Mechanistic insights into the alternative translation termination by ArfA and RF2


During cellular translation of messenger RNAs by ribosomes, the translation apparatus sometimes pauses or stalls at the elongation and termination steps1,2,3,4,5,6. With the exception of programmed stalling, which is usually used by cells for regulatory purposes5,7,8, ribosomes stalled on mRNAs need to be terminated and recycled to maintain adequate translation capacity9. Much ribosome stalling originates in aberrant mRNAs that lack a stop codon. Transcriptional errors, misprocessing of primary transcripts, and undesired mRNA cleavage all contribute to the formation of non-stop mRNAs. Ribosomes stalled at the 3′ end of non-stop mRNAs do not undergo normal termination owing to the lack of specific stop-codon recognition by canonical peptide release factors at the A-site decoding centre. In bacteria, the transfer-messenger RNA (tmRNA)–SmpB-mediated trans-translation rescue system reroutes stalled ribosomes to the normal elongation cycle and translation termination3,4,10,11,12. Two additional rescue systems, ArfA–RF2 (refs 13, 14, 15, 16) and ArfB (formerly known as YaeJ)17,18, are also present in many bacterial species, but their mechanisms are not fully understood. Here, using cryo-electron microscopy, we characterize the structure of the Escherichia coli 70S ribosome bound with ArfA, the release factor RF2, a short non-stop mRNA and a cognate P-site tRNA. The C-terminal loop of ArfA occupies the mRNA entry channel on the 30S subunit, whereas its N terminus is sandwiched between the decoding centre and the switch loop of RF2, leading to marked conformational changes in both the decoding centre and RF2. Despite the distinct conformation of RF2, its conserved catalytic GGQ motif is precisely positioned next to the CCA-end of the P-site tRNA. These data illustrate a stop-codon surrogate mechanism for ArfA in facilitating the termination of non-stop ribosomal complexes by RF2.

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Figure 1: The structure of non-stop ribosomal complex bound with ArfA and RF2.
Figure 2: Distinctly arranged decoding centre in the non-stop complex.
Figure 3: Interactions between ArfA and RF2 at the decoding centre.
Figure 4: Conformational signal propagation at the decoding centre of the non-stop complex.

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We thank the National Center for Protein Sciences (Beijing, China) for providing resources for data collection and computation. Part of the computation was performed on the Computing Platform of the Center for Life Science, Peking Univesity. We also thank S. Goto for providing expression vectors and the staff of Gene Research Center of Hirosaki University for the use of the facility. This work was supported by the National Natural Science Foundation of China (31422016, 31470722 and 31630087 to N.G.), the Ministry of Science and Technology of China (2016YFA0500700 and 2013CB910404 to N.G.) and by the Japan Society for the Promotion of Science (16K18684 to D.K.). N.L. is supported by a postdoctoral fellowship from the Peking-Tsinghua Center for Life Sciences.

Author information




H.H. and N.G. conceived the study; D.K. prepared proteins and performed peptide release assays; C.M., N.L., and Y.C. performed ribosome purification, sample preparation, data collection and image processing; N.G. wrote the paper, and all authors edited the paper.

Corresponding authors

Correspondence to Hyouta Himeno or Ning Gao.

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The authors declare no competing financial interests.

Additional information

Reviewer Information Nature thanks 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 Cryo-EM structural determination of the non-stop complex.

a, A representative raw cryo-EM micrograph collected with a Titan Krios 300 kV transmission electron microscope (TEM) (Falcon II camera). b, Representative 2D class averages of cryo-EM particles. c, Gold-standard Fourier shell correlation (FSC) of the final cryo-EM map and the FSC curve between the refined model and the final map. d, e, Local resolution estimation of the cryo-EM density map. The density map of the non-stop complex is displayed in surface representation, and coloured according to the scale bar. f, Atomic model cross-validation. Three FSC curves were calculated between the refined model (against half1 map) and the final map (black curve), between the refined model with half1 map (FSCwork, purple), and between the refined model with half2 map (FSCtest, red) (see Methods for details).

Extended Data Figure 2 Representative regions of the final density map.

ac, Segmented densities of ArfA, superimposed on the atomic model. di, Segmented densities of representative segments of ribosomal proteins from the two subunits, superimposed on respective atomic models. j, Interactions of the N terminus of ArfA with uS12. k, Interactions of ArfA with the β5 strand of the SPF-containing domain of RF2. l, Cognate P-tRNA interaction with the AUG codon of mRNA.

Extended Data Figure 3 ArfA has minimal contact with the terminal loop of H69.

a, Overall orientation of ArfA relative to the 50S subunit. CP, central protuberance; L1, L1 stalk; L7/L12, L7/L12 stalk. b, Zoomed-in view showing the interaction of ArfA with the terminal loop of H69. The base of C1914 interacts with the backbone of K12. Also, a possible interaction is seen between the side chain of H21 and the base of A1913 (probably mediated by a negatively charged ion).

