Mechanism of ribosome rescue by ArfA and RF2

Gabriel Demo; Egor Svidritskiy; Rohini Madireddy; Ruben Diaz-Avalos; Timothy Grant; Nikolaus Grigorieff; Duncan Sousa; Andrei A. Korostelev

doi:10.7554/eLife.23687

Mechanism of ribosome rescue by ArfA and RF2

Gabriel Demo, Egor Svidritskiy, Rohini Madireddy, Ruben Diaz-Avalos, Timothy Grant, Nikolaus Grigorieff, Duncan Sousa, Andrei A. Korostelev

Research output: Contribution to journal › Article › peer-review

25 Scopus citations

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 A 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.

Original language	English (US)
Article number	e23687
Journal	eLife
Volume	6
DOIs	https://doi.org/10.7554/eLife.23687
State	Published - Mar 16 2017
Externally published	Yes

ASJC Scopus subject areas

General Neuroscience
General Immunology and Microbiology
General Biochemistry, Genetics and Molecular Biology

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10.7554/eLife.23687

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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 A 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 {\textquoteleft}senses{\textquoteright} the vacant mRNA tunnel and activates RF2 to mediate peptide release without a stop codon, allowing stalled ribosomes to be recycled.",

author = "Gabriel Demo and Egor Svidritskiy and Rohini Madireddy and Ruben Diaz-Avalos and Timothy Grant and Nikolaus Grigorieff and Duncan Sousa and Korostelev, {Andrei A.}",

note = "Funding Information: We thank the National Institute of Genetics, Shizouka, Japan for providing ArfA- and RF2-overex- pressing strains of E. coli (ASKA-library); Yevheniya Stepanyuk for assistance with ArfA subcloning; Michael Spilman (Direct Electron, LP) for assistance with data collection and processing; Anna B Loveland for assistance with data processing; Darryl Conte Jr for assistance with manuscript preparation; members of the Korostelev laboratory for helpful discussions and comments on the manuscript. This study was supported by NIH Grants R01 GM106105 and GM107465 (to AAK). All authors contributed to manuscript finalization in the acknowledgements section. Publisher Copyright: {\textcopyright} Demo et al.",

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AU - Demo, Gabriel

AU - Svidritskiy, Egor

AU - Madireddy, Rohini

AU - Diaz-Avalos, Ruben

AU - Grant, Timothy

AU - Grigorieff, Nikolaus

AU - Sousa, Duncan

AU - Korostelev, Andrei A.

N1 - Funding Information: We thank the National Institute of Genetics, Shizouka, Japan for providing ArfA- and RF2-overex- pressing strains of E. coli (ASKA-library); Yevheniya Stepanyuk for assistance with ArfA subcloning; Michael Spilman (Direct Electron, LP) for assistance with data collection and processing; Anna B Loveland for assistance with data processing; Darryl Conte Jr for assistance with manuscript preparation; members of the Korostelev laboratory for helpful discussions and comments on the manuscript. This study was supported by NIH Grants R01 GM106105 and GM107465 (to AAK). All authors contributed to manuscript finalization in the acknowledgements section. Publisher Copyright: © Demo et al.

PY - 2017/3/16

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N2 - 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 A 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.

AB - 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 A 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.

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Mechanism of ribosome rescue by ArfA and RF2

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