Positive allosteric feedback regulation of the stringent response enzyme RelA by its product

Shyp, Viktoriya; Tankov, Stoyan; Ermakov, Andrey; Kudrin, Pavel; English, Brian P.; Ehrenberg, Måns; Tenson, Tanel; Elf, Johan; Hauryliuk, Vasili

doi:10.1038/embor.2012.106

Cited by 89 publications

(98 citation statements)

References 31 publications

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“…6D). As shown previously (36), ppGpp had a strong activating effect on RelA Ec synthetase activity in a reconstituted translation system containing 70S ribosomes, model mRNA, and deacylated tRNA. Interestingly, pGpp also had a strong activating effect on RelA Ec activity and, in contrast to ppGpp, did not inhibit the enzyme at higher concentrations (Fig.…”

Section: Relq Ef Prefers Gdp Over Gtp As a Substrate And Is Insensitimentioning

confidence: 87%

“…Recently, ppGpp was shown to stimulate the synthetase activity of E. coli RelA Ec (36). Importantly, when high concentrations of RelA Ec were used in the system, this product-mediated activation was masked by the in situproduced ppGpp.…”

Section: Relq Ef Prefers Gdp Over Gtp As a Substrate And Is Insensitimentioning

confidence: 99%

“…Feedforward or autocatalytic stimulation of RelQ synthetase activity by ppGpp raises the possibility that RelQ or Rel products serve as a signal amplifier for (pp)pGpp production in a manner homologous to the direct activation of RelA Ec by (p)ppGpp (36). Multiple attempts to test the activating effects of (pp)pGpp on Rel Ef activity were unsuccessful, due in part to the enzyme's poor in vitro activity, even in the presence of ribosomes.…”

Section: Figmentioning

confidence: 99%

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From (p)ppGpp to (pp)pGpp: Characterization of Regulatory Effects of pGpp Synthesized by the Small Alarmone Synthetase of Enterococcus faecalis

et al. 2015

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The bacterial stringent response (SR) is a conserved stress tolerance mechanism that orchestrates physiological alterations to enhance cell survival. This response is mediated by the intracellular accumulation of the alarmones pppGpp and ppGpp, collectively called (p)ppGpp. In Enterococcus faecalis, (p)ppGpp metabolism is carried out by the bifunctional synthetase/hydrolase E. faecalis Rel (Rel Ef ) and the small alarmone synthetase (SAS) RelQ Ef . Although Rel is the main enzyme responsible for SR activation in Firmicutes, there is emerging evidence that SASs can make important contributions to bacterial homeostasis. Here, we showed that RelQ Ef synthesizes ppGpp more efficiently than pppGpp without the need for ribosomes, tRNA, or mRNA. In addition to (p)ppGpp synthesis from GDP and GTP, RelQ Ef also efficiently utilized GMP to form GMP 3=-diphosphate (pGpp). Based on this observation, we sought to determine if pGpp exerts regulatory effects on cellular processes affected by (p)ppGpp. We found that pGpp, like (p)ppGpp, strongly inhibits the activity of E. faecalis enzymes involved in GTP biosynthesis and, to a lesser extent, transcription of rrnB by Escherichia coli RNA polymerase. Activation of E. coli RelA synthetase activity was observed in the presence of both pGpp and ppGpp, while RelQ Ef was activated only by ppGpp. Furthermore, enzymatic activity of RelQ Ef is insensitive to relacin, a (p)ppGpp analog developed as an inhibitor of "long" RelA/SpoT homolog (RSH) enzymes. We conclude that pGpp can likely function as a bacterial alarmone with target-specific regulatory effects that are similar to what has been observed for (p)ppGpp. IMPORTANCEAccumulation of the nucleotide second messengers (p)ppGpp in bacteria is an important signal regulating genetic and physiological networks contributing to stress tolerance, antibiotic persistence, and virulence. Understanding the function and regulation of the enzymes involved in (p)ppGpp turnover is therefore critical for designing strategies to eliminate the protective effects of this molecule. While characterizing the (p)ppGpp synthetase RelQ of Enterococcus faecalis (RelQ Ef ), we found that, in addition to (p)ppGpp, RelQ Ef is an efficient producer of pGpp (GMP 3=-diphosphate). In vitro analysis revealed that pGpp exerts complex, target-specific effects on processes known to be modulated by (p)ppGpp. These findings provide a new regulatory feature of RelQ Ef and suggest that pGpp may represent a new member of the (pp)pGpp family of alarmones. In order to survive under adverse environmental conditions, such as nutrient starvation, bacteria have evolved complex, interconnected regulatory networks that sense and integrate internal and external metabolic cues to activate cellular responses that enhance bacterial survival. One critical and highly conserved mechanism employed by bacteria to cope with nutritional as well as a variety of environmental and chemical stresses is the stringent response (SR). The SR is mediated by the accumulation of two guanine analog...