Extended Data Figure 4 Extensive interactions between ArfA and the 16S rRNA.

a, Zoomed-in view of ArfA binding to the decoding centre of the 30S subunit. The 16S rRNA (gold) and ribosomal proteins (yellow) are displayed in surface models. ArfA (red), H69 (cyan), mRNA (blue) and P-tRNA (purple) are displayed in ribbon models. The C-terminal loop of ArfA is positioned in the mRNA entry channel. The overall orientation of ArfA relative to the 30S subunit is shown in the left thumbnail. b, Interactions of ArfA with rRNA elements of the 16S rRNA. Helices of h1, h18, h28, h34 and h44 are separately coloured and labelled.

Extended Data Figure 5 In vitro peptide release assay using ArfA and RF2 mutants.

a, b, Activity of in vitro peptidyl-tRNA hydrolysis for ArfA (a) and RF2 (b) variants. The values obtained from experiments without ArfA or RF2 were subtracted as backgrounds. Data are mean and s.e.m. and are representative of at least three independent experiments. The reactions were quenched after 5 s incubation to measure the initial velocities (see Methods). c, Zoomed-in view showing the interaction between Y39–R41 of ArfA and h1 of the 16S rRNA. d, Zoomed-in view showing the interaction between ArfA/RF2 and h18/uS12 of the 30S subunit.

Extended Data Figure 6 Global and local conformational difference of the 16S rRNA in the non-stop complex.

a, Structural superimposition of the 16S rRNAs from the non-stop and elongation complexes. Coordinates of the 16S rRNA are from a previous crystal structure of the T. thermophilus 70S ribosome bound with cognate P-site and A-site tRNAs (PDB code 4V87)24. Structural alignment is done using the 23S rRNA as reference. b, Structural superimposition of the 16S rRNAs from the non-stop and standard termination complexes. Coordinates of the 16S rRNA are from a previous crystal structure of the T. thermophilus 70S ribosome bound with cognate P-site tRNA and RF2 (PDB code 4V67)26. c, d, Same as in a and b, but showing the top views. The clockwise movement of the head domain of the 16S rRNA is indicated by an arrow. eh, The local density of A1492–A1493 in the non-stop complex is displayed in varying contour levels with the atomic model superimposed. As shown, compared to A1492, the flipped base of A1493 is with considerable flexibility.

Extended Data Figure 7 Conformational difference in RF2 in the non-stop and termination complexes.

a, Zoomed-in view of the interaction of RF2 with the 70S ribosome in the non-stop complex. The 30S, 50S and individual domains of RF2 are separately coloured. b, Same as in a, but with the crystal structure of the T. thermophilus standard termination complex (grey) (PDB code 4V67)26 superimposed. Note that there is a 14-Å displacement of one end of RF2 domain 1, which results in the loss of contact with the 30S subunit as seen in a. The alignment is done with the 23S rRNA as reference. c, Same as in b, but showing a zoomed-in view for domains 2 and 4 of RF2. Note that the SPF-containing domain 2 of RF2 moves approximately 2–3 Å towards the head of the 30S subunit. d, Same as c, but showing a zoomed-in view for the GGQ motif of RF2. Note that the GGQ motifs are in nearly identical positions in the two complexes. ei, Structural superimposition of RF2 in the non-stop and termination complexes, showing the differential displacements of individual domains of RF2. Displacements of individual domains of RF2 are indicated by red arrows.

Extended Data Figure 8 Comparison of the switch loop of RF2 in different structures.

a, Structural superimposition between E. coli RF2 (individual domains separately coloured) in the non-stop complex and T. thermophilus RF2 (coloured grey) in a standard termination complex. Coordinates of T. thermophilus RF2 (grey) are from a crystal structure of the T. thermophilus standard termination complex (PDB code 4V67)26. The alignment is done using domain 2 of RF2 as reference. b, Same as in a, but showing a zoomed-in view of the switch loops of RF2 in the E. coli non-stop (orange) and T. thermophilus termination (yellow) complexes. c, Same as in a, but showing the comparison between E. coli RF2 (individual domains separately coloured) in the non-stop complex and E. coli RF2 (coloured pink) in a hybrid termination complex (T. thermophilus 70S plus E. coli RF2) (PDB code 5DFE)47. The alignment is done using domain 2 of RF2 as reference. d, Same as in c, but showing a zoomed-in view of the switch loops of RF2 in the two structures.

Extended Data Figure 9 Structural and multiple sequence alignment of RF1 and RF2.

a, b, Structural alignment of E. coli RF1 and RF2 in 70S ribosome-bound conformations. For clarification, only domains 2–4 are shown. The switch loops are highlighted in gold. The coordinates of E. coli RF1 and RF2 are from previous crystal structures of hybrid standard termination complexes (T. thermophilus 70S plus E. coli RF1/RF2) (PDB codes 5J30 and 5DFE)47. c, Multiple sequence alignment of RF1 and RF2 from different bacterial species. Only the C-terminal halves of these sequences are shown. The switch loops, and SPF/PxT and GGQ tripepetide motifs of RF2 and RF1 are indicated by thick red lines. The region of the β5 element of RF2 (F217–F221) that contributes to the hydrophobic interface between RF2 and ArfA (Fig. 3f) is indicated by a thick blue line.

Extended Data Table 1 Statistics of data collection and model refinement

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Ma, C., Kurita, D., Li, N. et al. Mechanistic insights into the alternative translation termination by ArfA and RF2. Nature 541, 550–553 (2017).

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