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Structural basis for ArfA–RF2-mediated translation termination on mRNAs lacking stop codons


In bacteria, ribosomes stalled on truncated mRNAs that lack a stop codon are rescued by the transfer-messenger RNA (tmRNA), alternative rescue factor A (ArfA) or ArfB systems1. Although tmRNA–ribosome and ArfB–ribosome structures have been determined2,3,4,5,6,7, how ArfA recognizes the presence of truncated mRNAs and recruits the canonical termination release factor RF2 to rescue the stalled ribosomes is unclear. Here we present a cryo-electron microscopy reconstruction of the Escherichia coli 70S ribosome stalled on a truncated mRNA in the presence of ArfA and RF2. The structure shows that the C terminus of ArfA binds within the mRNA entry channel on the small ribosomal subunit, and explains how ArfA distinguishes between ribosomes that bear truncated or full-length mRNAs. The N terminus of ArfA establishes several interactions with the decoding domain of RF2, and this finding illustrates how ArfA recruits RF2 to the stalled ribosome. Furthermore, ArfA is shown to stabilize a unique conformation of the switch loop of RF2, which mimics the canonical translation termination state by directing the catalytically important GGQ motif within domain 3 of RF2 towards the peptidyl-transferase centre of the ribosome. Thus, our structure reveals not only how ArfA recruits RF2 to the ribosome but also how it promotes an active conformation of RF2 to enable translation termination in the absence of a stop codon.

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Figure 1: Cryo-EM structure of ArfA-RF2-SRC.
Figure 2: Interaction of ArfA with the small subunit.
Figure 3: Interaction of ArfA with RF2 on the ribosome.
Figure 4: ArfA stabilizes a unique conformation of the RF2 switch loop.

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We thank H. Sieber, S. Rieder and C. Ungewickell for technical assistance and L. Bischoff and R. Green for providing expression plasmids for E. coli ArfAΔ17 and RF2, respectively. This research was supported by grants from the Deutsche Forschungsgemeinschaft WI3285/4-1, SPP-1879 (to D.N.W.), GRK 1721 and FOR1805 (to R.B. and D.N.W.).

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Authors and Affiliations



D.N.W. designed the study. C.M. and P.H. prepared the cryo-EM sample. P.H., C.M. and B.B. processed the cryo-EM data. P.H., S.A. and D.N.W. built and refined the molecular models. O.B. collected the cryo-EM data. P.H., C.M., R.B. and D.N.W. interpreted the results and D.N.W. wrote the paper.

Corresponding author

Correspondence to Daniel N. Wilson.

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Competing interests

The authors declare no competing financial interests.

Additional information

Reviewer Information Nature thanks T. Abo, Y. Hashem, K. Keiler and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Figure 1 Recycling of ribosomes stalled on truncated mRNA by ArfA and RF2.

In vitro translation assay of the truncated nlpD template was performed in the presence of ArfA, RF2 or RF2-GAQ, revealing a peptidyl-tRNA band (nlpD_ns*P-tRNA), whereas the peptidyl-tRNA was absent and free nlpD peptide (nlpD_ns) was observed when the reaction was performed with ArfA and RF2. Replacing wild-type RF2 with the inactive RF2-GAQ mutant led to the reappearance of the peptidyl-tRNA band and loss of the free nlpD peptide.

Extended Data Figure 2 Classification of the ArfA-RF2-SRC.

The complete dataset of 227,608 particles was initially aligned against a vacant E. coli 70S ribosome, refined with RELION using 3D auto-refine and the movie particles were extracted. The polished particles were then subjected to a 3D refinement and 3D classification using FREALIGN. The class 2 (138,582 particles) resulting from the 100 rounds of 3D classification with 3× binned images using a ribosomal mask was then further refined and classified with 2× binned images. The remaining 69,089 particles containing ArfA-RF2-SRC were then 3D-refined, resulting in a final reconstruction of 3.1 Å (0.143 Fourier shell correlation (FSC)) average resolution.

Extended Data Figure 3 Resolution of the ArfA-RF2-SRC.

a, Overview of the final refined cryo-EM map of the ArfA-RF2-SRC with separated densities for small (yellow) and large (grey) ribosomal subunit, as well as ArfA (red), RF2 (orange) and P-tRNA (green). b, Same view as in a but coloured according to local resolution. c, Transverse section of b showing local resolution in the core of the ribosomal subunits. d, FSC curve of the refined final map, indicating that the average resolution of the ArfA-RF2-SRC is 3.1 Å (at 0.143). e, Fit of models to maps. FSC curves calculated between the refined model and the final map (blue), with the self- and cross-validated correlations in orange and black, respectively. Information beyond 3.2 Å was not used during refinement and preserved for validation. f, g, Selected examples illustrating the quality of fit of the molecular models to the unsegmented cryo-EM map (grey mesh) for the ArfA (red) interaction with S12 (blue), related to Fig. 2b (f), and with RF2 (orange), related to Fig. 3c (g).

