The C-Terminal RRM/ACT Domain Is Crucial for Fine-Tuning the Activation of ‘Long’ RelA-SpoT Homolog Enzymes by Ribosomal Complexes
Takada, Hiraku ; Roghanian, Mohammad ; Murina, Victoriia ; Dzhygyr, Ievgen ; Murayama, Rikinori ; Akanuma, Genki ; Atkinson, Gemma C. LU ; Garcia-Pino, Abel and Hauryliuk, Vasili LU
(2020)
In Frontiers in Microbiology
11.
p.1-16
- Abstract
The (p)ppGpp-mediated stringent response is a bacterial stress response implicated in virulence and antibiotic tolerance. Both synthesis and degradation of the (p)ppGpp alarmone nucleotide are mediated by RelA-SpoT Homolog (RSH) enzymes which can be broadly divided in two classes: single-domain ‘short’ and multi-domain ‘long’ RSH. The regulatory ACT (Aspartokinase, Chorismate mutase and TyrA)/RRM (RNA Recognition Motif) domain is a near-universal C-terminal domain of long RSHs. Deletion of RRM in both monofunctional (synthesis-only) RelA as well as bifunctional (i.e., capable of both degrading and synthesizing the alarmone) Rel renders the long RSH cytotoxic due to overproduction of (p)ppGpp. To probe the molecular mechanism underlying... (More)
The (p)ppGpp-mediated stringent response is a bacterial stress response implicated in virulence and antibiotic tolerance. Both synthesis and degradation of the (p)ppGpp alarmone nucleotide are mediated by RelA-SpoT Homolog (RSH) enzymes which can be broadly divided in two classes: single-domain ‘short’ and multi-domain ‘long’ RSH. The regulatory ACT (Aspartokinase, Chorismate mutase and TyrA)/RRM (RNA Recognition Motif) domain is a near-universal C-terminal domain of long RSHs. Deletion of RRM in both monofunctional (synthesis-only) RelA as well as bifunctional (i.e., capable of both degrading and synthesizing the alarmone) Rel renders the long RSH cytotoxic due to overproduction of (p)ppGpp. To probe the molecular mechanism underlying this effect we characterized Escherichia coli RelA and Bacillus subtilis Rel RSHs lacking RRM. We demonstrate that, first, the cytotoxicity caused by the removal of RRM is counteracted by secondary mutations that disrupt the interaction of the RSH with the starved ribosomal complex – the ultimate inducer of (p)ppGpp production by RelA and Rel – and, second, that the hydrolytic activity of Rel is not abrogated in the truncated mutant. Therefore, we conclude that the overproduction of (p)ppGpp by RSHs lacking the RRM domain is not explained by a lack of auto-inhibition in the absence of RRM or/and a defect in (p)ppGpp hydrolysis. Instead, we argue that it is driven by misregulation of the RSH activation by the ribosome.
(Less)
- author
- Takada, Hiraku
; Roghanian, Mohammad
; Murina, Victoriia
; Dzhygyr, Ievgen
; Murayama, Rikinori
; Akanuma, Genki
; Atkinson, Gemma C.
LU
; Garcia-Pino, Abel
and Hauryliuk, Vasili
LU
- publishing date
- 2020
- type
- Contribution to journal
- publication status
- published
- keywords
- ACT, ppGpp, Rel, ribosome, RRM, stringent response
- in
- Frontiers in Microbiology
- volume
- 11
- article number
- 277
- pages
- 1 - 16
- publisher
- Frontiers Media S. A.
- external identifiers
-
- scopus:85082552430
- pmid:32184768
- ISSN
- 1664-302X
- DOI
- 10.3389/fmicb.2020.00277
- language
- English
- LU publication?
