Regulation of (p)ppGpp hydrolysis by a conserved archetypal regulatory domain

Ronneau, Séverin; Caballero-Montes, Julien; Coppine, Jérôme; Mayard, Aurélie; García-Pino, Abel; Hallez, Régis

doi:10.1093/nar/gky1201

Cited by 46 publications

(35 citation statements)

References 38 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Progressive deletion of both RRM and ZFD leads to induction of the hydrolysis activity ( Figure 4F ), while the synthesis activity is compromised ( Figure 4C ). Reduction of Rel to an NTD fragment lacking the regulatory CTD near-completely abrogates the hydrolysis activity, in good agreement with our microbiological (31) and ITC experiments ( Figure 5 ), as well as biochemical studies of S. equisimilis (15) and C. crescentus (19) Rel enzymes. The inhibitory effect of tRNA Val is lost only when Rel is reduced to its hydrolytically near-inactive NTD.…”

Section: Resultssupporting

confidence: 87%

“…The activation by initiation complexes was compromised, but not abrogated, and the synthetic activity of the Rel ΔRRM alone was weaker than that of the wild type. These results are seemingly at odds with mis-regulation and over-production of (p)ppGpp by both Rel ΔRRM (19) and RelA ΔRRM (32) in live cells, which could be attributed to the loss of autoinhibition in the mutant protein (15,18,44). This interpretation is not supported by our biochemical results, and follow-up experiments established that the toxicity of both B. subtilis Rel ΔRRM and E. coli RelA ΔRRM is strictly dependent on the interaction with starved ribosomes (31).…”

Section: Resultsmentioning

confidence: 98%

“…Experiments with Rel from Streptococcus dysgalactiae subsp. equisimilis (15,16), Mycobacterium tuberculosis (17,18), Caulobacter crescentus (19), Staphylococcus aureus (20) and Thermus thermophilus (21) have established that, first, the synthetic and hydrolytic activities of the NTD are mutually exclusive and, second, the CTD is essential for regulation by the ribosome.…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Ribosome association primes the stringent factor Rel for recruitment of deacylated tRNA to ribosomal A-site

Takada

Roghanian

Caballero-Montes

et al. 2020

Preprint

Self Cite

View full text Add to dashboard Cite

20 Hiraku Takada: hiraku.takada@umu.se, +46 761 023 344 21 Abel Garcia-Pino: agarciap@ulb.ac.be, +32 2 650 53 77 22 Vasili Hauryliuk: vasili.hauryliuk@umu.se, +46 907 850 807 23 24 ABSTRACT: 25In the Gram-positive Firmicute bacterium Bacillus subtilis, amino acid starvation induces synthesis of 26 the alarmone (p)ppGpp by the multi-domain RelA/SpoT Homolog factor Rel. This bifunctional enzyme 27 is capable of both synthesizing and hydrolysing (p)ppGpp. To detect amino acid deficiency, Rel monitors 28 the aminoacylation status of the ribosomal A-site tRNA by directly inspecting the tRNA's CCA end. Here 29 we uncover the molecular mechanism of Rel-mediated stringent response. Off the ribosome, Rel 30 assumes a 'closed' conformation which has predominantly (p)ppGpp hydrolysis activity. This state does 31 not specifically inspect tRNA and the interaction is only moderately affected by tRNA aminoacylation. 32Once bound to the vacant ribosomal A-site, Rel assumes an 'open' conformation, which primes its TGS 33 and Helical domains for specific recognition and recruitment of cognate deacylated tRNA to the 34 ribosome. The tRNA locks Rel on the ribosome in a hyperactivated state that processively synthesises 35 (p)ppGpp while the hydrolysis is suppressed. In stark contrast to non-specific tRNA interactions off the 36 ribosome, tRNA-dependent Rel locking on the ribosome and activation of (p)ppGpp synthesis are highly 37 specific and completely abrogated by tRNA aminoacylation. Binding pppGpp to a dedicated allosteric 38 site located in the N-terminal catalytic domain region of the enzyme further enhances its synthetase 39 activity.40 42 referred to as (p)ppGpp, regulate metabolism, virulence, stress responses and antibiotic tolerance in 43 the vast majority of bacterial species (for reviews see (1-4)). The cellular levels of (p)ppGpp are 44 controlled by members of the RelA/SpoT Homolog (RSH) protein family (5). These enzymes both 45 synthesise (p)ppGpp by transferring the pyrophosphate group of ATP onto the 3' position of either GDP 46 or GTP, and degrade the alarmone by hydrolysing the nucleotide back to GDP (or GTP), releasing 47 inorganic pyrophosphate in the process. 48RSH factors fall into two categories: 'small' single domain RSHs and 'long' multi-domain RSHs (5). In 49 the majority of bacterial species, long RSHs are represented by a single ribosome-associated 50 bifunctional enzyme called Rel that is capable of both synthesizing and degrading (p)ppGpp. In the 51 lineage to Beta-and Gammaproteobacteria, duplication and functional divergence of the ancestral 52 ribosome-associated Rel gave rise to a pair of specialized factors -the namesakes of the RSH protein 53 family -RelA and SpoT (5,6). RelA is a synthesis-only enzyme incapable of hydrolysing (p)ppGpp (7), 54while SpoT and Rel are bifunctional (8,9). The (p)ppGpp synthesis activity of RelA and Rel is induced 55 by so-called 'starved' ribosomal complexes (i.e. ribosomes accommodating deacylated tRNA in the A-56 site) (8,10). The association of SpoT with the ribo...

