Novel (p)ppGpp Binding and Metabolizing Proteins of
            <i>Escherichia coli</i>

Zhang, Yong; Zborníková, Eva; Rejman, Dominik; Gerdes, Kenn

doi:10.1128/mbio.02188-17

Cited by 133 publications

(178 citation statements)

References 72 publications

(113 reference statements)

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“…Given these observations, we wished to identify the component(s) of the translation machinery that is (are) targeted by ppGpp. The translational GTPases EF-Tu, EF-G and IF2 as well as the ribosome associated GTPases including Obg (Buglino et al, 2002; Feng et al, 2014), RsgA (Corrigan et al, 2016; Zhang et al, 2018), RbgA (Corrigan et al, 2016; Pausch et al, 2018), Era (Corrigan et al, 2016), and HflX (Corrigan et al, 2016; Zhang et al, 2018) have all been reported to bind (p)ppGpp. However, (p)ppGpp inhibits protein synthesis by the PURExpress system (Figure 4), which contains only IF2, EF-Tu, and EF-G, so inhibition of one or more of these proteins is likely sufficient to account for the in vivo inhibitory effect of (p)ppGpp on translation.…”

Section: Resultsmentioning

confidence: 99%

(p)ppGpp directly regulates translation initiation during entry into quiescence

Diez

Ryu

Caban

et al. 2019

Preprint

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12Many bacteria exist in a state of metabolic quiescence where they must minimize energy 13 consumption so as to maximize available resources over a potentially extended period of time. 14 As protein synthesis is the most energy intensive metabolic process in a bacterial cell, it would be 15 an appropriate target for downregulation during the transition from growth to quiescence. We find 16 that when Bacillus subtilis exits growth, a subpopulation of cells emerges with very low levels of 17 protein synthesis dependent on synthesis of the nucleotides (p)ppGpp. We show that (p)ppGpp 18 inhibits protein synthesis in vivo and in vitro by preventing the allosteric activation of the essential 19 GTPase Initiation Factor 2 (IF2) during translation initiation. Finally, we demonstrate that IF2 is 20 an authentic in vivo target of (p)ppGpp during the entry into quiescence, thus providing a 21 mechanistic basis for the observed attenuation of protein synthesis. 22 al., 2016), DNA primase (Wang et al., 2007) and the GTP biosynthetic enzymes HprT and Gmk 51 (Kriel et al., 2012), respectively. 52 Overexpression of a truncated RelA protein that synthesizes (p)ppGpp in the absence of 53 amino acid limitation results in a rapid decrease in 35 S-methionine incorporation (Svitil et al., 54 1993), consistent with an inhibitory effect of (p)ppGpp on translation. However, further 55investigation of a direct effect of (p)ppGpp on translation has been complicated by several factors. 56 First, (p)ppGpp affects synthesis of ribosomal proteins in E. coli (Lindahl et al., 1976). Whether 57 this effect is direct has been difficult to assess given the well-established effect of (p)ppGpp on 58 transcription by E. coli polymerase. Second, studies examining the effect of (p)ppGpp produced 59 by RelA use conditions that produce uncharged tRNAs in order to stimulate RelA (Arenz et al., 60 2016; Brown et al., 2016; Haseltine and Block, 1973; Loveland et al., 2016; O'Farrell, 1978). Since 61 uncharged tRNAs directly arrest translation, it has been difficult to differentiate this effect from a 62 direct effect of (p)ppGpp on translation. 63 In vitro, (p)ppGpp inhibits translation in a manner similar to that observed with non-64 hydrolyzable GTP analogs (Wagner and Kurland, 1980), suggesting that (p)ppGpp is targeting a 65 translational GTPase. Consistently, (p)ppGpp inhibits the GTPase activity of IF2 and EF-Tu 66 (Hamel and Cashel, 1974) as well as of GTPases that mediate other aspects of translation such 67 as ribosome assembly (Corrigan et al., 2016; Pausch et al., 2018), by acting as competitive 68 inhibitors. (p)ppGpp is capable of binding translational GTPases including EF-Tu, EF-G (Rojas et 69 al., 1984), IF2 (Milon et al., 2006; Mitkevich et al., 2010) and the ribosome assembly GTPase 70 ObgE (Persky et al., 2009) at affinities that are commensurate with the in vivo levels of (p)ppGpp 71 observed following stringent response induction, consistent with the enzymes being in vivo 72 targets. Of note, the affinity of IF2 f...

