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| 2. |
- Dzhygyr, Ievgen, 1985-
(författare)
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Functional studies of Escherichia coli stringent response factor RelA
- 2018
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- RelA is a ribosome associated multi-domain enzyme, which plays a crucial role in adaptation ofEscherichia coli to nutritional stress as such as amino acid deficiency. It detects the deficiency of aminoacids in the cell by monitoring whether a tRNA at the acceptor site (A-site) of the ribosome is chargedwith amino acid or not. When RelA detects uncharged, i.e. deacylated tRNA, it starts to producealarmone guanosine penta- or tetraphosphate, collectively referred to as (p)ppGpp. (p)ppGpp is aglobal metabolism regulator in bacteria. Increase in (p)ppGpp concentration alters crucial metabolicprocesses, such as DNA replication, gene expression, cell wall synthesis and translation. Thesechanges also include activation of different virulence factors and are proposed to drive formation of abacterial sub-population that is highly resilient to antibiotic treatment, the so-called persisters.For a long time the molecular mechanism of RelA’s activation by and interaction with the ribosomedeacylatedtRNA complex was unknown. Only recently several cryo-EM structures of RelA-ribosomecomplex have shed light on how C-terminal domains of RelA interact with ribosome-deacylated tRNAcomplex. Guided by these structures we investigated the role of RelA’s domains in this interaction byconstructing a set of RelA C-terminal truncates and subjecting these to biochemical and microbiologicalexperimentation. These experiments were complemented with mutations in ribosomal RNA atpositions that interact with RelA, namely A-site finger and sarcin-ricin loop.We have shown that only the full-length wild type RelA can be activated by ribosome-tRNA complex,whereas, the set of truncated proteins missing either one, two or three C-terminal domains do notrespond to the presence of uncharged tRNA in the A-site of the ribosome. However, these truncatedversions can still be activated by vacant 70S ribosome as well as pppGpp, suggesting that N-terminaldomain of RelA has an allosteric regulation site for (p)ppGpp and is able to interact with the ribosome.The mechanism of this interaction is yet to be elucidated.We have shown that A-site finger of the ribosome is required for RelA activation and recruitment tothe ribosome. Using EMSA assays, we have shown that RelA and deacylated tRNA do not form a stablecomplex off the ribosome. His432 located in TGS domain of RelA is crucial for recognition of deacylatedtRNA and a mutation of this histidine to glycine abolishes RelA activation by deacylated tRNA.Since (p)ppGpp plays an important role in bacterial survival and pathogenicity we have also testedseveral strategies for RelA inhibition by antibiotics, which target ribosomes and the interactionbetween RelA and ribosome-deacylated tRNA complex. We have shown that antibiotic thiostreptoninhibits (p)ppGpp synthesis by preventing RelA-tRNA interaction on the ribosome. (p)ppGppproduction is also inhibited by chloramphenicol and tetracycline.
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- Kudrin, Pavel, et al.
(författare)
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Subinhibitory Concentrations of Bacteriostatic Antibiotics Induce relA-Dependent and relA-Independent Tolerance to beta-Lactams
- 2017
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Ingår i: Antimicrobial Agents and Chemotherapy. - : AMER SOC MICROBIOLOGY. - 0066-4804 .- 1098-6596. ; 61:4
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Tidskriftsartikel (refereegranskat)abstract
- The nucleotide (p) ppGpp is a key regulator of bacterial metabolism, growth, stress tolerance, and virulence. During amino acid starvation, the Escherichia coli (p) ppGpp synthetase RelA is activated by deacylated tRNA in the ribosomal A-site. An increase in (p) ppGpp is believed to drive the formation of antibiotic-tolerant persister cells, prompting the development of strategies to inhibit (p) ppGpp synthesis. We show that in a biochemical system from purified E. coli components, the antibiotic thiostrepton efficiently inhibits RelA activation by the A-site tRNA. In bacterial cultures, the ribosomal inhibitors thiostrepton, chloramphenicol, and tetracycline all efficiently abolish accumulation of (p) ppGpp induced by the Ile-tRNA synthetase inhibitor mupirocin. This abolishment, however, does not reduce the persister level. In contrast, the combination of dihydrofolate reductase inhibitor trimethoprim with mupirocin, tetracycline, or chloramphenicol leads to ampicillin tolerance. The effect is independent of RelA functionality, specific to beta-lactams, and not observed with the fluoroquinolone norfloxacin. These results refine our understanding of (p) ppGpp's role in antibiotic tolerance and persistence and demonstrate unexpected drug interactions that lead to tolerance to bactericidal antibiotics.
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| 4. |
- Kudrin, Pavel, et al.
