Dissection of the Mechanism for the Stringent Factor RelA

112

172

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...

mentioning

confidence: 99%

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

Steinchen

Schuhmacher

Altegoer

et al. 2015

112

172

“…Using overexpressed Escherichia coli RelA, the most extensive in vitro analysis to date of the mechanism was undertaken in 2002 (10), arriving at the so-called hopping model. In this model, RelA binds to a stalled ribosome, senses the deacylated tRNA in the ribosome A site, becomes catalytically active, and synthesizes one ppGpp molecule.…”

mentioning

confidence: 99%

“…At the very same time, in vitro biochemical methods, however powerful when applied to other ribosome-associated enzymes, have so far failed to characterize these events as well. The original hopping model (10) was inferred within a very general conceptual framework that encompasses a broad range of ribosome-interacting enzymes, be it translational GTPases (11), ribosome-inactivating toxins (12), or ribosome RNA modification enzymes (13). Direct experimental evidence for hopping, for the synthesis of one ppGpp alarmone per one dissociation event, and also for the reaction being performed on the ribosome, is lacking (10).…”

mentioning

confidence: 99%

Single-molecule investigations of the stringent response machinery in living bacterial cells

English

Hauryliuk

Sanamrad

et al. 2011

262

294

The RelA-mediated stringent response is at the heart of bacterial adaptation to starvation and stress, playing a major role in the bacterial cell cycle and virulence. RelA integrates several environmental cues and synthesizes the alarmone ppGpp, which globally reprograms transcription, translation, and replication. We have developed and implemented novel single-molecule tracking methodology to characterize the intracellular catalytic cycle of RelA. Our single-molecule experiments show that RelA is on the ribosome under nonstarved conditions and that the individual enzyme molecule stays off the ribosome for an extended period of time after activation. This suggests that the catalytically active part of the RelA cycle is performed off, rather than on, the ribosome, and that rebinding to the ribosome is not necessary to trigger each ppGpp synthesis event. Furthermore, we find fast activation of RelA in response to heat stress followed by RelA rapidly being reset to its inactive state, which makes the system sensitive to new environmental cues and hints at an underlying excitable response mechanism. cytosolic diffusion | single particle tracking | photoactivated localization microscopy | stroboscopic illumination

“…2) and diauxie is abolished in both the wild-type and mutant strains when amino acids are present (data not shown). The possibility that RelA, along with SpoT, can sense carbon starvation independently of amino acid pool fluctuations seems unlikely given the strong evidence for physical association of RelA with the ribosome, which allows it to monitor translational pausing and hence the amino acyl-tRNA pool (38). Thus, the model shown in Fig.…”

mentioning

confidence: 99%

Guanosine 3′,5′-bispyrophosphate coordinates global gene expression during glucose-lactose diauxie in Escherichia coli

Traxler

Chang

Conway

2006

105

128

Guanosine 3 ,5 -bispyrophosphate (ppGpp), also known as ''magic spot,'' has been shown to bind prokaryotic RNA polymerase to down-regulate ribosome production and increase transcription of amino acid biosynthesis genes during the stringent response to amino acid starvation. Because many environmental growth perturbations cause ppGpp to accumulate, we hypothesize ppGpp to have an overarching role in regulating the genetic program that coordinates transitions between logarithmic growth (feast) and growth arrest (famine). We used the classic glucose-lactose diauxie as an experimental system to investigate the temporal changes in transcription that accompany growth arrest and recovery in wildtype Escherichia coli and in mutants that lack RelA (ppGpp synthetase) and other global regulators, i.e., RpoS and Crp. In particular, diauxie was delayed in the relA mutant and was accompanied by a 15% decrease in the number of carbon sources used and a 3-fold overall decrease in the induction of RpoS and Crp regulon genes. Thus the data significantly expand the previously known role of ppGpp and support a model wherein the ppGpp-dependent redistribution of RNA polymerase across the genome is the driving force behind control of the stringent response, general stress response, and starvation-induced carbon scavenging. Our conceptual model of diauxie describes these global control circuits as dynamic, interconnected, and dependent upon ppGpp for the efficient temporal coordination of gene expression that programs the cell for transitions between feast and famine.catabolite repression ͉ stringent response ͉ conceptual model T he fitness of free-living organisms depends on their ability to withstand environmental insults and grow as rapidly as possible when conditions allow. Consequently, the coordination of growth control processes constitutes a fundamental level of regulation in prokaryotes. For this reason, the bacterial existence is often thought to be one of ''feast and famine'' (1). In the laboratory, nutritional conditions that cause biphasic growth provide a unique opportunity to investigate this most basic of bacterial behaviors. When cultured on a mixture of glucose and lactose, Escherichia coli grows preferentially on glucose until the glucose is exhausted, resulting in growth arrest while the cells adjust to growth on lactose, i.e., diauxie. The genetic basis for biphasic sugar catabolism, elucidated by Jacob and Monod (2), is exemplified by lac operon induction, which is a textbook paradigm for illustrating genetic control. However, transcriptome analysis revealed that diauxie involves much more than induction of the lac operon, and that diauxie is accompanied by a global response to growth arrest that apparently ensures recovery when conditions allow growth to resume (3). The purpose of this study is to dissect the regulatory networks that govern diauxie as a means for understanding how the cell integrates the response to growth arrest.We showed previously (3) that during steady-state logarithmic growth, gene expressi...