ppGpp Binding to a Site at the RNAP-DksA Interface Accounts for Its Dramatic Effects on Transcription Initiation during the Stringent Response

Ross, Wilma; Sanchez-Vazquez, Patricia; Chen, Albert Y.; Lee, Jeong‐Hyun; Burgos, Hector L.; Gourse, Richard L.

doi:10.1016/j.molcel.2016.04.029

Cited by 224 publications

(331 citation statements)

References 40 publications

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“…Nutrient limitation triggers alterations in the activity and promoter preferences of RNA polymerase, and major changes in the cell's gene expression program prime the cell for maintenance and survival rather than growth. 58 Several starvation-regulated factors can directly influence transcription by RNA polymerase, including the alarmone ppGpp (binding at the β′-ω and the β′-DksA interfaces of RNA polymerase), 59,60 the small RNA 6S (binding the σ 70 -RNA polymerase holoenzyme) 61,62 and a number of protein factors (DksA, 63 alternative sigma factors, 64 and anti-sigma factors 65 ) (reviewed in ref. 66).…”

Section: Why Degrade Trna?mentioning

confidence: 99%

Transfer RNA instability as a stress response inEscherichia coli: Rapid dynamics of the tRNA pool as a function of demand

2018

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Production of the translation apparatus of E. coli is carefully matched to the demand for protein synthesis posed by a given growth condition. For example, the fraction of RNA polymerases that transcribe rRNA and tRNA drops from 80% during rapid growth to 24% within minutes of a sudden amino acid starvation. We recently reported in Nucleic Acids Research that the tRNA pool is more dynamically regulated than previously thought. In addition to the regulation at the level of synthesis, we found that tRNAs are subject to demand-based regulation at the level of their degradation. In this point-of-view article we address the question of why this phenomenon has not previously been described. We also present data that expands on the mechanism of tRNA degradation, and we discuss the possible implications of tRNA instability for the ability of E. coli to cope with stresses that affect the translation process.

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Section: Why Degrade Trna?mentioning

confidence: 99%

Transfer RNA instability as a stress response inEscherichia coli: Rapid dynamics of the tRNA pool as a function of demand

2018

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“…The number of publications showing a role for (p)ppGpp in the survival of bacteria under changing environmental conditions is continuously increasing. Alarmones can dramatically influence transcription during the stringent response in different bacteria (26,27). Recent publications show that ppGpp is robustly, but transiently, induced in response to DNA damage so as to allow efficient nucleotide excision repair in E. coli (28).…”

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confidence: 99%

Absence of ppGpp Leads to Increased Mobilization of Intermediately Accumulated Poly(3-Hydroxybutyrate) in Ralstonia eutropha H16

