title: Mechanism of ribosome rescue by ArfA and RF2.
abstract: ArfA rescues ribosomes stalled on truncated mRNAs by recruiting release factor RF2, which normally binds stop codons to catalyze peptide release. We report two 3.2 Å resolution cryo-EM structures - determined from a single sample - of the 70S ribosome with ArfA•RF2 in the A site. In both states, the ArfA C-terminus occupies the mRNA tunnel downstream of the A site. One state contains a compact inactive RF2 conformation. Ordering of the ArfA N-terminus in the second state rearranges RF2 into an extended conformation that docks the catalytic GGQ motif into the peptidyl-transferase center. Our work thus reveals the structural dynamics of ribosome rescue. The structures demonstrate how ArfA 'senses' the vacant mRNA tunnel and activates RF2 to mediate peptide release without a stop codon, allowing stalled ribosomes to be recycled.
authors.affiliation: RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, United States.
authors.forename: Gabriel
authors.identifier: 0000-0002-5472-9249
authors.initials: G
authors.lastname: Demo
authors.affiliation: RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, United States.
authors.forename: Egor
authors.initials: E
authors.lastname: Svidritskiy
authors.affiliation: Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, United States.
authors.forename: Rohini
authors.initials: R
authors.lastname: Madireddy
authors.affiliation: Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, United States.
authors.forename: Ruben
authors.initials: R
authors.lastname: Diaz-Avalos
authors.affiliation: Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, United States.
authors.forename: Timothy
authors.initials: T
authors.lastname: Grant
authors.affiliation: Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, United States.
authors.forename: Nikolaus
authors.identifier: 0000-0002-1506-909X
authors.initials: N
authors.lastname: Grigorieff
authors.affiliation: Department of Biological Science, Florida State University, Tallahassee, United States.
authors.forename: Duncan
authors.initials: D
authors.lastname: Sousa
authors.affiliation: RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, United States.|Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, United States.
authors.forename: Andrei A
authors.identifier: 0000-0003-1588-717X
authors.initials: AA
authors.lastname: Korostelev
mesh_terms: D020285:Cryoelectron Microscopy; D029968:Escherichia coli Proteins / Q000378:metabolism* / Q000648:ultrastructure*; D010454:Peptide Termination Factors / Q000378:metabolism* / Q000648:ultrastructure*; D011485:Protein Binding; D016601:RNA-Binding Proteins / Q000378:metabolism* / Q000648:ultrastructure*; D012270:Ribosomes / Q000378:metabolism* / Q000648:ultrastructure*
chemical_list: C556014:ArfA protein, E coli; D029968:Escherichia coli Proteins; D010454:Peptide Termination Factors; D016601:RNA-Binding Proteins; C489006:prfB protein, E coli
keywords: ArfA; E. coli; biochemistry; biophysics; release factor 2; ribosome rescue; stalled ribosome; stop-codon-independent termination; structural biology