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The complex of tmRNA–SmpB and EF-G on translocating ribosomes


Bacterial ribosomes stalled at the 3′ end of malfunctioning messenger RNAs can be rescued by transfer-messenger RNA (tmRNA)-mediated trans-translation1,2. The SmpB protein forms a complex with the tmRNA, and the transfer-RNA-like domain (TLD) of the tmRNA then enters the A site of the ribosome. Subsequently, the TLD–SmpB module is translocated to the P site, a process that is facilitated by the elongation factor EF-G, and translation is switched to the mRNA-like domain (MLD) of the tmRNA. Accurate loading of the MLD into the mRNA path is an unusual initiation mechanism. Despite various snapshots of different ribosome–tmRNA complexes at low to intermediate resolution3,4,5,6,7, it is unclear how the large, highly structured tmRNA is translocated and how the MLD is loaded. Here we present a cryo-electron microscopy reconstruction of a fusidic-acid-stalled ribosomal 70S–tmRNA–SmpB–EF-G complex (carrying both of the large ligands, that is, EF-G and tmRNA) at 8.3 Å resolution. This post-translocational intermediate (TIPOST) presents the TLD–SmpB module in an intrasubunit ap/P hybrid site and a tRNAfMet in an intrasubunit pe/E hybrid site. Conformational changes in the ribosome and tmRNA occur in the intersubunit space and on the solvent side. The key underlying event is a unique extra-large swivel movement of the 30S head, which is crucial for both tmRNA–SmpB translocation and MLD loading, thereby coupling translocation to MLD loading. This mechanism exemplifies the versatile, dynamic nature of the ribosome, and it shows that the conformational modes of the ribosome that normally drive canonical translation can also be used in a modified form to facilitate more complex tasks in specialized non-canonical pathways.

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Figure 1: Overall view of the 70S–tmRNA–EF-G complex.
Figure 2: Ligand positions and conformation of the 30S subunit.
Figure 3: The EF-G–SmpB interaction.
Figure 4: Structural basis of MLD loading.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

The electron density map and model of the 70S–tmRNA–SmpB–EF-G complex have been deposited in the 3DEM database with the accession number EMD-5386 and in the Protein Data Bank database with accession numbers 3J18 (30S and ligands) and 3J19 (50S).


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We thank M. Muhs, T. Budkevich, M. Sommer and A. Korostelev for discussions. The strain for tmRNA overexpression and the plasmid for SmpB were provided by A. Muto. We also thank J. Buerger for his assistance during the cryo-EM data collection, E. Einfeldt for tRNA preparation and R. Albrecht for the preparation of the reassociated 70S ribosomes. This work was supported by grants from the Deutsche Forschungsgemeinschaft DFG (SP 1130/2-1 to C.M.T.S. and K.H.N., and SFB740 to C.M.T.S.), the German Academic Exchange Service (D/09/42768 to K.R.) and the European Union and Senatsverwaltung für Wissenschaft, Forschung und Kultur Berlin (UltraStructureNetwork, Anwenderzentrum) and the Alexander-von-Humboldt grant (GAN 1127366 STP-2 to H.Y.).

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Authors and Affiliations



H.Y., D.W., M.P., P.I., Y.T. and O.S. established the in vitro system for the reconstitution of ribosome–tmRNA complexes. H.Y. prepared the 70S–tmRNA–SmpB–EF-G complex. D.J.F.R. and T.M. collected the cryo-EM data. D.J.F.R., J.L. and C.M.T.S. carried out the image processing. D.J.F.R., K.R., P.S. and C.M.T.S. carried out the modelling of the full-length tmRNA. D.J.F.R., H.Y., K.R., K.H.N. and C.M.T.S. discussed the results and wrote the paper.

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Correspondence to Christian M. T. Spahn.

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The authors declare no competing financial interests.

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Ramrath, D., Yamamoto, H., Rother, K. et al. The complex of tmRNA–SmpB and EF-G on translocating ribosomes. Nature 485, 526–529 (2012).

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