The ribosomal A-site finger is crucial for binding and activation of the stringent factor RelA

Kudrin, Pavel; Dzhygyr, Ievgen; Ishiguro, Kensuke; Beljantseva, Jelena; Maksimova, Elena; Oliveira, Sofia Raquel Alves; Varik, Vallo; Payoe, Roshani; Konevega, Andrey L.; Tenson, Tanel; Suzuki, Tsutomu; Hauryliuk, Vasili

doi:10.1093/nar/gky023

Cited by 48 publications

(84 citation statements)

References 66 publications

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“…We propose that self-amplification of (p)ppGpp is required for spontaneous persister formation from a small input signal, such as gratuitous (p)ppGpp synthesis by YjbM or potentially RelA (Fig S7B). It was shown that RelA activity is also stimulated by pppGpp in E. coli (Kudrin et al, 2018). We hypothesize that this cooperativity may also contribute to spontaneous persister formation.…”

Section: Discussionmentioning

confidence: 99%

A shared alarmone-GTP switch underlies triggered and spontaneous persistence

Fung

Barra

Schroeder

et al. 2020

Preprint

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SummaryPhenotypically-switched, antibiotic-refractory persisters may prevent pathogen eradication. Although how triggered persistence via starvation-induced (p)ppGpp is well characterized, generation of persisters without starvation are poorly understood. Here we visualized the formation of spontaneous persisters in a small fraction of cells from growing wild type bacteria, revealing a striking single cell rapid switch from growth to dormancy. This switch-like entrance is triggered by GTP dropping beneath a threshold due to stochastic production and self-amplification of (p)ppGpp via allosteric enzyme activation. In addition, persisters are induced by lethal and sublethal concentrations of cell wall antibiotics by inducing (p)ppGpp via cell wall stress response. Thus spontaneous, triggered and antibiotic-induced persisters can all stem from a common metabolic switch: GTP depletion by (p)ppGpp induction, and each pathway of persister formation is activated by different (p)ppGpp synthetases. These persistence pathways are likely conserved in pathogens which may be exploited to potentiate antibiotic efficacy.

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Section: Discussionmentioning

confidence: 99%

A shared alarmone-GTP switch underlies triggered and spontaneous persistence

Fung

Barra

Schroeder

et al. 2020

Preprint

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“…The association of SpoT with the ribosome is still a matter of debate (11,12). In the case of RelA, the pppGpp product is a potent inducer of the enzyme’s synthesis activity (13). The mechanistic basis of this regulatory mechanism is currently unexplored, and it is unknown whether bifunctional RSHs Rel and SpoT are similarly regulated by pppGpp.…”

Section: Introductionmentioning

confidence: 99%

“…The former model suggests that upon activation by starved ribosomes RelA processively synthesises (p)ppGpp off the ribosome and the latter states that upon activation, RelA spends prolonged periods of time catalytically active in association with starved ribosomal complexes. Finally, there are two ‘flavours’ of the ‘short hopping time’ model: RelA could first bind the vacant A-site and then recruit the deacylated tRNA (13,14) or form the RelA:tRNA complex off the ribosome and deliver deacylated tRNA to the A-site, similarly to how elongation factor EF-Tu delivers aminoacylated tRNA (25,26). The proposed delivery of deacylated tRNA to the ribosome by RelA/Rel is a controversial topic.…”

Section: Introductionmentioning

confidence: 99%

“…The proposed delivery of deacylated tRNA to the ribosome by RelA/Rel is a controversial topic. While in the case of M. tuberculosis , Rel binding to tRNA is readily detectible (8,17), binding of tRNA to E. coli RelA is not detected by EMSA (13,26), unless the complex is crosslinked by formaldehyde (26). On the ribosome, the TGS and Helical domains of RelA form multiple contacts with deacylated A-site tRNA (14,27,28), but it is unclear if these domains are accessible when RelA/Rel is off the ribosome.…”

Section: Introductionmentioning

confidence: 99%

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

Takada

Roghanian

Caballero-Montes

et al. 2020

Preprint

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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...

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“…However, given that a C-terminal His 6 -tagged protein was used in that 434 study and RelA's C-terminus resides well inside the ribosomal complex, it was likely that the 435 functionality of the protein was compromised. In a recent report using untagged RelA 436 protein, it was shown that pppGpp as an allosteric regulator and GDP as substrate synergize 437 for the maximum enzymatic activity of RelA in vitro(Kudrin et al, 2018). Allosteric activation of Rel protein by pppGpp was not restricted to full-length RelA, it has also been 439 noted for RelQ, an RNA binding small alarmone synthase from Enterococcus faecalis 440(Beljantseva et al, 2017) and as well as the small alarmone synthase, SAS1, from Bacillus 441 subtilis(Steinchen et al, 2015).…”

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confidence: 99%

SpoT and GppA hydrolases prevent the gratuitous activation of RelA by pppGpp inEscherichia coli

Harinarayanan

Sanyal

2018

Preprint

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20Stringent response, a conserved regulation seen in bacteria, is effected through the modified 21 nucleotides (p)ppGpp. The metabolic cycle of these molecules is driven by the synthase 22 activity of RelA and SpoT and the hydrolase activity of SpoT and GppA which together sets 23 the basal (p)ppGpp pool. Growth arrest due to (p)ppGpp accumulation from basal RelA 24 activity apparently explained the essentiality of SpoT hydrolase function. We found, pppGpp 25 degradation was enhanced when the SpoT hydrolase activity was lowered or eliminated and 26 when this was alleviated by inactivation of the GppA hydrolase, gratuitous synthesis of 27 (p)ppGpp by RelA was activated, leading to growth arrest. The RelA-ribosome interaction 28 was not mandatory for these phenotypes. Our results show, for the first time, elevated 29 pppGpp promoted the amplification of RelA-mediated stringent response in the absence of 30 established RelA activating signals in the cell and the SpoT and GppA hydrolases prevented 31 this. The accumulation of pppGpp inhibited the SpoT hydrolase activity. We propose this 32 autocatalytic activation of RelA by pppGpp is likely to be an allosteric regulation and can 33 result in a bistable switch. 34 pyrophosphate moiety from ATP to GDP or GTP respectively (Potrykus and Cashel, 2008; 44

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The ribosomal A-site finger is crucial for binding and activation of the stringent factor RelA

Cited by 48 publications

References 66 publications

A shared alarmone-GTP switch underlies triggered and spontaneous persistence

A shared alarmone-GTP switch underlies triggered and spontaneous persistence

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

SpoT and GppA hydrolases prevent the gratuitous activation of RelA by pppGpp inEscherichia coli

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