The stringent response, which leads to persistence of nutrient-starved mycobacteria, is induced by activation of the RelA/SpoT homolog (Rsh) upon entry of a deacylated-tRNA in a translating ribosome. However, the mechanism by which Rsh identifies such ribosomes in vivo remains unclear. Here, we show that conditions inducing ribosome hibernation result in loss of intracellular Rsh in a Clp protease–dependent manner. This loss is also observed in nonstarved cells using mutations in Rsh that block its interaction with the ribosome, indicating that Rsh association with the ribosome is important for Rsh stability. The cryo-EM structure of the Rsh-bound 70S ribosome in a translation initiation complex reveals unknown interactions between the ACT domain of Rsh and components of the ribosomal L7/L12 stalk base, suggesting that the aminoacylation status of A-site tRNA is surveilled during the first cycle of elongation. Altogether, we propose a surveillance model of Rsh activation that originates from its constitutive interaction with the ribosomes entering the translation cycle.

The stringent response, which leads to persistence of nutrient-starved mycobacteria, is induced by activation of RelA/SpoT homologue (Rsh) upon entry of a deacylated-tRNA in a translating ribosome. However, the mechanism by which Rsh finds such ribosomes in vivo remains unclear. Here we show that conditions inducing ribosome hibernation cause loss of intracellular Rsh in a Clp protease-dependent manner. The loss is also observed in non-starved cells when Rsh-ribosome interaction is abolished by mutation in Rsh, indicating that interaction with translating ribosomes is necessary for physiological stability of Rsh. The cryo-EM structure of the Rsh-bound 70S ribosome in a translation initiation complex reveals hitherto unknown interactions between the ACT domain of Rsh and components of the ribosomal L7/L12-stalk base, suggesting that the aminoacylation status of A-site tRNA is surveyed during the first cycle of elongation. Altogether, we propose a surveillance model of Rsh activation that originates from its constitutive interaction with translating ribosomes.