show abstract

Section: Resultssupporting

confidence: 87%

Section: Resultsmentioning

confidence: 98%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Ribosome association primes the stringent factor Rel for recruitment of deacylated tRNA to ribosomal A-site

Takada

Roghanian

Caballero-Montes

et al. 2020

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…In alpha-proteobacteria, the PTS mediates the transfer of a phosphoryl group from phosphoenolpyruvate to stimulate the accumulation of the alarmone guanosine (penta) tetra-phosphate commonly referred to as (p)ppGpp. The phosphoryltransfer proceeds via EI Ntr /PtsP→HPr→EII culminating in the regulation of the bifunctional (p)ppGpp synthase/hydrolase SpoT 14, 15 (Fig. 3A).…”

Section: Resultsmentioning

confidence: 99%

Bypass of an epigenetic S-phase transcriptional module by convergent nutrient stress signals

Delaby

Panis

Fumeaux

et al. 2019

Preprint

View full text Add to dashboard Cite

14The signals feeding into bacterial S-phase transcription are poorly understood. Cellular cycling 15 in the alpha-proteobacterium Caulobacter crescentus is driven by a complex circuit of at least 16 three transcriptional modules that direct sequential promoter firing during the G1, early and late 17 S cell cycle phases. In alpha-proteobacteria, the transcriptional regulator GcrA and the CcrM 18 methyltransferase epigenetically activate promoters of cell division and polarity genes that fire 19 in S-phase. By evolving Caulobacter crescentus cells to cycle and differentiate in the absence 20 of the GcrA/CcrM module, we discovered that phosphate deprivation and (p)ppGpp alarmone 21 stress signals converge on S-phase transcriptional activation. The cell cycle oscillations of the 22CtrA protein, the transcriptional regulator that implements G1 and late S-phase transcription, 23 are essential in our evolved mutants, but not in wild-type cells, showing that the periodicity in 24CtrA abundance alone can sustain cellular cycling without GcrA/CcrM. While similar 25 nutritional sensing occurs in other alpha-proteobacteria, GcrA and CcrM are not encoded in the 26 reduced genomes of obligate intracellular relatives. We thus propose that the nutritional stress 27 response induced during intracellular growth obviated the need for an S-phase transcriptional 28 regulator. 29 to a subclass of σ 70 -dependent promoters 6,7 . Since binding of GcrA to such a target promoter 64 had been recapitulated in vitro without σ 70 and RNAP, GcrA can bind DNA specifically. The 65 basis for the promoter selectivity was shown to reside in the epigenetic (methylation) mark in 66 the context of the 5'-GANTC-3' sequence found in many, but not all, σ 70 -dependent promoters. 67The adenine (N6) methylation mark is introduced by the CcrM adenine methyltransferase 7,8 . 68Preferential promoter binding of GcrA is no longer observed 6,7 in the absence of CcrM. Since 69 preferential binding of methylated target promoters by GcrA is not commensurate with general 70 σ 70 RNAP promoter associations, the σ 70 RNAP interaction likely serves to recruit GcrA to 71 σ 70 -dependent promoters. This would allow a firm association of GcrA at promoters having a 72 CcrM-dependent methylation mark to stabilize σ 70 RNAP at the promoter and induce promoter 73 firing. 74 Loss of GcrA and/or CcrM lead to similar severe pleiotropic defects, rendering cells 75 non-motile, unsynchronizable (because of a defect in capsule control), strongly elongated with 76 multiple chromosomes 7,9 . Selection of efficiently dividing ΔgcrA cells by transposon (Tn) 77 mutagenesis had yielded promoter-up mutations in the GcrA-target promoter of the ftsN cell 78 division gene conferred by an outward directed promoter within the himar1 Tn (PftsN::Tn). 79 Although PftsN::Tn directs FtsN expression independently of GcrA 7 and ameliorates division, 80 other defects of ΔgcrA PftsN::Tn cells are not corrected, indicating that cellular cycling is not 81 restored. Thus, the question whether a proper ...

show abstract

“…The Sa-RelQ (SAS1), Sa-RelP (SAS2), full-length Sa-Rel protein, and N-terminal domain of Sa-Rel (Sa-Rel trunc , residues 9392), were expressed and purified in a soluble form. The Sa-Rel trunc protein encodes the SYNTH and HD domains that are respectively responsible for the synthesis and hydrolysis of alarmones; but lacks the putative regulatory domains found in the C-terminal region (Fig 1) [16,26,27,31,[47][48][49][50]. Its composition is analogous to the previously-characterized truncated form of the Rel protein from Streptococcus dysgalactiae subsp.…”

Section: Multimeric Arrangements Of the Sa-rel Sa-rel Trunc Sa-relq mentioning

confidence: 99%

The Ps and Qs of alarmone synthesis inStaphylococcus aureus

Yang

Xie

Choi

et al. 2019

Preprint

View full text Add to dashboard Cite

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...

show abstract

Regulation of (p)ppGpp hydrolysis by a conserved archetypal regulatory domain

Cited by 46 publications

References 38 publications

Ribosome association primes the stringent factor Rel for recruitment of deacylated tRNA to ribosomal A-site

Ribosome association primes the stringent factor Rel for recruitment of deacylated tRNA to ribosomal A-site

Bypass of an epigenetic S-phase transcriptional module by convergent nutrient stress signals

The Ps and Qs of alarmone synthesis inStaphylococcus aureus

Contact Info

Product

Resources

About