(p)ppGpp controls stringent factors by exploiting antagonistic allosteric coupling between catalytic domains

Summary Amino acid starvation is sensed by Escherichia coli RelA and Bacillus subtilis Rel through monitoring the aminoacylation status of ribosomal A-site tRNA. These enzymes are positively regulated their product—the alarmone nucleotide (p)ppGpp—through an unknown mechanism. The (p)ppGpp-synthetic activity Rel/RelA controlled via auto-inhibition hydrolase/pseudo-hydrolase (HD/pseudo-HD) domain within enzymatic N-terminal region (NTD). We localize allosteric pppGpp site to interface between SYNTH pseudo-HD/HD domains, with stimulating exploiting intra-NTD autoinhibition dynamics. show that without stimulation pppGpp, starved ribosomes cannot efficiently activate Rel/RelA. Compromised activation ablates function in vivo, suggesting regulation second messenger (p)ppGpp necessary for mounting acute response coordinated individual molecules. Control lacking in E. synthetase SpoT, thus explaining its weak activity.