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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.

Superhelical and partially relaxed DNAs of simian virus 40 were allowed to react in vitro with (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BaP diol epoxide). The modified DNA contained N2 guanine and N6 adenine hydrocarbon adducts in the ratio 86:14. Superhelical simian virus 40 DNA was approximately 6% more susceptible to modification than was partially relaxed viral DNA. Counterions inhibited DNA alkylation by up to 90%, Mg2+ being 50-fold more effective than Na+. The sensitivity of covalent binding to helix stability is consistent with a reaction complex in which BaP diol epoxide is intercalated. The superhelical density of the modified DNA substrates was determined electrophoretically relative to partially relaxed standards, and an unwinding angle for the hydrocarbon adducts was calculated. The angle was dependent upon the superhelicity of the DNA molecule and ranged from 330 degrees to 30 degrees. These data indicate that the modified base pairs are disrupted and, in the presence of torsional strain, act as centers for the further denaturation of up to eight adjacent base pairs. In the absence of such strain the alkylation sites have an ordered structure, with the attached hydrocarbon probably oriented in the minor or major groove of the helix.