LETTER doi:10.1038/nature11006 The complex of tmRNA–SmpB and EF-G on translocating ribosomes David J. F. Ramrath 1 *, Hiroshi Yamamoto 1,2 *, Kristian Rother 3 , Daniela Wittek 2 , Markus Pech 1,2 , Thorsten Mielke 1,4 , Justus Loerke 1 , Patrick Scheerer 1 , Pavel Ivanov 2,5 {, Yoshika Teraoka 2 , Olga Shpanchenko 2,5 , Knud H. Nierhaus 1,2 & Christian M. T. Spahn 1 Bacterial ribosomes stalled at the 39 end of malfunctioning messenger RNAs can be rescued by transfer-messenger RNA (tmRNA)-mediated trans-translation 1,2 . 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 dif- ferent ribosome–tmRNA complexes at low to intermediate resolu- tion 3–7 , 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 A ˚ resolution. This post-translocational intermediate (TI POST ) presents the TLD– SmpB module in an intrasubunit ap/P hybrid site and a tRNA fMet 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 transloca- tion to MLD loading. This mechanism exemplifies the versatile, dynamic nature of the ribosome, and it shows that the conforma- tional modes of the ribosome that normally drive canonical trans- lation can also be used in a modified form to facilitate more complex tasks in specialized non-canonical pathways. The tmRNA–SmpB-mediated trans-translation is a bacterial emer- gency system that rescues ribosomes stalled at the 39 end of defective mRNAs that lack a stop codon. The tmRNA is a highly structured bifunc- tional molecule that acts as tRNA and mRNA through its TLD and MLD 1,2 (Fig. 1a). SmpB interacts with the TLD and mimics the tRNA anticodon stem that is lacking in the TLD 8–10 . After the TLD–SmpB module has entered the A site of the ribosome in a decoding-like reaction, the immature polypeptide is transferred to Ala-tmRNA (tmRNA with the amino acid alanine loaded at its 39 end). Subsequently, the TLD–SmpB module is translocated to the P site of the ribosome, and the template for translation is switched to the MLD. The MLD encodes a tag, which marks the immature peptide for protease-dependent degradation and ends with a stop codon allowing canonical termination 1,2 (Fig. 1a). Several cryo-electron microscopy (cryo-EM) maps of ribosomal complexes with bound tmRNA have provided important information about the passage of tmRNA through the ribosome. At low to inter- mediate resolution, these maps display the TLD–SmpB module in the A/T site of the ribosome when the tmRNA is delivered to the ribosome as an Ala-tmRNA–SmpB–EF-Tu–GTP complex 3,4 . The maps also show the complexes after accommodation 5,6 and first translocation 6,7 , with the TLD–SmpB module occupying the ribosomal A and P sites, respectively. However, as discussed in previous reports 11,12 , for a detailed interpretation of cryo-EM maps in molecular terms, sub- nanometre resolution is required so that secondary structural elements can be directly observed. Moreover, the interplay between the ribo- some and its ligands during translocation, and the process by which the large and highly structured tmRNA passes through the ribosome and leads to correct MLD loading, remains unclear. Here we present a cryo-EM reconstruction of an in vitro-reconstituted ribosomal 70S–tmRNA–EF-G complex from Escherichia coli (Fig. 1). Pre-translocated (PRE) ribosomes carrying fMet-Ala-tmRNA–SmpB in the A site and initiator tRNA fMet in the P site were translocated by EF-G, which was stalled on the complex using the antibiotic fusidic acid. Because P-site-bound initiator tRNA fMet has a slower rate of transloca- tion than P-site-bound elongator tRNA 13,14 , the usage of a tRNA fMet might increase the time window for fusidic acid to trap the reaction. The resultant complex was analysed by multiparticle cryo-EM 15 to over- come sample heterogeneity caused by substoichiometric binding of the ligands. The final map, reconstructed from a subpopulation of 68,842 particle images (9.5% of the total data set), reached the subnanometre resolution of 8.3 A ˚ (Supplementary Fig. 1). The structure shows well- defined densities for the ligands fMet-Ala-tmRNA–SmpB, deacylated tRNA fMet and EF-G. In agreement with the subnanometre resolution estimate, the structure contains clearly visible structured RNA or rod- shaped a-helical elements, and it readily allows the docking of atomic models of the ribosome 16 , EF-G–GDP–fusidic acid 17 , tRNA fMet (ref. 18) and the TLD–SmpB module 9 (Figs 1c and 2a). Moreover, we attempted RNA modelling of major tmRNA regions for which no high-resolution structure was available (Supplementary Table 1). Compared with the ribosome in the classical state 16 , the 70S–tmRNA– EF-G complex has undergone large-scale conformational changes. The body/platform domains of the 30S subunit are rotated by ,5u, and the head is swivelled by an additional ,19u (Fig. 2b). Thus, the conforma- tion of the presented 70S–tmRNA–EF-G complex is similar overall to the recently described translocation intermediate TI POST , which is char- acterized by an intermediate (,4u) 30S rotation and a large (,18u) head swivel 19 . However, the overall movement of the head is unique: com- pared with the head position of the previously reported TI POST (ref. 19), there is an additional ,12u tilt movement around an axis that is approxi- mately parallel to the mRNA, around the A- and P-site codons (Fig. 2b). This tilt moves the head away from the central protuberance of the 50S subunit. The combination of intersubunit rotation, head swivel and head tilt lead to a displacement of peripheral elements of up to ,50 A ˚ com- pared with the classical state 16 (Fig. 2b). The two tRNA/tRNA-like entities are trapped in intermediate states during translocation. From the perspective of the 50S subunit and the 1 Institut fu ¨ r Medizinische Physik und Biophysik, Charite – Universita ¨ tsmedizin Berlin, Ziegelstrasse 5-9, 10117 Berlin, Germany. 2 Max Planck Institute for Molecular Genetics, Abteilung Vingron, AG Ribosomen, Ihnestrasse 73, 14195 Berlin, Germany. 3 Institute of Biology and Molecular Biotechnology, Collegium Biologicum, Adam Mickiewicz University, Ulica Umultowska 89, 61-614 Poznan, Poland. 4 UltraStrukturNetzwerk, Max Planck Institute for Molecular Genetics, Ihnestrasse 73, 14195 Berlin, Germany. 5 Department of Chemistry, M. V. Lomonosov Moscow State University, 119899 Moscow, Russia. {Present address: Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA. *These authors contributed equally to this work. 526 | NATURE | VOL 485 | 24 MAY 2012 Macmillan Publishers Limited. All rights reserved ©2012