Starvation sensing by mycobacterial RelA/SpoT homologue through constitutive surveillance of translation

The stringent response, which leads to persistence of nutrient-starved mycobacteria, is induced by activation of the RelA/SpoT homologue (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 non-starved 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 surveyed 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. Significance Bacteria persist under nutrient starvation by activating RelA/SpoT homologue (Rsh), which synthesizes a growth regulating alarmone, ppGpp. Rsh is activated specifically upon recognizing a translation elongation complex with deacylated tRNA at the A-site. It is however unclear how Rsh identifies such a complex in vivo. We show here that conditions inducing ribosome hibernation in mycobacteria cause loss of intracellular Rsh, implying that association with translating ribosomes is necessary for intracellular stability of Rsh. Using structural analysis of Rsh-bound 70S translation initiation complex, we propose here that mycobacterial Rsh identifies a Rsh-activating ribosomal complex by constitutively surveying the ribosome entering the translation cycle at the early elongation stage.