Intramolecular interactions dominate the autoregulation of escherichia coli stringent factor RelA
Turnbull, Kathryn Jane ; Dzhygyr, Ievgen ; Lindemose, Søren ; Hauryliuk, Vasili LU
and Roghanian, Mohammad
(2019)
In Frontiers in Microbiology
10.
- Abstract
Amino acid starvation in Escherichia coli activates the enzymatic activity of the stringent factor RelA, leading to accumulation of the alarmone nucleotide (p)ppGpp. The alarmone acts as an intercellular messenger to regulate transcription, translation and metabolism to mediate bacterial stress adaptation. The enzymatic activity of RelA is subject to multi-layered allosteric control executed both by ligands – such as “starved” ribosomal complexes, deacylated tRNA and pppGpp – and by individual RelA domains. The auto-regulation of RelA is proposed to act either in cis (inhibition of the enzymatic activity of the N-terminal region, NTD, by regulatory C-terminal region, CTD) or in trans (CTD-mediated dimerization leading to enzyme... (More)
Amino acid starvation in Escherichia coli activates the enzymatic activity of the stringent factor RelA, leading to accumulation of the alarmone nucleotide (p)ppGpp. The alarmone acts as an intercellular messenger to regulate transcription, translation and metabolism to mediate bacterial stress adaptation. The enzymatic activity of RelA is subject to multi-layered allosteric control executed both by ligands – such as “starved” ribosomal complexes, deacylated tRNA and pppGpp – and by individual RelA domains. The auto-regulation of RelA is proposed to act either in cis (inhibition of the enzymatic activity of the N-terminal region, NTD, by regulatory C-terminal region, CTD) or in trans (CTD-mediated dimerization leading to enzyme inhibition). In this report, we probed the regulatory roles of the individual domains of E. coli RelA and our results are not indicative of RelA dimerization being the key regulatory mechanism. First, at growth-permitting levels, ectopic expression of RelA CTD does not interfere with activation of native RelA, indicating lack of regulation via inhibitory complex formation in the cell. Second, in our biochemical assays, increasing RelA concentration does not decrease the enzyme activity, as would be expected in the case of efficient auto-inhibition via dimerization. Third, while high-level CTD expression efficiently inhibits the growth, the effect is independent of native RelA and is mediated by direct inhibition of protein synthesis, likely via direct interaction with the ribosomal A-site. Finally, deletion of the RRM domain of the CTD region leads to growth inhibition mediated by accumulation of (p)ppGpp, suggesting de-regulation of the synthetic activity in this mutant.
(Less)
- author
- Turnbull, Kathryn Jane
; Dzhygyr, Ievgen
; Lindemose, Søren
; Hauryliuk, Vasili
LU
and Roghanian, Mohammad
- publishing date
- 2019
- type
- Contribution to journal
- publication status
- published
- keywords
- (p)ppGpp, RelA, Ribosome, RSH, Stringent response, Translation
- in
- Frontiers in Microbiology
- volume
- 10
- article number
- 1966
- publisher
- Frontiers Media S. A.
- external identifiers
-
- scopus:85071934247
- ISSN
- 1664-302X
- DOI
- 10.3389/fmicb.2019.01966
- language
- English
- LU publication?
- no
- additional info
- Funding Information: This work was carried out at both the Centre for Bacterial Stress Response and Persistence (BASP) at the University of Copenhagen, and the Department of Molecular Biology at Ume? University. This work was supported by a grant from the Danish National Research Foundation (DNFR120), the MIMS Excellence by Choice Postdoctoral Fellowship Programme to MR and the Swedish Research Council (grant 2017-03783 to VH). We would like to thank Professor Kenn Gerdes, Dr. Anurag Sinha, and Dr. Farshid Jalalvand for helpful discussions and comments on the manuscript, Dr. Szabolcs Semsey for kindly providing plasmid pSEM3034UR2, and the Protein Expertise Platform (PEP) at Ume? University and Mikael Lindberg for constructing pET24d:His10-SUMO and purifying His6-Ulp1. This manuscript was deposited as a preprint to bioRxiv (Turnbull et al., 2019). Funding Information: This work was carried out at both the Centre for Bacterial Stress Response and Persistence (BASP) at the University of Copenhagen, and the Department of Molecular Biology at Umeå University. This work was supported by a grant from the Danish National Research Foundation (DNFR120), the MIMS Excellence by Choice Postdoctoral Fellowship Programme to MR and the Swedish Research Council (grant 2017-03783 to VH). Publisher Copyright: © 2019 Turnbull, Dzhygyr, Lindemose, Hauryliuk and Roghanian. Copyright: Copyright 2019 Elsevier B.V., All rights reserved.
- id
- 4b6c2446-29de-48fb-8b73-e966288d5cb7
- date added to LUP
- 2021-09-24 20:33:48
- date last changed
- 2022-04-27 04:15:45
@article{4b6c2446-29de-48fb-8b73-e966288d5cb7,
abstract = {{<p>Amino acid starvation in Escherichia coli activates the enzymatic activity of the stringent factor RelA, leading to accumulation of the alarmone nucleotide (p)ppGpp. The alarmone acts as an intercellular messenger to regulate transcription, translation and metabolism to mediate bacterial stress adaptation. The enzymatic activity of RelA is subject to multi-layered allosteric control executed both by ligands – such as “starved” ribosomal complexes, deacylated tRNA and pppGpp – and by individual RelA domains. The auto-regulation of RelA is proposed to act either in cis (inhibition of the enzymatic activity of the N-terminal region, NTD, by regulatory C-terminal region, CTD) or in trans (CTD-mediated dimerization leading to enzyme inhibition). In this report, we probed the regulatory roles of the individual domains of E. coli RelA and our results are not indicative of RelA dimerization being the key regulatory mechanism. First, at growth-permitting levels, ectopic expression of RelA CTD does not interfere with activation of native RelA, indicating lack of regulation via inhibitory complex formation in the cell. Second, in our biochemical assays, increasing RelA concentration does not decrease the enzyme activity, as would be expected in the case of efficient auto-inhibition via dimerization. Third, while high-level CTD expression efficiently inhibits the growth, the effect is independent of native RelA and is mediated by direct inhibition of protein synthesis, likely via direct interaction with the ribosomal A-site. Finally, deletion of the RRM domain of the CTD region leads to growth inhibition mediated by accumulation of (p)ppGpp, suggesting de-regulation of the synthetic activity in this mutant.</p>}},
author = {{Turnbull, Kathryn Jane and Dzhygyr, Ievgen and Lindemose, Søren and Hauryliuk, Vasili and Roghanian, Mohammad}},
issn = {{1664-302X}},
keywords = {{(p)ppGpp; RelA; Ribosome; RSH; Stringent response; Translation}},
language = {{eng}},
publisher = {{Frontiers Media S. A.}},
series = {{Frontiers in Microbiology}},
title = {{Intramolecular interactions dominate the autoregulation of escherichia coli stringent factor RelA}},
url = {{http://dx.doi.org/10.3389/fmicb.2019.01966}},
doi = {{10.3389/fmicb.2019.01966}},
volume = {{10}},
year = {{2019}},
}