The ribosome triggers the stringent response by RelA via a highly distorted tRNA

Agirrezabala, Xabier; Fernandez, I.S.; Kelley, A.C.; Gil, David; Ramakrishnan, V.; Valle, Mikel

doi:10.1038/embor.2013.106

Cited by 51 publications

(84 citation statements)

References 38 publications

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“…1B). The structure of RelA-Syn ends with α16 (which has no equivalent in SAS1) and proceeds into the as yet structurally unresolved C-terminal part of RelA that is involved in ribosome binding (21).…”

Section: Resultsmentioning

confidence: 99%

“…1A). Best understood is the RelA protein, which senses amino acid starvation during the stringent response by detecting ribosomes blocked by cognate, uncharged tRNAs at the A-site (20,21). RelA's synthetase activity is stimulated upon binding to stalled ribosomes, and alarmone synthesis decreases the affinity of this binding.…”

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confidence: 99%

“…RelA's synthetase activity is stimulated upon binding to stalled ribosomes, and alarmone synthesis decreases the affinity of this binding. Besides a 40-kDa C-terminal domain that is involved in ribosome binding (21), RelA from Bacillus subtilis also contains an N-terminal hydrolase domain that recycles (p)ppGpp by removing the 3′-diphosphate moiety to generate GDP or GTP (Fig. 1A).…”

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confidence: 99%

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Catalytic mechanism and allosteric regulation of an oligomeric (p)ppGpp synthetase by an alarmone

Steinchen

Schuhmacher

Altegoer

et al. 2015

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

109

172

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Nucleotide-based second messengers serve in the response of living organisms to environmental changes. In bacteria and plant chloroplasts, guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp) [collectively named "(p)ppGpp"] act as alarmones that globally reprogram cellular physiology during various stress conditions. Enzymes of the RelA/SpoT homology (RSH) family synthesize (p)ppGpp by transferring pyrophosphate from ATP to GDP or GTP. Little is known about the catalytic mechanism and regulation of alarmone synthesis. It also is unclear whether ppGpp and pppGpp execute different functions. Here, we unravel the mechanism and allosteric regulation of the highly cooperative alarmone synthetase small alarmone synthetase 1 (SAS1) from Bacillus subtilis. We determine that the catalytic pathway of (p)ppGpp synthesis involves a sequentially ordered substrate binding, activation of ATP in a strained conformation, and transfer of pyrophosphate through a nucleophilic substitution (S N 2) reaction. We show that pppGpp-but not ppGpp-positively regulates SAS1 at an allosteric site. Although the physiological significance remains to be elucidated, we establish the structural and mechanistic basis for a biological activity in which ppGpp and pppGpp execute different functional roles. stringent response | (p)ppGpp | hydrogen-deuterium exchange mass spectrometry | alarmone | crystallography T he ability of living organisms to adapt their metabolism to nutrient limitation or environmental changes is of utmost importance to survival. The stringent response is a highly conserved mechanism that enables bacteria (1-3) and plant chloroplasts (4-6) to respond to nutrient (i.e., amino acid) limitations. However, recent work has indicated that guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp) [collectively named "(p)ppGpp"] also impact other, nonstringent response processes such as virulence (7-9) as well as persister (10, 11) and biofilm formation (12). Realization of the importance of (p)ppGpp has also opened new avenues for the design of antimicrobial agents (13,14).Central to these processes is the synthesis of two alarmones, pppGpp and ppGpp, which globally reprogram the transcription and translation associated with a variety of cellular processes (summarized in refs. 9 and 15) and which also control the elongation of DNA replication (16). Until now, both alarmones have been collectively named "(p)ppGpp," because knowledge of their individual roles remained mysterious. Only recently has a study indicated that either alarmone might execute disparate biological functions (17).Alarmone synthesis is carried out by synthetases of RelA/SpoT homology (RSH) (18) that catalyze the transfer of pyrophosphate (β-, γ-phosphates) from ATP to the ribose 3′-OH of GDP or GTP to synthesize ppGpp or pppGpp, respectively. An in-depth analysis of the catalytic mechanism for this reaction is currently not available. Only one structure describes the GDP-bound state of an RSH synthetase domain, bearing remarkable simil...

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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

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Catalytic mechanism and allosteric regulation of an oligomeric (p)ppGpp synthetase by an alarmone

Steinchen

Schuhmacher

Altegoer

et al. 2015

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

109

172

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“…Bacterial SR, as a cellular adaptation and survival system, is activated in response to a number of stresses, including amino acid starvation (51,52), fatty acid starvation (53), carbon source starvation (54), phosphorous limitation (55,56), iron limitation (57), hyperosmotic shock (58), oxidative stress (59), and diauxic shifts (60,61). Additionally, bacterial SR has been shown to be important to the persistent survival (62,63) and virulence (64,65) of bacterial pathogens.…”

Section: Discussionmentioning

confidence: 99%

(p)ppGpp, a Small Nucleotide Regulator, Directs the Metabolic Fate of Glucose in Vibrio cholerae

Lee

Bari

et al. 2015

Journal of Biological Chemistry

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Background: Bacteria respond to nutrient starvation by (p)ppGpp that activates the stringent response. Results: V. cholerae mutants defective in (p)ppGpp production lost their viability during glucose-supplemented growth because of overproduction of organic acids. Conclusion: (p)ppGpp regulates energy metabolism, contributing to the successful proliferation of V. cholerae under glucoserich environments. Significance: We report a previously unexplored role of (p)ppGpp in V. cholerae glucose metabolism.

