title: The complex of tmRNA-SmpB and EF-G on translocating ribosomes.
abstract: Bacterial ribosomes stalled at the 3' end of malfunctioning messenger RNAs can be rescued by transfer-messenger RNA (tmRNA)-mediated trans-translation. 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 resolution, 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 (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 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.
authors.affiliation: Institut für Medizinische Physik und Biophysik, Charite - Universitätsmedizin Berlin, Ziegelstrasse 5-9, 10117 Berlin, Germany.
authors.forename: David J F
authors.initials: DJ
authors.lastname: Ramrath
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Yamamoto
authors.forename: Kristian
authors.initials: K
authors.lastname: Rother
authors.forename: Daniela
authors.initials: D
authors.lastname: Wittek
authors.forename: Markus
authors.initials: M
authors.lastname: Pech
authors.forename: Thorsten
authors.initials: T
authors.lastname: Mielke
authors.forename: Justus
authors.initials: J
authors.lastname: Loerke
authors.forename: Patrick
authors.initials: P
authors.lastname: Scheerer
authors.forename: Pavel
authors.initials: P
authors.lastname: Ivanov
authors.forename: Yoshika
authors.initials: Y
authors.lastname: Teraoka
authors.forename: Olga
authors.initials: O
authors.lastname: Shpanchenko
authors.forename: Knud H
authors.initials: KH
authors.lastname: Nierhaus
authors.forename: Christian M T
authors.initials: CM
authors.lastname: Spahn
mesh_terms: D001483:Base Sequence; D020285:Cryoelectron Microscopy; D004926:Escherichia coli / Q000737:chemistry*; D005672:Fusidic Acid / Q000378:metabolism; D008024:Ligands; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D020653:Peptide Elongation Factor G / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D011485:Protein Binding; D014176:Protein Biosynthesis; D011487:Protein Conformation; D012329:RNA, Bacterial / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D054657:Ribosome Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D012270:Ribosomes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure
chemical_list: D008024:Ligands; D020653:Peptide Elongation Factor G; D012329:RNA, Bacterial; D016601:RNA-Binding Proteins; C430328:small protein B; C096222:tmRNA; D005672:Fusidic Acid