Research ArticleSTRUCTURAL BIOLOGY

Structures of ribosome-bound initiation factor 2 reveal the mechanism of subunit association

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Science Advances  04 Mar 2016:
Vol. 2, no. 3, e1501502
DOI: 10.1126/sciadv.1501502
  • Fig. 1 Overview of the cryo-EM reconstruction of the two distinct 70S initiation complexes.

    (A to F) Overview of the cryo-EM reconstruction of the 70S-IC I complex (A to C) and of the 70S-IC II complex (D to F). The cryo-EM maps (blue, 23S and 5S rRNA; orange, L proteins; yellow, 16S rRNA; gray, S proteins; green, fMet-tRNAiMet; red, IF2) (A and D) and mesh representation of the cryo-EM maps separated into ribosomal subunits with docked models (blue, 23S and 5S rRNA; orange, L proteins; yellow, 16S rRNA; gray, S proteins; green, fMet-tRNAiMet; red, IF2; purple, mRNA) (B and E) are shown. (C and F) View from the intersubunit space onto the models of 30S subunit depicted as ribbons. Elements are colored according to their structural displacement compared to the classical (13) conformation upon 50S alignment. The directions and magnitudes of the intersubunit rotation and head swivel for the 70S-IC I complex (C) and the 70S-IC II complex (F) are indicated. (G to I) Enlarged regions of the cryo-EM map of the 70S-IC II at 3.7-Å resolution, showing bL34 (G), bS16 (H), and IF2 (I) (gray, cryo-EM density; orange, bL34; gray, bS16; red, IF2).

  • Fig. 2 Binding state of the fMet-tRNAfMet.

    (A and B) Comparison of the position of P/pi-tRNA (green) and P/ei-tRNA (orange) with classical P-site and hybrid P/E-site (gray) (13) [Protein Data Bank (PDB) ID 4V9D] tRNAs upon 30S (A) and 50S alignment (B). Compared to the P/P tRNA, the elbow of the P/pi tRNA is shifted by 22 Å toward the E-site, and the codon-anticodon duplex of the P/ei-tRNA is shifted by 4 Å toward the E-site. Compared to the P/pi tRNA, the elbow of the P/ei-tRNA is displaced by additional 16 Å toward the E-site to allow interaction with the oncoming L1 stalk. (C) Conformation of the P/pi tRNA (green) compared to the P/E and P/P (gray) conformations upon anticodon stem loop (ASL) alignment; highlighted is the hinge region of the tRNAs.

  • Fig. 3 IF2 on the ribosome.

    (A) Superposition of IF2 and initiator tRNA in 70S-IC I (gray) and 70S-IC II complexes upon 50S alignment. The distances between both substates are color-coded (capped at 5 Å). (B) Model for the interaction of the 3′-CCA of the fMet-tRNAiMet (green) with domain IV of IF2 (red). Important residues for the binding interaction are indicated. (C) Comparison of the switch regions in Thermus thermophilus apo IF2 (7) (PDB ID 4KJZ, chain B) (gray) and Escherichia coli IF2-GDPNP (red) upon G-domain alignment.

  • Fig. 4 Scheme of the late steps of initiation.

    IF2 (red)–induced subunit joining of the 30S IC (yellow) with the 50S (blue) subunit occurs in a rotated conformation and leads to the formation of the 70S-IC I. The initiator tRNA (green) is positioned in the P/ei state through interactions with the L1 stalk and domain IV of IF2. Partial back rotation and unswiveling facilitate the P/pi state of initiator tRNA and reorient the G-domain of IF2 to trigger GTP hydrolysis. To reach the elongation-competent 70S complex, the 30S subunit completes back rotation, IF2-GDP dissociates, and the initiator tRNA completes the partial reverse translocation on the 50S subunit to reach the P/P-site state. Movements of elbow and acceptor stem are indicated.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/2/3/e1501502/DC1

    Materials and Methods

    Fig. S1. Overview of three-dimensional classification.

    Fig. S2. Resolution estimations of the 70S-IC complexes.

    Fig. S3. Comparison of the present E. coli IF2 with previous models.

    Fig. S4. Comparison of the tRNA positions.

    Fig. S5. Comparison of the present ribosome-bound IF2 with ribosome-bound EF-Tu, ribosome-bound eIF5b, and the x-ray structure of IF2.

    Fig. S6. Comparison of the tRNA binding domains II of EF-Tu and IV of IF2.

    Fig. S7. Back rotation of the 30S subunits positions the G-domain of IF2 onto the SRL.

    Fig. S8. Subunit joining requires repositioning of the initiator tRNA.

    Table S1. Model statistics.

    Reference (49)

  • Supplementary Materials

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Overview of three-dimensional classification.
    • Fig. S2. Resolution estimations of the 70S-IC complexes.
    • Fig. S3. Comparison of the present E. coli IF2 with previous models.
    • Fig. S4. Comparison of the tRNA positions.
    • Fig. S5. Comparison of the present ribosome-bound IF2 with ribosome-bound EF-Tu, ribosome-bound eIF5b, and the x-ray structure of IF2.
    • Fig. S6. Comparison of the tRNA binding domains II of EF-Tu and IV of IF2.
    • Fig. S7. Back rotation of the 30S subunits positions the G-domain of IF2 onto the SRL.
    • Fig. S8. Subunit joining requires repositioning of the initiator tRNA.
    • Table S1. Model statistics.
      Reference (49)

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