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

大肠杆菌RelA 和枯草芽孢杆菌检测到氨基酸饥饿Rel 通过监测核糖体 A 位 tRNA 的氨酰化状态。这些酶通过一种未知机制受到它们的产物——alarmone 核苷酸 (p)ppGpp 的正向调节。Rel/RelA 的 (p)ppGpp 合成活性通过酶促 N 末端结构域 (NTD) 内的水解酶/假水解酶 (HD/假-HD) 结构域的自动抑制进行控制。我们将变构 pppGpp 位点定位到 SYNTH 和伪 HD/HD 域之间的界面，警报器通过利用 NTD 内自动抑制动力学来刺激 Rel/RelA。我们表明，如果没有 pppGpp 的刺激，饥饿的核糖体不能有效地激活 Rel/RelA。pppGpp 的受损激活在体内消融 Rel/RelA 功能，表明第二信使 (p)ppGpp 的调节对于通过个体 Rel/RelA 分子的协调酶活性启动急性饥饿反应是必要的。大肠杆菌(p)ppGpp 合成酶 SpoT缺乏(p)ppGpp 的控制，因此解释了其弱合成酶活性。