Activation of the Stringent Response by Loading of RelA-tRNA Complexes at the Ribosomal A-Site

RelA/SpoT homologs (RSHs) are ubiquitous bacterial enzymes that synthesize and hydrolyze (p)ppGpp in response to environmental challenges. Bacteria cannot survive in hosts and produce infection without activating the (p)ppGpp-mediated stringent response, but it is not yet understood how the enzymatic activities of RSHs are controlled. Using UV crosslinking and deep sequencing, we show that Escherichia coli RelA ((p)ppGpp synthetase I) interacts with uncharged tRNA without being activated. Amino acid starvation leads to loading of cognate tRNA⋅RelA complexes at vacant ribosomal A-sites. In turn, RelA is activated and synthesizes (p)ppGpp. Mutation of a single, conserved residue in RelA simultaneously prevents tRNA binding, ribosome binding, and activation of RelA, showing that all three processes are interdependent. Our results support a model in which (p)ppGpp synthesis occurs by ribosome-bound RelA interacting with the Sarcin-Ricin loop of 23S rRNA. [Display omitted] •RelA interacts with uncharged tRNA off the ribosome•RelA loads uncharged tRNA at hungry A-site codons•RelA binds tRNA before loading at the A-site•Activation of RelA entails interaction with the Sarcin-Ricin loop of 23S rRNA Using in vivo crosslinking combined with deep sequencing, Winther et al. investigate the activation mechanism of RelA ((p)ppGpp synthetase I) of Escherichia coli. The study reveals that RelA interacts with uncharged tRNA off the ribosome and that this interaction is prerequisite to ribosome binding and activation of RelA’s (p)ppGpp synthetic activity.