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

Abstract

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...

The C-Terminal RRM/ACT Domain Is Crucial for Fine-Tuning the Activation of ‘Long’ RelA-SpoT Homolog Enzymes by Ribosomal Complexes

“…A dedicated follow-up study is necessary to clarify this question. E. coli RelA RRM displays a similar -although more pronounced -defect in activation by starved ribosomal complexes (Takada et al, 2020).…”

The stringent response and physiological roles of (pp)pGpp in bacteria

“…Cryo-EM structures reveal that upon ribosome binding, RelA adopts an open conformation where (p)ppGpp synthesis is favoured [23][24][25] . Uncharged tRNA is not required for this initial RelA/Rel binding event, but it does stabilise the interaction and promotes synthesis 22,25,26 . When bound, the TGS, ACT/RRM and ZFD/CC domains of the C-terminal region of RelA interact with the Asite finger element and the uncharged tRNA, while the enzymatic region extends away from the ribosome producing (p)ppGpp [23][24][25][26] (FIG.…”

“…The SYNTH and HD domains work in concert to maintain an optimum level of (pp)pGpp depending on the environmental conditions [18][19][20] , with a functional HD domain required to avoid toxic accumulation of (pp)pGpp 21 . The switch between enzymatic activities is controlled by binding of the C-terminal region with interaction partners such as the starved ribosome [22][23][24][25][26] , as well as by substrate interactions 27 . Here, the binding of GDP and ATP to the SYNTH domain opens the structure of the enzyme, activating synthetase activity and inhibiting hydrolase activity 27 .…”

“…The way in which Rel and RelA sense starved ribosomes has long been disputed, with theories suggesting that RelA from E. coli could 'hop' between different ribosomes to sense the charged status of tRNAs 38 or that (p)ppGpp was produced following dissociation of active RelA from the ribosome 40 , as opposed to only when bound 41 . In recent years, much of how long RSH enzymes sense amino acid starvation was clarified, with publications of biochemical studies and a number of cryo-EM structures of RelA from E. coli in complex with the stalled ribosome [22][23][24][25] (FIG. 1c).…”

(p)ppGpp: Magic Modulators of Bacterial Physiology and Metabolism

“…Enzymes of fatty acid metabolism exhibit weak binding affinities (Polakis et al, 1973;Stein and Bloch, 1976), suggesting regulation by (p)ppGpp only to take place under full stringent control (i.e., 1,000 μM (p)ppGpp). Some enzymes partaking in (p)ppGpp metabolism, i.e., the (p)ppGpp synthetases RelQ (Gaca et al, 2015;Steinchen et al, 2015) and Rel/RelA (Shyp et al, 2012;Kudrin et al, 2018;Takada et al, 2020a), are stimulated in their activity by pppGpp and to a lesser extent by ppGpp. Furthermore, the enzymes GdpP, Pde2, and PgpH involved in the degradation pathway of the second messenger cyclic-diadenosine-monophosphate (c-di-AMP) display K i values for inhibition by ppGpp between 35 and 400 μM.…”