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

par Takada, Hiraku;Roghanian, Mohammad;Caballero Montes, Julien ;Van Nerom, Katleen ;Jimmy, Steffi;Kudrin, Pavel;Trebini, Fabio;Murayama, Rikinori;Akanuma, Genki;Garcia-Pino, Abel ;Hauryliuk, Vasili
Référence Nucleic acids research, 49, 1, page (444-457)
Publication Publié, 2021-06-01

Article révisé par les pairs

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Résumé :

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.