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Section: Relq Ef Prefers Gdp Over Gtp As a Substrate And Is Insensitimentioning

confidence: 87%

Section: Relq Ef Prefers Gdp Over Gtp As a Substrate And Is Insensitimentioning

confidence: 99%

Section: Figmentioning

confidence: 99%

See 1 more Smart Citation

From (p)ppGpp to (pp)pGpp: Characterization of Regulatory Effects of pGpp Synthesized by the Small Alarmone Synthetase of Enterococcus faecalis

et al. 2015

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“…Biochemical in vitro translation system from purified E. coli components and TLC measurements of 3 H ppGpp synthesis have been described previously (9). Enzymatic assays with E. faecalis RelQ were performed following the method of Gaca et al (19).…”

Section: Methodsmentioning

confidence: 99%

“…The enzymatic activities of the two E. coli RSH enzymes are regulated allosterically. Synthesis of (p)ppGpp by RelA is strongly induced on amino acid limitation by so-called "starved" ribosomal complexes loaded with cognate deacylated tRNA in the A-site (8), and RelA activation is further potentiated by the product of the reaction, ppGpp (9). SpoT has both (p)ppGpp synthesis and hydrolysis activities and is regulated by numerous stress signals, including fatty acid (10), iron (11), and carbon source (6) limitations.…”

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confidence: 99%

Negative allosteric regulation of Enterococcus faecalis small alarmone synthetase RelQ by single-stranded RNA

Beljantseva

Kudrin

Andresen

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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The alarmone nucleotides guanosine pentaphosphate (pppGpp) and tetraphosphate (ppGpp), collectively referred to as (p)ppGpp, are key regulators of bacterial growth, stress adaptation, pathogenicity, and antibiotic tolerance. We show that the tetrameric small alarmone synthetase (SAS) RelQ from the Gram-positive pathogen Enterococcus faecalis is a sequence-specific RNA-binding protein. RelQ's enzymatic and RNA binding activities are subject to intricate allosteric regulation. (p)ppGpp synthesis is potently inhibited by the binding of single-stranded RNA. Conversely, RelQ's enzymatic activity destabilizes the RelQ:RNA complex. pppGpp, an allosteric activator of the enzyme, counteracts the effect of RNA. Tetramerization of RelQ is essential for this regulatory mechanism, because both RNA binding and enzymatic activity are abolished by deletion of the SAS-specific C-terminal helix 5α. The interplay of pppGpp binding, (p)ppGpp synthesis, and RNA binding unites two archetypal regulatory paradigms within a single protein. The mechanism is likely a prevalent but previously unappreciated regulatory switch used by the widely distributed bacterial SAS enzymes. stringent response | (p)ppGpp | RNA-protein interaction | allosteric regulation | nucleotide signaling

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Mechanisms of Stress‐Activated Persister Formation inEscherichia Coli

Amato

Brynildsen

2016

Stress and Environmental Regulation of Gene Expression and Adaptation in Bacteria

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Positive allosteric feedback regulation of the stringent response enzyme RelA by its product

Cited by 89 publications

References 31 publications

From (p)ppGpp to (pp)pGpp: Characterization of Regulatory Effects of pGpp Synthesized by the Small Alarmone Synthetase of Enterococcus faecalis

From (p)ppGpp to (pp)pGpp: Characterization of Regulatory Effects of pGpp Synthesized by the Small Alarmone Synthetase of Enterococcus faecalis

Negative allosteric regulation of Enterococcus faecalis small alarmone synthetase RelQ by single-stranded RNA

Mechanisms of Stress‐Activated Persister Formation inEscherichia Coli

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