(författare)
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The ribosomal A-site finger is crucial for binding and activation of the stringent factor RelA
- 2018
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Ingår i: Nucleic Acids Research. - : OXFORD UNIV PRESS. - 0305-1048 .- 1362-4962. ; 46:4, s. 1973-1983
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Tidskriftsartikel (refereegranskat)abstract
- During amino acid starvation the Escherichia coli stringent response factor RelA recognizes deacylated tRNA in the ribosomal A-site. This interaction activates RelA-mediated synthesis of alarmone nucleotides pppGpp and ppGpp, collectively referred to as (p)ppGpp. These two alarmones are synthesized by addition of a pyrophosphate moiety to the 3' position of the abundant cellular nucleotide GTP and less abundant nucleotide GDP, respectively. Using untagged native RelA we show that allosteric activation of RelA by pppGpp increases the efficiency of GDP conversion to achieve the maximum rate of (p) ppGpp production. Using a panel of ribosomal RNA mutants, we show that the A-site finger structural element of 23S rRNA helix 38 is crucial for RelA binding to the ribosome and consequent activation, and deletion of the element severely compromises (p) ppGpp accumulation in E. coli upon amino acid starvation. Through binding assays and enzymology, we show that E. coli RelA does not form a stable complex with, and is not activated by, deacylated tRNA off the ribosome. This indicates that in the cell, RelA first binds the empty A-site and then recruits tRNA rather than first binding tRNA and then binding the ribosome.
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| 5. |
- Moparthi, Vamsi, et al.
(författare)
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The two Dps proteins, NpDps2 and NpDps5, are involved in light-induced oxidative stress tolerance in the N-2-fixing cyanobacterium Nostoc punctiforme
- 2016
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Ingår i: Biochimica et Biophysica Acta - Bioenergetics. - : Elsevier. - 0005-2728 .- 1879-2650. ; 1857:11, s. 1766-1776
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Tidskriftsartikel (refereegranskat)abstract
- Cyanobacteria are photosynthetic prokaryotes that are considered biotechnologically prominent organisms for production of high-value compounds. Cyanobacteria are subject to high-light intensities, which is a challenge that needs to be addressed in design of efficient bio-engineered photosynthetic organisms. Dps proteins are members of the ferritin superfamily and are omnipresent in prokaryotes. They play a major role in oxidative stress protection and iron homeostasis. The filamentous, heterocyst-forming Nostoc punctiforme, has five Dps proteins. In this study we elucidated the role of these Dps proteins in acclimation to high light intensity, the gene loci organization and the transcriptional regulation of all five dps genes in N. punctiforme was revealed, and dps-deletion mutant strains were used in physiological characterization. Two mutants defective in Dps2 and Dps5 activity displayed a reduced fitness under increased illumination, as well as a differential Photosystem (PS) stoichiometry, with an elevated Photosystem II to Photosystem I ratio in the dps5 deletion strain. This work establishes a Dps-mediated link between light tolerance, H2O2 detoxification, and iron homeostasis, and provides further evidence on the non-redundant role of multiple Dps proteins in this multicellular cyanobacterium.
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| 6. |
- Roghanian, Mohammad, et al.
(författare)
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(p)ppGpp controls stringent factors by exploiting antagonistic allosteric coupling between catalytic domains
- 2021
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Ingår i: Molecular Cell. - : Elsevier. - 1097-2765 .- 1097-4164. ; 81:16, s. 3310-3322.e6
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Tidskriftsartikel (refereegranskat)abstract
- Amino acid starvation is sensed by Escherichia coli RelA and Bacillus subtilis Rel through monitoring the aminoacylation status of ribosomal A-site tRNA. These enzymes are positively regulated by their product—the alarmone nucleotide (p)ppGpp—through an unknown mechanism. The (p)ppGpp-synthetic activity of Rel/RelA is controlled via auto-inhibition by the hydrolase/pseudo-hydrolase (HD/pseudo-HD) domain within the enzymatic N-terminal domain region (NTD). We localize the allosteric pppGpp site to the interface between the SYNTH and pseudo-HD/HD domains, with the alarmone stimulating Rel/RelA by exploiting intra-NTD autoinhibition dynamics. We show that without stimulation by pppGpp, starved ribosomes cannot efficiently activate Rel/RelA. Compromised activation by pppGpp ablates Rel/RelA function in vivo, suggesting that regulation by the second messenger (p)ppGpp is necessary for mounting an acute starvation response via coordinated enzymatic activity of individual Rel/RelA molecules. Control by (p)ppGpp is lacking in the E. coli (p)ppGpp synthetase SpoT, thus explaining its weak synthetase activity.
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| 7. |
- Takada, Hiraku, et al.
(författare)
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The C-Terminal RRM/ACT Domain Is Crucial for Fine-Tuning the Activation of 'Long' RelA-SpoT Homolog Enzymes by Ribosomal Complexes
- 2020
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Ingår i: Frontiers in Microbiology. - : Frontiers Media S.A.. - 1664-302X. ; 11
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Tidskriftsartikel (refereegranskat)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 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.
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| 8. |
- Turnbull, Kathryn Jane, et al.
(författare)
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Intramolecular Interactions Dominate the Autoregulation of Escherichia coli Stringent Factor RelA
- 2019
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Ingår i: Frontiers in Microbiology. - : Frontiers Media S.A.. - 1664-302X. ; 10
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Tidskriftsartikel (refereegranskat)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 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 ReIA, 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.
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