Translational termination without a stop codon

Abstract

Ribosomes stall when they encounter the end of messenger RNA (mRNA) without an in-frame stop codon. In bacteria, these "nonstop" complexes can be rescued by alternative ribosome-rescue factor A (ArfA). We used electron cryomicroscopy to determine structures of ArfA bound to the ribosome with 3'-truncated mRNA, at resolutions ranging from 3.0 to 3.4 angstroms. ArfA binds within the ribosomal mRNA channel and substitutes for the absent stop codon in the A site by specifically recruiting release factor 2 (RF2), initially in a compact preaccommodated state. A similar conformation of RF2 may occur on stop codons, suggesting a general mechanism for release-factor-mediated translational termination in which a conformational switch leads to peptide release only when the appropriate signal is present in the A site.

Fig. 1. Structures of nonstop complexes recognized by ArfA and ArfA(A18T).

(A) Overview of the bacterial ribosome with a 3′-truncated mRNA in complex with ArfA and RF2 (left). ArfA and RF2 occupy the A site with a nonhydrolyzable aminoacyl-tRNA in the P site. RF2 adopts a similar conformation on the ribosome with ArfA as with a UAA stop codon (right). The catalytic GGQ motif of RF2 domain 3 is accommodated within the PTC (inset). (B) Overview of a nonstop complex recognized by ArfA(A18T) (left). Pre-accommodated RF2 resembles the isolated RF2 crystal structure (PDB accession code 1GQE) (right). The GGQ motif faces the P-site tRNA. Movement of domain 1 results from contacts with the L7/L12 stalk base.

Fig. 2. Conformations of the decoding nucleotides.

(A) Conformation of the decoding center with an unoccupied A site. A1493 is disordered in this and all ArfA-containing structures. (B) ArfA(A18T) recognizes a vacant A site and does not remodel the decoding center. (C) Closer contacts between wild-type ArfA, RF2 and the ribosome cause A1492 to switch from a syn to an anti configuration. (D) Canonical termination on a UAA stop codon involves remodeling of the decoding center that does not occur with ArfA.

Fig. 3. Interactions between ArfA, RF2 and the ribosome.

(A) The N-terminal half of ArfA binds RF2 while the C-terminal tail occupies the mRNA channel. The path of ArfA as it emerges from the channel entrance, which can be traced in unfiltered maps, is indicated with a dashed line. (B) Interactions between ArfA and the rRNA lining the mRNA channel for the region highlighted in panel C as viewed from the channel entrance. (C) Both wild-type ArfA and ArfA(A18T) form an antiparallel β-sheet with domain 2 of RF2. F25 packs against an RF2-specific hydrophobic pocket formed by V198 and F217. With wild-type ArfA, this pocket is also recognized by W319 from the switch loop of accommodated RF2.

Fig. 4. Switch-loop stabilization and RF2 accommodation.

(A) In the structure of the nonstop complex recognized by ArfA(A18T), the first 14 residues of ArfA(A18T) and the switch loop between domains 3 and 4 of RF2 are disordered. (B) In the structure with wild-type ArfA, the ordered N-terminus of ArfA helps to stabilize the switch loop of RF2, which extends the α7-helix. A1913 from helix 69 of the 23S rRNA stacks with A1492 from helix 44 of the 16S rRNA, while C1914 from helix 69 stabilizes residues 10–14 of ArfA. (C) RF2 recruited by ArfA(A18T) adopts a compact conformation with the GGQ loop disordered and facing the P-site tRNA. Superposition with accommodated RF2 (outlined) reveals that all four domains move during accommodation. (D) TtRF2 has a switch-loop composition (top) incompatible with ArfA and adopts a pre-accommodated conformation on the ribosome (bottom) similar to E. coli RF2 recruited by ArfA(A18T). (E) During stop-codon recognition, the switch loop of TtRF2 is stabilized by interactions that are dependent on the stop-codon-induced remodeling of the decoding center.