A New Mechanism for Ribosome Rescue Can Recruit RF1 or RF2 to Nonstop Ribosomes

ABSTRACT Bacterial ribosomes frequently translate to the 3′ end of an mRNA without terminating at an in-frame stop codon. In all bacteria studied to date, these “nonstop” ribosomes are rescued using trans-translation. Genes required for trans-translation are essential in some species, but other spec...

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Autores principales:	Tyler D. P. Goralski, Girish S. Kirimanjeswara, Kenneth C. Keiler
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2018
Materias:	Francisella tularensis ribosomes Tn-seq trans-translation Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/60f46cf7423c4dac9874bbae57cd928f
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spelling	oai:doaj.org-article:60f46cf7423c4dac9874bbae57cd928f2021-11-15T15:52:19ZA New Mechanism for Ribosome Rescue Can Recruit RF1 or RF2 to Nonstop Ribosomes10.1128/mBio.02436-182150-7511https://doaj.org/article/60f46cf7423c4dac9874bbae57cd928f2018-12-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.02436-18https://doaj.org/toc/2150-7511ABSTRACT Bacterial ribosomes frequently translate to the 3′ end of an mRNA without terminating at an in-frame stop codon. In all bacteria studied to date, these “nonstop” ribosomes are rescued using trans-translation. Genes required for trans-translation are essential in some species, but other species can survive without trans-translation because they express an alternative ribosome rescue factor, ArfA or ArfB. Francisella tularensis cells lacking trans-translation are viable, but F. tularensis does not encode ArfA or ArfB. Transposon mutagenesis followed by deep sequencing (Tn-seq) identified a new alternative ribosome rescue factor, now named ArfT. arfT can be deleted in wild-type (wt) cells but not in cells that lack trans-translation activity. Overexpression of ArfT suppresses the slow-growth phenotype in cells lacking trans-translation and counteracts growth arrest caused by trans-translation inhibitors, indicating that ArfT rescues nonstop ribosomes in vivo. Ribosome rescue assays in vitro show that ArfT promotes hydrolysis of peptidyl-tRNA on nonstop ribosomes in conjunction with F. tularensis release factors. Unlike ArfA, which requires RF2 for activity, ArfT can function with either RF1 or RF2. Overall, these results indicate that ArfT is a new alternative ribosome rescue factor with a distinct mechanism from ArfA and ArfB. IMPORTANCE Francisella tularensis is a highly infectious intracellular pathogen that kills more than half of infected humans if left untreated. F. tularensis has also been classified as a potential bioterrorism agent with a great risk for deliberate misuse. Recently, compounds that inhibit ribosome rescue have been shown to have antibiotic activity against F. tularensis and other important pathogens. Like all bacteria that have been studied, F. tularensis uses trans-translation as the main pathway to rescue stalled ribosomes. However, unlike most bacteria, F. tularensis can survive without any of the known factors for ribosome rescue. Our work identified a F. tularensis protein, ArfT, that rescues stalled ribosomes in the absence of trans-translation using a new mechanism. These results indicate that ribosome rescue activity is essential in F. tularensis and suggest that ribosome rescue activity might be essential in all bacteria.Tyler D. P. GoralskiGirish S. KirimanjeswaraKenneth C. KeilerAmerican Society for MicrobiologyarticleFrancisella tularensisribosomesTn-seqtrans-translationMicrobiologyQR1-502ENmBio, Vol 9, Iss 6 (2018)
institution	DOAJ
collection	DOAJ
language	EN
topic	Francisella tularensis ribosomes Tn-seq trans-translation Microbiology QR1-502
spellingShingle	Francisella tularensis ribosomes Tn-seq trans-translation Microbiology QR1-502 Tyler D. P. Goralski Girish S. Kirimanjeswara Kenneth C. Keiler A New Mechanism for Ribosome Rescue Can Recruit RF1 or RF2 to Nonstop Ribosomes
description	ABSTRACT Bacterial ribosomes frequently translate to the 3′ end of an mRNA without terminating at an in-frame stop codon. In all bacteria studied to date, these “nonstop” ribosomes are rescued using trans-translation. Genes required for trans-translation are essential in some species, but other species can survive without trans-translation because they express an alternative ribosome rescue factor, ArfA or ArfB. Francisella tularensis cells lacking trans-translation are viable, but F. tularensis does not encode ArfA or ArfB. Transposon mutagenesis followed by deep sequencing (Tn-seq) identified a new alternative ribosome rescue factor, now named ArfT. arfT can be deleted in wild-type (wt) cells but not in cells that lack trans-translation activity. Overexpression of ArfT suppresses the slow-growth phenotype in cells lacking trans-translation and counteracts growth arrest caused by trans-translation inhibitors, indicating that ArfT rescues nonstop ribosomes in vivo. Ribosome rescue assays in vitro show that ArfT promotes hydrolysis of peptidyl-tRNA on nonstop ribosomes in conjunction with F. tularensis release factors. Unlike ArfA, which requires RF2 for activity, ArfT can function with either RF1 or RF2. Overall, these results indicate that ArfT is a new alternative ribosome rescue factor with a distinct mechanism from ArfA and ArfB. IMPORTANCE Francisella tularensis is a highly infectious intracellular pathogen that kills more than half of infected humans if left untreated. F. tularensis has also been classified as a potential bioterrorism agent with a great risk for deliberate misuse. Recently, compounds that inhibit ribosome rescue have been shown to have antibiotic activity against F. tularensis and other important pathogens. Like all bacteria that have been studied, F. tularensis uses trans-translation as the main pathway to rescue stalled ribosomes. However, unlike most bacteria, F. tularensis can survive without any of the known factors for ribosome rescue. Our work identified a F. tularensis protein, ArfT, that rescues stalled ribosomes in the absence of trans-translation using a new mechanism. These results indicate that ribosome rescue activity is essential in F. tularensis and suggest that ribosome rescue activity might be essential in all bacteria.
format	article
author	Tyler D. P. Goralski Girish S. Kirimanjeswara Kenneth C. Keiler
author_facet	Tyler D. P. Goralski Girish S. Kirimanjeswara Kenneth C. Keiler
author_sort	Tyler D. P. Goralski
title	A New Mechanism for Ribosome Rescue Can Recruit RF1 or RF2 to Nonstop Ribosomes
title_short	A New Mechanism for Ribosome Rescue Can Recruit RF1 or RF2 to Nonstop Ribosomes
title_full	A New Mechanism for Ribosome Rescue Can Recruit RF1 or RF2 to Nonstop Ribosomes
title_fullStr	A New Mechanism for Ribosome Rescue Can Recruit RF1 or RF2 to Nonstop Ribosomes
title_full_unstemmed	A New Mechanism for Ribosome Rescue Can Recruit RF1 or RF2 to Nonstop Ribosomes
title_sort	new mechanism for ribosome rescue can recruit rf1 or rf2 to nonstop ribosomes
publisher	American Society for Microbiology
publishDate	2018
url	https://doaj.org/article/60f46cf7423c4dac9874bbae57cd928f
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A New Mechanism for Ribosome Rescue Can Recruit RF1 or RF2 to Nonstop Ribosomes

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