Juengert

Borisova

Mayer

et al. 2017

Appl Environ Microbiol

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In this study, we constructed a set of Ralstonia eutropha H16 strains with single, double, or triple deletions of the (p)ppGpp synthase/hydrolase (spoT1), (p)ppGpp synthase (spoT2), and/or polyhydroxybutyrate (PHB) depolymerase (phaZa1 or phaZa3) gene, and we determined the impact on the levels of (p)ppGpp and on accumulated PHB. Mutants with deletions of both the spoT1 and spoT2 genes were unable to synthesize detectable amounts of (p)ppGpp and accumulated only minor amounts of PHB, due to PhaZa1-mediated depolymerization of PHB. In contrast, unusually high levels of PHB were found in strains in which the (p)ppGpp concentration was increased by the overexpression of (p)ppGpp synthase (SpoT2) and the absence of (p)ppGpp hydrolase. Determination of (p)ppGpp levels in wild-type R. eutropha under different growth conditions and induction of the stringent response by amino acid analogs showed that the concentrations of (p)ppGpp during the growth phase determine the amount of PHB remaining in later growth phases by influencing the efficiency of the PHB mobilization system in stationary growth. The data reported for a previously constructed ΔspoT2 strain (C. J. Brigham, D. R. Speth, C. Rha, and A. J. Sinskey, Appl Environ Microbiol 78:8033-8044, 2012, https://doi.org/ 10.1128/AEM.01693-12) were identified as due to an experimental error in strain construction, and our results are in contrast to the previous indication that the spoT2 gene product is essential for PHB accumulation in R. eutropha. IMPORTANCE Polyhydroxybutyrate (PHB) is an important intracellular carbon and energy storage compound in many prokaryotes and helps cells survive periods of starvation and other stress conditions. Research activities in several laboratories over the past 3 decades have shown that both PHB synthase and PHB depolymerase are constitutively expressed in most PHB-accumulating bacteria, such as Ralstonia eutropha. This implies that PHB synthase and depolymerase activities must be well regulated in order to avoid a futile cycle of simultaneous PHB synthesis and PHB degradation (mobilization). Previous reports suggested that the stringent response in Rhizobium etli and R. eutropha is involved in the regulation of PHB metabolism. However, the levels of (p)ppGpp and the influence of those levels on PHB accumulation and PHB mobilization have not yet been determined for any PHB-accumulating species. In this study, we optimized a (p)ppGpp extraction procedure and a highperformance liquid chromatography-mass spectrometry (HPLC-MS)-based detection method for the quantification of (p)ppGpp in R. eutropha. This enabled us to study the relationship between the concentrations of (p)ppGpp and the accumulated levels of PHB in the wild type and in several constructed mutant strains. We show that overproduction of the alarmone (p)ppGpp correlated with reduced growth and mas-

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“…rRNA synthesis rates are coordinated with the cell's translational capacity following nutritional shifts such as amino acid starvation. The primary signal molecules responsible for adjusting ribosome synthesis rates to changing nutritional conditions are ppGpp, an "alarmone" whose synthesis is induced by uncharged transfer RNAs (tRNAs) in the ribosomal A site (Paul et al 2004;Potrykus and Cashel 2008;Ross et al 2016), and the concentration of the initial NTP responsible for forming the rRNA transcript (Murray et al 2003).…”

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confidence: 99%

Colocalization of distant chromosomal loci in space in E. coli: a bacterial nucleolus

et al. 2016

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The spatial organization of DNA within the bacterial nucleoid remains unclear. To investigate chromosome organization in Escherichia coli, we examined the relative positions of the ribosomal RNA (rRNA) operons in space. The seven rRNA operons are nearly identical and separated from each other by as much as 180°on the circular genetic map, a distance of ≥2 million base pairs. By inserting binding sites for fluorescent proteins adjacent to the rRNA operons and then examining their positions pairwise in live cells by epifluorescence microscopy, we found that all but rrnC are in close proximity. Colocalization of the rRNA operons required the rrn P1 promoter region but not the rrn P2 promoter or the rRNA structural genes and occurred with and without active transcription. Non-rRNA operon pairs did not colocalize, and the magnitude of their physical separation generally correlated with that of their genetic separation. Our results show that E. coli bacterial chromosome folding in three dimensions is not dictated entirely by genetic position but rather includes functionally related, genetically distant loci that come into close proximity, with rRNA operons forming a structure reminiscent of the eukaryotic nucleolus.

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ppGpp Binding to a Site at the RNAP-DksA Interface Accounts for Its Dramatic Effects on Transcription Initiation during the Stringent Response

Cited by 224 publications

References 40 publications

Transfer RNA instability as a stress response inEscherichia coli: Rapid dynamics of the tRNA pool as a function of demand

Transfer RNA instability as a stress response inEscherichia coli: Rapid dynamics of the tRNA pool as a function of demand

Absence of ppGpp Leads to Increased Mobilization of Intermediately Accumulated Poly(3-Hydroxybutyrate) in Ralstonia eutropha H16

Colocalization of distant chromosomal loci in space in E. coli: a bacterial nucleolus

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