comparison of the positions of fMet-tRNAfMet in the 30S IC (red) with the atomic coordinates for A-, P-, and E-site tRNAs (pdb code: 2HGP; [41]). The arrow indicates a tilt of the fMet-tRNAfMet by around 10 degrees compared to the tRNA in the P site. (B) Top view showing the 15 Å rotation of fMet-tRNAfMet in the complex with IF2 (semitransparent green). (C and D) Comparison between the P/I1 and P/I sites. In (C) densities for IF2 and tRNA from the 30S IC are shown semitransparent, allowing us to visualize fitted coordinates for tRNA (red ribbons) and IF2 (green). For comparison, coordinates fitted in the 70S IC (pdb code: 1ZO1 [12]) are represented for IF2 (purple ribbons) and tRNA in the P/I site (blue). In (D) density for IF2 and tRNA is taken from the 70S IC (EM database code 1249) and depicted semi-transparent. Atomic coordinates are as in (C).
Figure 6
Figure 6. IF3 on the 30S IC.
(A) Density for IF3 (semitransparent orange) fitted using the atomic coordinates for IF3 domains, IF3N (pdb code: 1TIF; [36]) and IF3C (pdb code: 2IFE; [37]). Thumbnail shows the orientation. (B) Same orientation as in (A) with semitransparent 30S subunit. Regions on 16S rRNA and tRNA in the proximity of IF3, based on hydroxyl radical footprinting , are shown in navy blue. Arrow indicates helix h24 and the loop around nucleotide 790 in 16S rRNA.
Figure 7
Figure 7. Docking of the 50S subunit onto the 30S IC.
(A) Steric clash between IF3C and helix H69 of 23S rRNA at intersubunit bridge B2b. The present map for the 30S IC was aligned with the 70S IC . (B) IF2 as an important determinant for the 50S subunit association. The position of the sarcin-ricin loop (SRL) at the cleft formed by domains G2 and C1 of IF2 is indicated. In both panels, the thumbnails show the 30S IC docked onto the 50S subunit. Labels on the 50S subunit indicate helices in 23S rRNA (H69, H89, and H95) and the stalk region for protein L1.
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