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![[Figure 3]](id5003fig3_combined_mag.jpg)
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Figure 3
The ribosomal exit tunnel and NCs visualized by cryo-EM. (a) The active ribosome comprises 30S (yellow) and 50S (blue) subunits. The exit tunnel site is shown in the central section of the large 50S subunit. The tunnel starts at the PTC and is lined with 23S rRNA nucleotides (purple), the L4 and L22 loops (cyan and green), forming a constriction site, the L23 (violet) loop, 23S rRNA nucleotides (purple) and the L24 (pink) loop at the vestibule region and is shown here with a nascent polypeptide chain (red). The dimensions of the exit tunnel are narrower at the top, ∼10 Å (starting at the C-terminus of the NC), and wider near the vestibule, ∼20 Å. 23S rRNA nucleotides and constriction-site residues (marked regions 1–3, respectively) interact equally with the NC. (b) A schematic representation of the three (bacterial) ribosome-stalling NCs visualized by cryo-EM. The left sides of (b) and (c) indicate different areas in the tunnel (starting at the PTC): upper, central tunnel and vestibule regions. Types of interactions between the tunnel components and the stalling NC residues and their relative interaction points are indicated in different colours (grey circle for non-electrostatic, green circle for electrostatic). L-Tryptophan-binding pockets and an antibiotic-binding pocket for ERY are shown in orange and red, respectively. In 70S–TnaC (shown in purple), the Pro24 and Val20 residues of the TnaC NC interact with U2585 (grey circle) of 23S rRNA, Lys18 interacts with A2058 (green circle), Phe11 interacts with A751 of 23S rRNA (grey circle) and Trp12 interacts with L22 Lys90 (green circle), requiring free L-tryptophan (W1 and W2, orange) molecules to induce ribosome stalling. 70S–SecM shows two SecM NC conformations: SecM-Pro (opaque brown) and SecM-Gly (brown) stalled forms. In SecM-Gly, Ala164 interacts with U2585 (grey circle), Arg163 interacts with the U2585 nucleotide of 23S rRNA (green circle) and Trp155 interacts with Arg64 of the L4 loop (green circle) or A751 of 23S rRNA (grey circle) in SecM-Pro, to induce ribosome stalling. In 70S–ErmBL, the NC (in blue) also adopts a unique conformation induced by bound antibiotic erythromycin (ERY, red) to induce ribosome stalling. The flexible N-terminal residues (shown in yellow, encircled in red) do not interact with ERY but instead adopt altered geometry to allow the critical C-terminal Arg7 residue to interact with U2586 of 23S rRNA (green circle) and cause a translational pause. (c) Three NCs co-translationally folding at the vestibule region on stalled ribosomes as visualized by cryo-EM. 70S–TnaC–R16 (TnaC in purple, GS linker in dark purple, R16 in pink) shows the R16 partially folded domain at the lower vestibule region. 70S–SecM–ADR1α (SecM in brown, ADR1α in red) shows the folded zinc-binding domain at the vestibule region of the tunnel. In 80S–RNC, on a non-stop codon mRNA stalled ribosome, the NC forms an α-helix (in yellow with the α-helix shown as a black line) at the start of the vestibule region. |
![[Figure 3]](id5003fig3_combined.jpg)
 | STRUCTURAL BIOLOGY |
ISSN: 2059-7983
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