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

In the Gram-positive Firmicute bacterium Bacillus subtilis, amino acid starvation induces synthesis of the alarmone (p)ppGpp by the multi-domain RelA/SpoT Homolog factor Rel. This bifunctional enzyme is capable of both synthesizing and hydrolysing (p)ppGpp. To detect amino acid deficiency, Rel monitors the aminoacylation status of the ribosomal A-site tRNA by directly inspecting the tRNA’s CCA end. Here we uncover the molecular mechanism of Rel-mediated stringent response. Off the ribosome, Rel assumes a ‘closed’ conformation which has predominantly (p)ppGpp hydrolysis activity. This state does not specifically inspect tRNA and the interaction is only moderately affected by tRNA aminoacylation. Once bound to the vacant ribosomal A-site, Rel assumes an ‘open’ conformation, which primes its TGS and Helical domains for specific recognition and recruitment of cognate deacylated tRNA to the ribosome. The tRNA locks Rel on the ribosome in a hyperactivated state that processively synthesises (p)ppGpp while the hydrolysis is suppressed. In stark contrast to non-specific tRNA interactions off the ribosome, tRNA-dependent Rel locking on the ribosome and activation of (p)ppGpp synthesis are highly specific and completely abrogated by tRNA aminoacylation. Binding pppGpp to a dedicated allosteric site located in the N-terminal catalytic domain region of the enzyme further enhances its synthetase activity.


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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.

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Once 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.

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Taken together, these results suggest that in the absence of ribosomes, complex formation between

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Rel and tRNA is dominated by non-specific interactions mediated by the protein's NTD region. This

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The isolated TGS-Helical region of Rel specifically recognises the tRNA 3' CCA end

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While we do not detect formation of a stable complex in the case of TGS alone, the TGS-Helical fragment 160 binds tRNA Val with EC50 of 0.8 μM ( Figure 2D). In stark contrast to the full-length protein, this interaction 161 is completely abrogated upon introduction of the H420E mutation, suggesting that for these isolated

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Our results demonstrate that, unlike the full-length protein, the isolated Rel TGS-Helical region is highly 174 specific in its recognition of the tRNA 3' CCA end. To rationalise this result, we hypothesise that the 175 association with the ribosome that drives the transition of Rel from the 'closed' conformation, unable to  Figure S7). The A-site tRNA-dependent Rel recruitment to starved complexes is 186 highly specific and is efficiently abrogated by tRNA aminoacylation.

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We tested the association of Rel with ribosomes in conditions that naturally activate the stringent 188 response, as opposed to acute non-physiological isoleucine starvation caused by mupirocin ( Figure   189 3D). As bacteria enter the stationary phase and nutrients become limiting, the ribosome-associated

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RSHs RelA/Rel are activated and the intracellular concentration of (p)ppGpp increases (35). We

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Recruitment of Rel to ribosomes by deacylated tRNA is not sufficient to induce synthesis activity in the 219 absence of L11

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To dissect the relationship between Rel recruitment to the ribosome and (p)ppGpp synthesis, we used 221 three mutant strains with compromised (p)ppGpp synthesis activity ( Figure 3A). First, we tested the 222 effect of mupirocin on Rel carrying the H420E substitution that abrogated ribosomal recruitment in the 223 reconstituted system. In contrast to wild type Rel, we detect no ribosomal recruitment of H420E Rel.

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Similarly, no tRNA-dependent ribosomal recruitment of H420E Rel was observed with reconstituted 70S 225 initiation complexes (Supplementary Figure S7). Next, we tested Rel with a D264G substitution in the 226 SYNTH domain, which abrogates (p)ppGpp synthesis by the enzyme (38). Surprisingly, D264G Rel 227 does not bind ribosomes regardless of the presence or absence of mupirocin ( Figure 3A).

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To further discriminate between 'hopping' and processive synthesis on the ribosome, we titrated wild 256 type Rel in our reconstituted biochemical system. When the reaction turnover is calculated per starved 257 ribosomal complex (Figure 3C, red trace), the enzymatic activity reaches a plateau when the 258 concentration of Rel is equal to that of the ribosomes (0.5 μM). At higher Rel concentrations the 259 efficiency of (p)ppGpp production in the reconstituted system does not increase -and when turnover is 260 calculated per Rel molecule, it decreases ( Figure 3C, black trace). This behaviour is consistent with Rel 261 processively synthesising (p)ppGpp while associated with starved complexes rather than the enzyme 262 spending prolonged periods off the ribosome in a catalytically active state upon departure from the 263 ribosome. In the latter case one could expect that, acting catalytically, one starved ribosomal complex 264 would fully activate several Rel molecules; this is not the case.

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The

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The synthetase activity of Rel is activated by pppGpp binding to an allosteric site in the NTD region

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While the ribosome stimulates Rel's synthesis activity 5-to 10-fold, and the ultimate activator -the 286 starved complex -has a significantly stronger effect, approximately 50-fold ( Figure 4AB)  RelA NTD with an affinity of 6.9 ± 0.9 μM, and is insensitive to the addition of GDP, AMPCPP and EDTA

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The ribosome and tRNA inhibit (p)ppGpp hydrolysis by Rel

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The synthetic and hydrolytic activities of Rel's NTD are mutually exclusive (15,16,21). This motivated us 349 to directly test the effects of the ligands that induce the synthetic activity (ribosomes, tRNA) on Rel's 350 hydrolysis activity. The hydrolase activity of the enzyme tested alone is strictly Mn 2+ -dependent and 351 peaks at 1 mM of Mn 2+ (Figure 4D). We tested the effects of tRNA, ribosomes and starved complexes 352 ( Figure 4E). In good agreement with earlier results with M. tuberculosis Rel (8) with the antagonistic allosteric coupling between SYNTH and HD domains (16,21). This activity is 358 insensitive to inhibition by ribosomes, in good agreement with the ribosomal recruitment being 359 compromised by the mutation (Figure 3A).

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Next, we tested the HD activity of our C-terminally truncated Rel variants, both in the presence and 361 absence of tRNA Val (Figure 4F). Progressive deletion of both RRM and ZFD leads to induction of the 362 hydrolysis activity (Figure 4F), while the synthesis activity is compromised (Figure 4C). Reduction of

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Rel to an NTD fragment lacking the regulatory CTD near-completely abrogates the hydrolysis activity,

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Here we tackle these questions through extensive comparative functional characterisation of B. subtilis

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Rel and E. coli RelA. We propose a model that summarises our results (Figure 6). Off the ribosome,

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Rel adopts a 'closed' conformation in which tRNA-binding TGS and Helical domains are sequestered.

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In this state the protein has net hydrolase activity, i.e. HD ON SYNTH OFF . Amino acid starvation depletes   526 527