Differential regulation by ppGpp versus pppGpp in Escherichia coli

Mechold, Undine; Potrykus, Katarzyna; Murphy, Helen; Murakami, Katsuhiko; Cashel, Michael

doi:10.1093/nar/gkt302

Cited by 184 publications

(241 citation statements)

References 65 publications

(72 reference statements)

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“…The ω subunit was also shown to participate in the binding of ppGpp, an alarmone that plays a key role in the stringent response in bacteria. The binding of ppGpp was demonstrated to affect the active site conformation through changes in the relative positions and mobility of the core and shelf/clamp RNAP modules, resulting in destabilization of promoter complexes and, probably, modification of the elongation properties of RNAP (28)(29)(30). Importantly, ppGpp stimulates intrinsic transcription termination and transcription pausing (refs.…”

Section: Discussionmentioning

confidence: 99%

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Distinct pathways of RNA polymerase regulation by a phage-encoded factor

Esyunina

Klimuk

Severinov

et al. 2015

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

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Transcription antitermination is a common strategy of gene expression regulation, but only a few transcription antitermination factors have been studied in detail. Here, we dissect the transcription antitermination mechanism of Xanthomonas oryzae virus Xp10 protein p7, which binds host RNA polymerase (RNAP) and regulates both transcription initiation and termination. We show that p7 suppresses intrinsic termination by decreasing RNAP pausing and increasing the transcription complex stability, in cooperation with host-encoded factor NusA. Uniquely, the antitermination activity of p7 depends on the ω subunit of the RNAP core and is modulated by ppGpp. In contrast, the inhibition of transcription initiation by p7 does not require ω but depends on other RNAP sites. Our results suggest that p7, a bifunctional transcription factor, uses distinct mechanisms to control different steps of transcription. We propose that regulatory functions of the ω subunit revealed by our analysis may extend to its homologs in eukaryotic RNAPs.RNA polymerase | transcription antitermination | transcription pausing | omega subunit | NusA T ranscription promoters and terminators are the main "punctuation marks" that control the expression of individual genes in the genome. In bacteria, transcription termination is an essential regulatory step that determines the relative levels of expression of promoter-proximal and distal genes within operons. Depending on the factors involved, transcription termination in bacteria can be classified as intrinsic (depends on RNAencoded signals) or factor-dependent (requires additional protein factors such as Rho helicase). Intrinsic terminators consist of a G/C-rich hairpin formed in the nascent RNA followed by a downstream oligo(U) tract. During intrinsic termination, RNA polymerase (RNAP) pauses after synthesizing an oligo(U) sequence, accompanied by hairpin formation, leading to subsequent dissociation of the transcription elongation complex (TEC) (1, 2). Despite the relatively simple overall scenario, the exact mechanisms of pausing and hairpin-induced TEC destabilization remain an issue of debate (1, 3).Transcription antitermination is a widespread phenomenon involved in regulation of bacterial and phage operons (2). Transcription antitermination is often mediated by specialized protein factors that target host RNAP and can suppress termination at multiple sites in the operon. The well-studied examples of phage antiterminators include proteins N and Q of Escherichia coli phage λ and the gp39 protein of Thermus thermophilus phage P23-45. These factors suppress RNAP pausing, thus inhibiting the first step of the termination pathway (4-6). The antitermination activity of N and Q, but not gp39, is enhanced by cell-encoded proteins, including transcription elongation factor NusA, which by itself stimulates termination but reverses its activity to additionally stabilize the TEC in cooperation with N and Q (5, 6). Curiously, the N, Q, gp39, and NusA proteins all target the flexible β flap domain of RNAP (2, 4...

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Section: Discussionmentioning

confidence: 99%

“…The ω subunit is gray; the β′ ZBD is black, with amino acid residues 70-88 deleted in this work shown in yellow; and the β flap (residues 885-921) is red. The position of ppGpp (turquoise) is superimposed from the E. coli RNAP holoenzyme ppGpp structure (4JK1) (28). NusA is schematically shown as a yellow oval.…”

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confidence: 99%

Distinct pathways of RNA polymerase regulation by a phage-encoded factor

Esyunina

Klimuk

Severinov

et al. 2015

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

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“…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).…”

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confidence: 99%

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.

108

168

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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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“…In contrast to classical transcription factors that bind at or near promoters, DksA and ppGpp regulate transcription by interacting with RNA polymerase (RNAP) without contacting DNA. Specifically, it has been shown in crystals of the E. coli RNAP holoenzyme that ppGpp binds in the interface between the ␤Ј and subunits of RNAP (13)(14)(15), whereas DksA has been structurally positioned in the RNAP secondary channel, Ն30 Å from the ppGpp-binding site (16,17). The mechanism by which ppGpp and DksA affect transcription is not entirely clear, but they are suggested to repress transcriptional output by affecting different kinetic steps on the pathway to open complex formation (18).…”

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confidence: 99%

A Proximal Promoter Element Required for Positive Transcriptional Control by Guanosine Tetraphosphate and DksA Protein during the Stringent Response

Gummesson¹,

Lovmar²,

Nyström

2013

Journal of Biological Chemistry

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Background: ppGpp/DksA regulate genes both positively and negatively during stringency. Results: Positive control by ppGpp/DksA is linked to RNA polymerase-promoter saturation kinetics dependent on the promoter's discriminator. Conclusion: Promoters harboring discriminators that make them easily saturated with RNA polymerase are controlled positively by ppGpp/DksA. Significance: The discriminator is a cis-acting element dictating the response of the promoter to alterations in ppGpp/DksA and RNA polymerase availability.

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Differential regulation by ppGpp versus pppGpp in Escherichia coli

Cited by 184 publications

References 65 publications

Distinct pathways of RNA polymerase regulation by a phage-encoded factor

Distinct pathways of RNA polymerase regulation by a phage-encoded factor

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

A Proximal Promoter Element Required for Positive Transcriptional Control by Guanosine Tetraphosphate and DksA Protein during the Stringent Response

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