Regulation of (p)ppGpp-metabolic activities of RelA, Rel and SpoT

严格应答是细菌适应的中心机制，在调节细菌毒力、宿主入侵存活、抗生素耐药性和持久性等方面发挥着重要作用。作为对各种应激刺激的响应，严格反应蛋白relA调节细胞内丙二醛核苷酸(P)ppGpp的浓度。RelA具有很强的核糖体依赖的(P)ppGpp合成活性，这种合成活性可以通过直接感应核糖体A位点上的脱酰化tRNA而被氨基酸饥饿激活。我们对关键的RelA和其他严格的反应蛋白机制的结构方面的了解的缺乏阻碍了SR领域本身的进展以及控制SR的有效分子工具的发展。我们计划通过确定RELA单独的和与底物的复合体的X射线结构来解决这一空白，以及确定RELA与细菌核糖体的复合体的冷冻电子显微镜结构。此外，我们将对RELA水解酶和合成酶功能在严格反应过程中的相互作用进行动力学描述。总而言之，我们的发现将为RelA通过产生(P)ppGpp来调节严格反应的过程提供基础和机制。此外，我们的发现还将为设计新型抗菌剂以对抗日益普遍的多重耐药病原菌提供结构基础。

The stringent response is a central bacterial adaptation mechanism, which plays an important role in regulation of bacterial virulence, survival during host invasion, antibiotic resistance and persistence. In response to various stress stimuli, the stringent response protein RelA modulates the intracellular concentration of the alarmone nucleotides (p)ppGpp. RelA possesses a strong ribosome-dependent (p)ppGpp synthetic activity that is activated by amino acid starvation via direct sensing of the deacylated tRNA in the ribosomal A-site. The paucity of our understanding of the structural aspects of the mechanism of key RelA and other stringent response proteins is hampering the progress of the SR field itself as well as the development of efficient molecular tools for controlling the SR. We plan to address this void by determination of X-ray structures of RelA alone and in complex with substrates as well as determine cryo-electron microscopy structures of RelA in complex with the bacterial ribosome. Moreover, we will provide a kinetic description of the interplay of the RelA hydrolase and synthetase functions during the stringent response. Collectively our findings will provide fundamental and mechanistic in the process by which RelA mediates the stringent response via production of (p)ppGpp. Moreover, our findings will also provide a structural basis for the design of novel antimicrobial agents to combat the ever-increasing prevalence of multi-drug resistant pathogenic bacteria.