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

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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.