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Section: Resultsmentioning

confidence: 99%

(p)ppGpp directly regulates translation initiation during entry into quiescence

Diez

Ryu

Caban

et al. 2019

Preprint

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“…There are several other recently identified (p)ppGpp targets that contain the PRPP binding motif, including the phosphoribosyltransferases XGPRT, UPRT, and PurF from E. coli 6,19,20 . All these enzymes bind PRPP as a substrate.…”

Section: Discussionmentioning

confidence: 99%

Molecular mechanism of regulation of the purine salvage enzyme XPRT by the alarmones pppGpp, ppGpp, and pGpp

Anderson

Hao

Satyshur

et al. 2020

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1The alarmones pppGpp and ppGpp mediate starvation response and maintain purine homeostasis 2 to protect bacterial species. Xanthine phosphoribosyltransferase (XPRT) is a purine salvage 3 enzyme that produces the nucleotide XMP from PRPP and xanthine. Combining structural, 4 biochemical and genetic analyses, we show that pppGpp and ppGpp, as well as a third putative 5 alarmone pGpp, all directly interact with XPRT and inhibit XPRT activity by competing with its 6 substrate PRPP. Structural analysis reveals that ppGpp binds the PRPP binding motif within the 7 XPRT active site. This motif is present in another (p)ppGpp target, the purine salvage enzyme 8HPRT, suggesting evolutionary conservation in different enzymes. However, XPRT oligomeric 9 interaction is distinct from HPRT in that XPRT forms a symmetric dimer with two (p)ppGpp 10 binding sites at the dimer interface. This results in two distinct regulatory features. First, XPRT 11 cooperatively binds (p)ppGpp with a Hill coefficient of 2. Also, XPRT displays differential 12 regulation by the alarmones as it is potently inhibited by both ppGpp and pGpp, but only 13 modestly by pppGpp. Lastly, we demonstrate that the alarmones are necessary for protecting 14 GTP homeostasis against excess environmental xanthine in Bacillus subtilis, suggesting that 15 regulation of XPRT is key for regulating the purine salvage pathway. 16 PRPP share the same pocket, and the 3′-phosphates of ppGpp overlap with the 1′-phosphates of 132 PRPP ( Figure 5B). 133Next, we examined whether (p)ppGpp competes with PRPP to inhibit XPRT using 134 steady-state kinetics. We measured initial velocities of XPRT enzymatic reaction by examining 135 the rate of synthesis of XMP at varied pppGpp and PRPP concentrations ( Figure 5C). The data 136 best fit a global competitive inhibition model, demonstrating that (p)ppGpp competes with PRPP 137 ( Figure 5C and 5D). In addition, the kinetic data yielded a Ki for pppGpp of 2.5 μM and a Km for 138

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“…Mutant strains lacking both relP and relQ have reduced survival activities in response to antibiotic agents that induce cell-envelope 'stresses', such as ampicillin and vancomycin [37]. In other Firmicutes taxa that contain SAS homologues whose alarmonesynthesizing activities have been determined, such as E. faecalis, S. mutans and B. subtilis; the rsh gene is non-essential for viability [34,63,64], suggesting that there may be alternative pathways for (pp)pGpp hydrolysis, such as small alarmone hydrolases (SAHs) [14,65], NUDIX hydrolases (Ndx8, MutT, NudG) [66,67], or phosphohydrolases belonging to a variety of families (e.g. Mesh1, TrmE, NadR, PhoA, UshA) [67,68].…”

