Activation of the stringent response by loading of tRNA-RelA 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 during steady-state cell growth 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. The relA locus of Escherichia coli was fused to a dual affinity His-TEV-FLAG tag. Untagged Control strain (named Control) and strains expressing relA-HTF (rela), relAΔRRM-HTF (RRM deletion, named RRM), relAΔZFDΔRRM-HTF (ZFDRRM deletion, named ZFDRRM) and relAH432E-HTF (H432E substitution, named H432E) were grown exponentially in MOPS minimal medium. Cell samples were UV-C irradiated (100sec of 1800mJ) before and after 30 min of isoleucine starvation (indicated by +val) and harvested. Cells were washed with ice-cold PBS before snap freezing in liquid nitrogen. RNA-RelA complexes were purified on M2-anti-FLAG resin and Ni-NTA resin. Sequencing libraries were prepared from purified complexes essentially per Winther et al. 2018. Amplified libraries were sequenced on an Illumina MiSeq instrument.