Biochemical studies on <i>Francisella tularensis</i> RelA in (p)ppGpp biosynthesis

Wilkinson, Rachael C.; Batten, Laura E.; Wells, Neil J.; Oyston, Petra C. F.; Roach, Peter L.

doi:10.1042/bsr20150229

Cited by 12 publications

(13 citation statements)

References 42 publications

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“…In agreement with our findings, deletion of the RRM domain of Caulobacter crescentus bifunctional ribosome-associated RSH, Rel, also leads to de-regulation of the protein and (p)ppGpp-mediated growth inhibition (Ronneau et al, 2019). Curiously, while Francisella tularensis RelA naturally lacks the RRM domain (Atkinson et al, 2011), the enzyme displays normal, wt-like enzymatic functionality in biochemical assays (Wilkinson et al, 2015). Further work is required to determine the exact regulatory role of the RRM domain that is near-universally conserved in “long” RSHs Rel, RelA and SpoT (Atkinson et al, 2011).…”

Section: Discussionmentioning

confidence: 99%

Intramolecular Interactions Dominate the Autoregulation of Escherichia coli Stringent Factor RelA

et al. 2019

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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.

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Section: Discussionmentioning

confidence: 99%

Intramolecular Interactions Dominate the Autoregulation of Escherichia coli Stringent Factor RelA

et al. 2019

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“…The method was adopted from Wilkinson et al . . The samples (40 μL) were injected and chromatographed on a reverse phase column (Agilent Eclipse XDB‐C18 4.6 × 150 mm, 5 μm, Santa Clara, CA, USA) at a flow rate of 1 mL·min −1 with UV detection at 260 nm.…”

Section: Methodsmentioning

confidence: 99%

“…The activity of MtRel NTD was determined using ion-pair reverse-phase high-performance liquid chromatography (IP-RP HPLC). The method was adopted from Wilkinson et al [11]. The samples (40 lL) were injected and chromatographed on a reverse phase column (Agilent Eclipse XDB-C18 4.6 9 150 mm, 5 lm, Santa Clara, CA, USA) at a flow rate of 1 mLÁmin À1 with UV detection at 260 nm.…”

Section: Activity Analysis Of the Mtrel Ntd Using Hplcmentioning

confidence: 99%

Crystallographic and solution structure of the N‐terminal domain of the Rel protein from Mycobacterium tuberculosis

et al. 2017

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Modulation of intracellular guanosine 3′,5′‐bispyrophosphate ((p)ppGpp) level, the effector of the stringent response, is crucial for survival as well as optimal growth of prokaryotes and, thus, for bacterial pathogenesis and dormancy. In Mycobacterium tuberculosis (Mtb), (p)ppGpp synthesis and degradation are carried out by the bifunctional enzyme MtRel, which consists of 738 residues, including an N‐terminal hydrolase‐ and synthetase‐domain (N‐terminal domain or NTD) and a C‐terminus with a ribosome‐binding site. Here, we present the first crystallographic structure of the enzymatically active MtRel NTD determined at 3.7 Å resolution. The structure provides insights into the residues of MtRel NTD responsible for nucleotide binding. Small‐angle X‐ray scattering experiments were performed to investigate the dimeric state of the MtRel NTD and possible substrate‐dependent structural alterations.

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“…The RRM is absent in RelA enzymes from Methylotenera mobilis, Elusimicrobium minutum, Francisella philomiraga, and Francisella tularensis (Atkinson et al, 2011). The only experimentally characterized representative amongst these is F. tularensis RelA (Wilkinson et al, 2015). In a reconstituted biochemical system, the factor behaves similarly to E. coli RelA, i.e., it has very low synthesis activity by itself and is potently activated by the ribosome.…”

Section: Introductionmentioning

confidence: 99%

The C-Terminal RRM/ACT Domain Is Crucial for Fine-Tuning the Activation of ‘Long’ RelA-SpoT Homolog Enzymes by Ribosomal Complexes

et al. 2020

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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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Biochemical studies on Francisella tularensis RelA in (p)ppGpp biosynthesis

Abstract: Francisella tularensis RelA shows significant sequence differences from other members of the RelA family of enzymes. In the present study, we describe the functional similarities and differences between F. tularensis RelA and the model RelA from Escherichia coli.

Cited by 12 publications

References 42 publications

Intramolecular Interactions Dominate the Autoregulation of Escherichia coli Stringent Factor RelA

Intramolecular Interactions Dominate the Autoregulation of Escherichia coli Stringent Factor RelA

Crystallographic and solution structure of the N‐terminal domain of the Rel protein from Mycobacterium tuberculosis

The C-Terminal RRM/ACT Domain Is Crucial for Fine-Tuning the Activation of ‘Long’ RelA-SpoT Homolog Enzymes by Ribosomal Complexes

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