Ribosome association primes the stringent factor Rel for tRNA-dependent locking in the A-site and activation of (p)ppGpp synthesis

Abstract

In the Gram-positive Firmicute bacterium Bacillus subtilis, amino acid starvation induces synthesis of the alarmone (p)ppGpp by the 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 dissect the molecular mechanism of B. subtilis Rel. Off the ribosome, Rel predominantly assumes a ‘closed’ conformation with dominant (p)ppGpp hydrolysis activity. This state does not specifically select deacylated tRNA since 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 stabilization of cognate deacylated tRNA on 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.

Dual role of a (p)ppGpp‐ and (p)ppApp‐degrading enzyme in biofilm formation and interbacterial antagonism

“…Pa SAH, MESH1 as well as Rel seq and Rel Tth harbor the amino acid residues critical for coordination of a divalent metal ion cofactor (i.e., HD motifs 2, 3, and the aspartate of HD5, Figure ) in similar structural arrangements. Likewise, Pa SAH exhibited a strong preference for Mn 2+ as a cofactor for (p)ppGpp hydrolysis over Mg 2+ in vitro, the same preference documented biochemically for C. glutamicum SAH (Ruwe et al., 2018), Dm MESH1 and Hs MESH1 (Sun et al., 2010) and the bifunctional Rel enzymes from S. equisimilis (Mechold et al., 2002), Mycobacterium tuberculosis (Avarbock et al., 2000), B. subtilis (Takada et al., 2020), T. thermophilus (Van Nerom et al., 2019), and Staphylococcus aureus (Yang et al., 2019). The concentration of Mn 2+ in vivo is estimated to be in the low micromolar range (Foster et al., 2014) and thus, 1,000‐fold less than the concentration necessary to promote hydrolytic activity in our in vitro assays (1–10 mM).…”

RqcH and RqcP catalyze processive poly-alanine synthesis in a reconstituted ribosome-associated quality control system

“…Experiments were performed as described earlier ( 58 ). Before performing the experiment, stock mRNA(MVF) (5′-GGC AAGGAGGA GAUAAGA AUGGUUUUCUAA UA-3′) was incubated for 2 min at 60°C to denature possible secondary structures.…”

(p)ppGpp controls stringent factors by exploiting antagonistic allosteric coupling between catalytic domains

“…Investigations of E. coli RelA (Agirrezabala et al, 2013;Arenz et al, 2016;Brown et al, 2016;Loveland et al, 2016;Turnbull et al, 2019) as well as Rel enzymes from B. subtilis (Pausch et al, 2020;Takada et al, 2020Takada et al, , 2021, Mycobacterium tuberculosis (Avarbock et al, 2005;Jain et al, 2006), Streptococcus equisimilis (Hogg et al, 2004;Mechold et al, 2002), and Thermus thermophilus (Tamman et al, 2020) have been instrumental for our understanding of the molecular regulation of long ribosome-associated RSHs. Collectively these studies have established that (1) the CTD contacts the rRNA and a highly distorted deacylated A-site tRNA of the ''starved '' ribosomal complex, (2) the CTD transmits the allosteric signal to regulate the synthetic activity of the NTD, and (3) the synthetic and hydrolytic activities of the SYNTH and HD domains of the NTD allosterically oppose each other, with HD inhibiting SYNTH through direct occlusion of the ATP-binding site preventing efficient accommodation of substrates.…”

“…RelA is an unusual enzyme in that it is positively regulated by its product (Shyp et al, 2012), with pppGpp being a more potent activator than ppGpp (Kudrin et al, 2018). We have recently localized the allosteric pppGpp binding site to the NTD region of E. coli RelA and B. subtilis Rel (Takada et al, 2021), but the exact location of the site within the NTD and the molecular details of this allosteric regulatory mechanism have remained elusive.…”