The C-terminal RRM/ACT domain is crucial for fine-tuning the activation of ‘long’ RelA-SpoT Homolog enzymes by ribosomal complexes

Takada, Hiraku; Roghanian, Mohammad; Murina, Victoriia; Dzhygyr, Ievgen; Murayama, Rikinori; Akanuma, Genki; Atkinson, Gemma C.; García-Pino, Abel; Hauryliuk, Vasili

doi:10.1101/849810

Cited by 19 publications

(43 citation statements)

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“…Taken together, these results suggest that in the absence of ribosomes, complex formation between Rel and tRNA is dominated by non-specific interactions mediated by the protein’s NTD region. This result is clearly at odds with the highly specific recognition of tRNA by the TGS domain in the cellular context as well as in in the presence of starved ribosomal complexes, both of which are abrogated by the H420E substitution (31). To deconvolute RNA binding by the NTD from that mediated by CTD, we next characterised the isolated B. subtilis Rel TGS-Helical region (amino acid positions 374-583) as well as the TGS domain alone (amino acid positions 374-469).…”

Section: Resultsmentioning

confidence: 99%

“…The corresponding histidine residue in E. coli RelA (H432) forms a stacking interaction with the 3’ CCA end of the uncharged A-site tRNA (27). The H432E substitution abrogates RelA’s functionality by abolishing RelA activation by deacylated tRNA (25,31). We have shown a similar loss-of-function effect of the H420E substitution in B. subtilis Rel (31).…”

Section: Resultsmentioning

confidence: 99%

“…The H432E substitution abrogates RelA’s functionality by abolishing RelA activation by deacylated tRNA (25,31). We have shown a similar loss-of-function effect of the H420E substitution in B. subtilis Rel (31). Therefore, we concluded that the H420E substitution is an excellent tool to probe the specificity of Rel’s interaction with deacylated tRNA.…”

Section: Resultsmentioning

confidence: 99%

“…All bacterial strains and plasmids used in this study are listed in Supplementary Tables 1 and 2 . All proteins were expressed, purified and characterised using SUMO-tagging strategy described earlier for E. coli RelA (32) and B. subtilis Rel (31); gel filtration profiles for mutant Rel variants are shown as ( Supplementary Figure S1 ). Radiochemistry experiments were performed as described for E. coli RelA (13), with modifications.…”

Section: Methodsmentioning

confidence: 99%

“…However, mechanistic understanding of B. subtilis Rel is lacking. Using our reconstituted biochemical system (31), we dissected the relationship between, on the one hand, Rel’s association with deacylated tRNA and the ribosome and, on the other hand, (p)ppGpp synthesis and hydrolysis by the enzyme, and probed the roles of individual domains. We establish that the specific recognition of deacylated tRNA takes place after Rel associates with the vacant ribosomal A-site, and the strength of the interaction with deacylated tRNA fine-tunes the stability of the interaction of Rel/RelA enzymes with starved ribosomes.…”

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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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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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Atomic structure of the regulatory TGS domain of Rel protein from Mycobacterium tuberculosis and its interaction with deacylated tRNA

et al. 2021

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The stringent response is critical for the survival of Mycobacterium tuberculosis (Mtb) under nutrient starvation. The mechanism is mediated by a GTP pyrophosphokinase known as Rel, containing N-terminal synthetase and hydrolase domains and C-terminal regulatory domains, which include the TGS domain (ThrRS, GTPase, and SpoT proteins) that has been proposed to activate the synthetase domain via interaction with deacylated tRNA. Here, we present the NMR solution structure of the Mtb Rel TGS domain (MtRel TGS), consisting of five antiparallel b-strands and one helix-loop-helix motif. The interaction of MtRel TGS with deacylated tRNA is shown, indicating the critical amino acids of MtRel TGS in tRNA binding, and presenting the first structural evidence of MtRel TGS binding to deacylated tRNA in solution in the absence of the translational machinery.

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Structural Basis for Regulation of the Opposing (p)ppGpp Synthetase and Hydrolase within the Stringent Response Orchestrator Rel

et al. 2020

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The C-terminal RRM/ACT domain is crucial for fine-tuning the activation of ‘long’ RelA-SpoT Homolog enzymes by ribosomal complexes

Abstract: 21

Cited by 19 publications

References 58 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

Atomic structure of the regulatory TGS domain of Rel protein from Mycobacterium tuberculosis and its interaction with deacylated tRNA

Structural Basis for Regulation of the Opposing (p)ppGpp Synthetase and Hydrolase within the Stringent Response Orchestrator Rel

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