Section: Discussionmentioning

confidence: 99%

The Ps and Qs of alarmone synthesis inStaphylococcus aureus

Yang

Xie

Choi

et al. 2019

Preprint

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During the stringent response, bacteria synthesize guanosine-3',5'-bis(diphosphate) (ppGpp) and guanosine-5'-triphosphate 3'-diphosphate (pppGpp), which act as secondary messengers to promote cellular survival and adaptation. (p)ppGpp 'alarmones' are synthesized and/or hydrolyzed by proteins belonging to the RelA/SpoT Homologue (RSH) family. Many bacteria also encode 'small alarmone synthetase' (SAS) proteins (e.g. RelP, RelQ) which may also be capable of synthesizing a third alarmone: guanosine-5'-phosphate 3'-diphosphate (pGpp). Here, we report the biochemical properties of the Rel (RSH), RelP and RelQ proteins from Staphylococcus aureus (Sa-Rel, Sa-RelP, Sa-RelQ, respectively). Sa-Rel synthesized pppGpp more efficiently than ppGpp, but lacked the ability to produce pGpp. However, Sa-Rel efficiently hydrolyzed all three alarmones in a Mn(II) ion-dependent manner. The removal of the C-terminal regulatory domain of Sa-Rel increased its rate of (p)ppGpp synthesis ca. 10fold, but had negligible effects on its rate of (pp)pGpp hydrolysis. Sa-RelP and Sa-RelQ efficiently synthesized pGpp in addition to pppGpp and ppGpp. The alarmone-synthesizing abilities of Sa-RelQ, but not Sa-RelP, were allosterically-stimulated by the addition of pppGpp, ppGpp or pGpp. The respective (pp)pGpp-synthesizing activities of Sa-RelP/Sa-RelQ were compared and contrasted with SAS homologues from Enterococcus faecalis (Ef-RelQ) andStreptococcus mutans (Sm-RelQ, Sm-RelP). Results indicated that EF-RelQ, Sm-RelQ and Sa-RelQ were functionally-equivalent; but exhibited considerable variations in their respective biochemical properties, and the degrees to which alarmones and single-stranded RNA molecules allosterically stimulated their respective alarmone-synthesizing activities. The respective (pp)pGpp-synthesizing capabilities of Sa-RelP and Sm-RelP proteins were inhibited by pGpp, ppGpp and pppGpp. Our results support the premise that RelP and RelQ proteins may synthesize pGpp in addition to (p)ppGpp within S. aureus and other Gram-positive bacterial species. intracellular messengers that directly or indirectly up-regulate cellular processes that promote the conservation, recycling and biosynthesis of important molecular building-blocks; initiate physiological mechanisms that preserve cellular integrity and tolerance in response to certain cytotoxic exogenous agents or harsh extracellular conditions; and down-regulate processes that are superfluous or non-essential for short-term viability (reviewed in [4][5][6][7][8][9][10][11][12]).Alarmones are synthesized and/or hydrolyzed by proteins belonging to the RelA/SpoT-Homologue (RSH) family [13,14] RelA proteins, such as the Escherichia coli RelA protein, are termed monofunctional or 'synthase-only', in that they lack a catalytic Histidine-Aspartate (HD) domain responsible for hydrolytic activities, and only catalyze (p)ppGpp production [15,16]. This synthase activity involves the transfer of a diphosphate unit from ATP to the 3'hydroxyl group of GTP or GDP in a Mg 2+ -dependent manner to pr...

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Novel (p)ppGpp Binding and Metabolizing Proteins of Escherichia coli

Cited by 133 publications

References 72 publications

(p)ppGpp directly regulates translation initiation during entry into quiescence

(p)ppGpp directly regulates translation initiation during entry into quiescence

Molecular mechanism of regulation of the purine salvage enzyme XPRT by the alarmones pppGpp, ppGpp, and pGpp

The Ps and Qs of alarmone synthesis inStaphylococcus aureus

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