Extended Data Figure 4 Hydroxyl radical probing of ArfA on the ribosome.

ad, Hydroxyl-radical probing data15 of ArfA in complex with RF2 on the ribosome reveal that tethers linked to the N-terminal region of ArfA, for example, residues S2 and R3 (magenta), cleave the 16S rRNA within the vicinity of helices h18, whereas tethers linked to the C-terminal region of ArfA, such as residues 33–34/38–39 and 46 (teal), cleave the 16S rRNA within the vicinity of helices h34 (ref. 15). These findings are in excellent agreement with the position of ArfA (red) within the ArfA-RF2-SRC structure reported here. In the overview panels a and c, P-tRNA (green) is shown for reference.

Extended Data Figure 5 Sequence alignment of E. coli RF1 and RF2 with secondary structure assignments.

Sequence alignment of E. coli RF1 and RF2 generated using ClustalX with secondary structure (helices and strands) and domain (I–IV) assignments based on the crystal structures of E. coli RF2 (ref. 22), except for the switch loop (yellow) and extension to helix α7 (purple), which was based on the ArfA-RF2-SRC structure. The pink boxes indicate regions of RF2 that form an interface with ArfA, with residues in bold predicted to prevent interaction of RF1 with ArfA. Asterisk (*) or colon (:) and full stop (.) indicate a single, fully conserved residue or residues with strong (>0.5 in the Gonnet PAM 250 matrix) and weakly (>0.5) similar properties, respectively.

Extended Data Figure 6 Potential specificity determinants for ArfA-mediated ribosome recycling.

a, b, ArfA (red) and E. coli RF2 (orange) compared to homology model of E. coli RF1 (blue) aligned to RF2 in the ArfA-RF2-SRC. a, The ArfA interface with β4 and β5 strands of E. coli RF2 (orange) consists of hydrophobic residues V198, F217 and F221, which are mutated to Gly, Ala and Ala, respectively, in RF1 (blue). b, The ArfA interface with α-helix α7 of RF2 (orange). Replacing negatively charged residues such as E311 and D312 in RF2 with Arg in RF1 is also likely to disrupt the interaction with ArfA. c, d, Sequence alignments for the regions of RF1 and RF2 corresponding to a and b, respectively. The pink boxes indicate regions of RF2 that form an interface with ArfA, including residues in bold predicted to prevent interaction of RF1 with ArfA and therefore could provide the basis for RF2-specificity of ArfA action. Organisms in bold contain ArfA, whereas others have no detectable ArfA homologue. Asterisk (*), colon (:) or full stop (.) indicate a single, fully conserved residue or residues with strong (>0.5 in the Gonnet PAM 250 matrix) and weakly (>0.5) similar properties, respectively.

Extended Data Figure 7 Location of the ArfA-A18T mutation relative to RF2.

a, Overview of ArfA (red) and RF2 (gold) on the ribosome (30S, grey; 50S, slate). b, c, Zoom of boxed region in a showing the environment of A18 (teal) of ArfA in close proximity to I11 and K8 in the N terminus of ArfA (red) (b), and A18T (teal) of ArfA in sterically clashing with I11 and K8 in the N terminus of ArfA (red) (c).

Extended Data Figure 8 Comparison of ArfA with other ribosome rescue systems.

ac, Relative orientation on the ribosome with truncated mRNAs and ArfA (red) and RF2 (orange) (a), ArfB (purple, PDB code 4V95)5 (b) or tmRNA (brown) and SmpB (yellow) (PDB code 4V8Q)6 (c). In all cases, the mRNA and P-tRNA are coloured cyan and green, respectively.

Extended Data Table 1 Data collection and refinement statistics

Supplementary information

Comparison of free and ArfA-bound conformations of RF2

Animation showing the conformation change in RF2 when comparing the crystal structure of the free (closed) form of RF2 (PDB ID 1GQE) with that when ArfA (red) is bound. RF2 is coloured orange except for the switch loop (purple). (MP4 5790 kb)

Comparison of decoding and ArfA-bound conformations of RF2

Animation showing the conformation change in RF2 when comparing the canonical termination form of RF2 (PDB ID 4V5E) with that when ArfA (red) is bound. RF2 is coloured orange except for the switch loop (purple). (MP4 7093 kb)

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Huter, P., Müller, C., Beckert, B. et al. Structural basis for ArfA–RF2-mediated translation termination on mRNAs lacking stop codons. Nature 541, 546–549 (2017).

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