title: Structural basis for ArfA-RF2-mediated translation termination on mRNAs lacking stop codons.
abstract: 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 systems. Although tmRNA-ribosome and ArfB-ribosome structures have been determined, 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.
authors.affiliation: Gene Center, Department of Biochemistry and Center for integrated Protein Science Munich (CiPSM), Ludwig-Maximilians-Universität München, Feodor-Lynen-Strasse 25, 81377 Munich, Germany.
authors.forename: Paul
authors.initials: P
authors.lastname: Huter
authors.affiliation: Gene Center, Department of Biochemistry and Center for integrated Protein Science Munich (CiPSM), Ludwig-Maximilians-Universität München, Feodor-Lynen-Strasse 25, 81377 Munich, Germany.
authors.forename: Claudia
authors.initials: C
authors.lastname: Müller
authors.affiliation: Gene Center, Department of Biochemistry and Center for integrated Protein Science Munich (CiPSM), Ludwig-Maximilians-Universität München, Feodor-Lynen-Strasse 25, 81377 Munich, Germany.|Institute for Biochemistry and Molecular Biology, University of Hamburg, Martin-Luther-King-Pl. 6, 20146 Hamburg, Germany.
authors.forename: Bertrand
authors.initials: B
authors.lastname: Beckert
authors.affiliation: Gene Center, Department of Biochemistry and Center for integrated Protein Science Munich (CiPSM), Ludwig-Maximilians-Universität München, Feodor-Lynen-Strasse 25, 81377 Munich, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Arenz
authors.affiliation: Gene Center, Department of Biochemistry and Center for integrated Protein Science Munich (CiPSM), Ludwig-Maximilians-Universität München, Feodor-Lynen-Strasse 25, 81377 Munich, Germany.
authors.forename: Otto
authors.initials: O
authors.lastname: Berninghausen
authors.affiliation: Gene Center, Department of Biochemistry and Center for integrated Protein Science Munich (CiPSM), Ludwig-Maximilians-Universität München, Feodor-Lynen-Strasse 25, 81377 Munich, Germany.
authors.forename: Roland
authors.initials: R
authors.lastname: Beckmann
authors.affiliation: Gene Center, Department of Biochemistry and Center for integrated Protein Science Munich (CiPSM), Ludwig-Maximilians-Universität München, Feodor-Lynen-Strasse 25, 81377 Munich, Germany.|Institute for Biochemistry and Molecular Biology, University of Hamburg, Martin-Luther-King-Pl. 6, 20146 Hamburg, Germany.
authors.forename: Daniel N
authors.initials: DN
authors.lastname: Wilson
mesh_terms: D018388:Codon, Terminator; D020285:Cryoelectron Microscopy; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000648:ultrastructure; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D008958:Models, Molecular; D010443:Peptide Chain Termination, Translational; D010454:Peptide Termination Factors / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D011485:Protein Binding; D011487:Protein Conformation; D055550:Protein Stability; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D012270:Ribosomes / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure
chemical_list: C556014:ArfA protein, E coli; D018388:Codon, Terminator; D029968:Escherichia coli Proteins; D010454:Peptide Termination Factors; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; C489006:prfB protein, E coli