Structural insight into nascent polypeptide chain-mediated translational stalling

Abstract

Expression of the Escherichia coli tryptophanase operon depends on ribosome stalling during translation of the upstream TnaC leader peptide, a process for which interactions between the TnaC nascent chain and the ribosomal exit tunnel are critical. We determined a 5.8 angstrom-resolution cryo-electron microscopy and single-particle reconstruction of a ribosome stalled during translation of the tnaC leader gene. The nascent chain was extended within the exit tunnel, making contacts with ribosomal components at distinct sites. Upon stalling, two conserved residues within the peptidyltransferase center adopted conformations that preclude binding of release factors. We propose a model whereby interactions within the tunnel are relayed to the peptidyltransferase center to inhibit translation. Moreover, we show that nascent chains adopt distinct conformations within the ribosomal exit tunnel.

Fig. 1

Cryo-EM reconstruction of the TnaC•70S complex. (A-B) Cryo-EM reconstruction of the control E. coli 70S ribosome at 6.6 Å resolution, with small and large subunit colored yellow and blue, respectively. (C-D) The 5.8 Å resolution cryo-EM density of the TnaC•70S complex, with density for the TnaC-tRNA shown in green. (E) Isolated density for the TnaCtRNA (green) and mRNA (red) from (C). The relative positions of ribosomal proteins L4 (purple), L22 (blue) and L23 (yellow) are indicated. (F) Fitting of molecular models for the TnaC-tRNA^Pro into the cryo-EM density from (E).

Fig. 2

Interaction between the TnaC nascent chain and the ribosomal tunnel. The central panel shows a section through the large subunit revealing contact points between the TnaC nascent chain (light green) and the ribosome (gray). Density attributed to the P-tRNA is coloured dark green, and for peptidyl-tRNA at a lower threshold is shown as a green mesh. (A)-(D) Different views of connections between the nascent chain (green ribbon) and the 23S rRNA (blue sticks) or ribosomal protein L22 (blue ribbon). Density for the TnaC•70S complex is shown as a transparent gray surface, whereas the isolated nascent chain at a lower threshold is shown as a green mesh. Important TnaC residues are highlighted yellow, orange or red.

Fig. 3

Silencing of the peptidyltransferase center (PTC). (A) Conformation of 23S rRNA nucleotides at the PTC when tRNA CCA-end mimics are bound to A- (cyan) and P-sites (green) (PDB1VQN). (B) Comparison of A2602 and U2585 conformations in various ribosome crystal structures (red, PDB1VQK; teal, PDB1S72; yellow, PDB2I2T; gold, PDB2JL5/6; blue, PDB1VQ9; green, PDB1VQN). (C) View into the PTC of the TnaC-70S complex, with the MDFF model of the TnaC-tRNA (green) and nucleotides of the 23S rRNA (blue). The cryo-EM density is shown as a transparent gray surface, with an asterisk indicating the connection between P-tRNA and nascent chain. (D) View into the PTC of 70SRNC complex, with fitted models as in (A). Note the lack of density (gray) for nucleotide A2602. (E) As in (A), but with the antibiotic sparsomycin (SPAR, red; PDB1VQ9) and the terminal A76 and aminoacyl moiety of an A-tRNA (cyan; PDB1VQN) included. (F) Comparison of A2602 and U2585 positions (arrowed) between TnaC-70S complex (blue) and RF2-70S complex (24); gold), with RF2 shown as surface representation (gold).

Fig. 4

Relay for PTC silencing and the path of the nascent chain. (A) Ribosomal components potentially involved in a relay mechanism to inactivate the PTC, with those implicated in stalling in bold. The TnaC nascent chain is in green, with residues essential for stalling colored yellow. The isolated TnaC-tRNA density is shown as a transparent gray surface. (B) Schematic indicating potential relay pathways from Trp¹² (W12) of TnaC to the PTC, either through the nascent chain itself (R1) or via networks of interconnected 23S rRNA nucleotides (R2 and R3). (C-D) Transverse section through the large subunit showing the path of (C) TnaC (dark green) and (D) DP120 (35); orange) nascent chains through the ribosomal tunnel. (E) Superposition of (A) and (B). (F) Schematic highlighting the similarities and differences between the TnaC and DP120 nascent chain in terms of contacts and passage through the tunnel.