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“…The amount of (p)ppGpp made following amino acid starvation is much higher than the amount of ribosome-bound RelA protein in the cell, indicating multiple rounds of catalysis by each RelA molecule. Based on in vitro and in vivo studies, some mechanistic models have been proposed for how this could be achieved (10)(11)(12)(13). SpoT-dependent (p)ppGpp synthesis has been documented in response to deprivations such as iron, phosphorus, fatty acid, and carbon sources (14)(15)(16)(17)(18)(19).…”

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Inactivation of Cell Division Protein FtsZ by SulA Makes Lon Indispensable for the Viability of a ppGpp ⁰ Strain of Escherichia coli

Nazir

Harinarayanan

2016

J Bacteriol

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The modified nucleotides (p)ppGpp play an important role in bacterial physiology. While the accumulation of the nucleotides is vital for adaptation to various kinds of stress, changes in the basal level modulates growth rate and vice versa. Studying the phenotypes unique to the strain lacking (p)ppGpp (ppGpp 0 ) under overtly unstressed growth conditions may be useful to understand functions regulated by basal levels of (p)ppGpp and its physiological significance. In this study, we show that the ppGpp 0 strain, unlike the wild type, requires the Lon protease for cell division and viability in LB. Our results indicate the decrease in FtsZ concentration in the ppGpp 0 strain makes cell division vulnerable to SulA inhibition. We did not find evidence for SOS induction contributing to the cell division defect in the ppGpp 0 ⌬lon strain. Based on the results, we propose that basal levels of (p)ppGpp are required to sustain normal cell division in Escherichia coli during growth in rich medium and that the basal SulA level set by Lon protease is important for insulating cell division against a decrease in FtsZ concentration and conditions that can increase the susceptibility of FtsZ to SulA. IMPORTANCEThe physiology of the stringent response has been the subject of investigation for more than 4 decades, with the majority of the work carried out using the bacterial model organism Escherichia coli. These studies have revealed that the accumulation of (p)ppGpp, the effector of the stringent response, is associated with growth retardation and changes in gene expression that vary with the intracellular concentration of (p)ppGpp. By studying a synthetic lethal phenotype, we have uncovered a function modulated by the basal levels of (p)ppGpp and studied its physiological significance. Our results show that (p)ppGpp and Lon protease contribute to the robustness of the cell division machinery in E. coli during growth in rich medium.A daptation in response to stress is a universal trait among living organisms. In eubacteria and plants, two modified nucleotides, namely, guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp), collectively referred to as (p)ppGpp, have been implicated in adaptation to various nutritional and environmental stresses (1-5). By far the best-studied stress response mediated by (p)ppGpp is the stringent response in the bacterial model organism Escherichia coli, and it is characterized by the repression of rRNA transcription and the activation of amino acid biosynthetic genes. This adaptation requires the RelA-mediated synthesis of (p)ppGpp in response to amino acid starvation and results in the extensive reprogramming of gene expression globally (6, 7). More commonly, the stringent response also refers to responses caused by elevating (p)ppGpp by other means. In E. coli, relA and spoT genes are involved in (p)ppGpp metabolism and are part of the RSH (Rel-Spo homolog) superfamily of genes conserved in eubacteria and plants (3,8). (p)ppGpp is synthesized in response to amino acid st...

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The ribosome triggers the stringent response by RelA via a highly distorted tRNA

Cited by 51 publications

References 38 publications

Catalytic mechanism and allosteric regulation of an oligomeric (p)ppGpp synthetase by an alarmone

Catalytic mechanism and allosteric regulation of an oligomeric (p)ppGpp synthetase by an alarmone

(p)ppGpp, a Small Nucleotide Regulator, Directs the Metabolic Fate of Glucose in Vibrio cholerae

Inactivation of Cell Division Protein FtsZ by SulA Makes Lon Indispensable for the Viability of a ppGpp ⁰ Strain of Escherichia coli

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The ribosome triggers the stringent response by RelA via a highly distorted tRNA

Cited by 51 publications

References 38 publications

Catalytic mechanism and allosteric regulation of an oligomeric (p)ppGpp synthetase by an alarmone

Catalytic mechanism and allosteric regulation of an oligomeric (p)ppGpp synthetase by an alarmone

(p)ppGpp, a Small Nucleotide Regulator, Directs the Metabolic Fate of Glucose in Vibrio cholerae

Inactivation of Cell Division Protein FtsZ by SulA Makes Lon Indispensable for the Viability of a ppGpp 0 Strain of Escherichia coli

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Inactivation of Cell Division Protein FtsZ by SulA Makes Lon Indispensable for the Viability of a ppGpp ⁰ Strain of Escherichia coli