- no
- additional info
- Funding Information: This work was supported by the European Regional Development Fund through the Centre of Excellence for Molecular Cell Technology (VH), the Molecular Infection Medicine Sweden (MIMS) (VH), Swedish Research Council (Grant Nos 2017-03783 to VH and 2015-04746 to GCA), Ragnar Söderberg Foundation (VH), Umeå Centre for Microbial Research (UCMR) (postdoctoral grant 2017 to HT), MIMS Excellence by Choice Postdoctoral Fellowship Programme (MR), the Fonds National de la Recherche Scientifique (Grant Nos FRFS-WELBIO CR-2017S-03, FNRS CDR J.0068.19, and FNRS-PDR T.0066.18 to AG-P) and the Fonds Jean Brachet and the Fondation Van Buuren (AG-P). Funding Information: We would like to thank the Protein Expertise Platform (PEP) at Ume? University and Mikael Lindberg for constructing pET24d:His10-SUMO -based expression constructs and purifying His6-Ulp1, we would also like to thank Tetiana Brodiazhenko for construction of the pMG25-based RelA and SpoT plasmids, and Julien Caballero-Montes for comments on the manuscript. The manuscript was deposited as a preprint to bioRxiv (Takada et al., 2019). The electron microscopy data was collected at the Ume? Core Facility for Electron Microscopy, a node of the Cryo-EM Swedish National Facility, funded by the Knut and Alice Wallenberg, Family Erling Persson and Kempe Foundations, SciLifeLab, Stockholm University, and Ume? University. Funding. This work was supported by the European Regional Development Fund through the Centre of Excellence for Molecular Cell Technology (VH), the Molecular Infection Medicine Sweden (MIMS) (VH), Swedish Research Council (Grant Nos 2017-03783 to VH and 2015-04746 to GCA), Ragnar S?derberg Foundation (VH), Ume? Centre for Microbial Research (UCMR) (postdoctoral grant 2017 to HT), MIMS Excellence by Choice Postdoctoral Fellowship Programme (MR), the Fonds National de la Recherche Scientifique (Grant Nos FRFS-WELBIO CR-2017S-03, FNRS CDR J.0068.19, and FNRS-PDR T.0066.18 to AG-P) and the Fonds Jean Brachet and the Fondation Van Buuren (AG-P). Publisher Copyright: © Copyright © 2020 Takada, Roghanian, Murina, Dzhygyr, Murayama, Akanuma, Atkinson, Garcia-Pino and Hauryliuk. Copyright: Copyright 2020 Elsevier B.V., All rights reserved.
- id
- 0eab15be-4d83-4cb0-98df-404872387285
- date added to LUP
- 2021-09-24 20:35:34
- date last changed
- 2024-06-30 20:23:49
@article{0eab15be-4d83-4cb0-98df-404872387285,
abstract = {{<p>The (p)ppGpp-mediated stringent response is a bacterial stress response implicated in virulence and antibiotic tolerance. Both synthesis and degradation of the (p)ppGpp alarmone nucleotide are mediated by RelA-SpoT Homolog (RSH) enzymes which can be broadly divided in two classes: single-domain ‘short’ and multi-domain ‘long’ RSH. The regulatory ACT (Aspartokinase, Chorismate mutase and TyrA)/RRM (RNA Recognition Motif) domain is a near-universal C-terminal domain of long RSHs. Deletion of RRM in both monofunctional (synthesis-only) RelA as well as bifunctional (i.e., capable of both degrading and synthesizing the alarmone) Rel renders the long RSH cytotoxic due to overproduction of (p)ppGpp. To probe the molecular mechanism underlying this effect we characterized Escherichia coli RelA and Bacillus subtilis Rel RSHs lacking RRM. We demonstrate that, first, the cytotoxicity caused by the removal of RRM is counteracted by secondary mutations that disrupt the interaction of the RSH with the starved ribosomal complex – the ultimate inducer of (p)ppGpp production by RelA and Rel – and, second, that the hydrolytic activity of Rel is not abrogated in the truncated mutant. Therefore, we conclude that the overproduction of (p)ppGpp by RSHs lacking the RRM domain is not explained by a lack of auto-inhibition in the absence of RRM or/and a defect in (p)ppGpp hydrolysis. Instead, we argue that it is driven by misregulation of the RSH activation by the ribosome.</p>}},
author = {{Takada, Hiraku and Roghanian, Mohammad and Murina, Victoriia and Dzhygyr, Ievgen and Murayama, Rikinori and Akanuma, Genki and Atkinson, Gemma C. and Garcia-Pino, Abel and Hauryliuk, Vasili}},
issn = {{1664-302X}},
keywords = {{ACT; ppGpp; Rel; ribosome; RRM; stringent response}},
language = {{eng}},
pages = {{1--16}},
publisher = {{Frontiers Media S. A.}},
series = {{Frontiers in Microbiology}},
title = {{The C-Terminal RRM/ACT Domain Is Crucial for Fine-Tuning the Activation of ‘Long’ RelA-SpoT Homolog Enzymes by Ribosomal Complexes}},
url = {{http://dx.doi.org/10.3389/fmicb.2020.00277}},
doi = {{10.3389/fmicb.2020.00277}},
volume = {{11}},
year = {{2020}},
}