Molecular principles of stringent response activation in bacteria

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PROJECT SUMMARY/ABSTRACT As the leading cause of childhood mortality and a major cause of adult mortality, bacterial infections remain the main threat to health worldwide. The emergence of antibiotic resistance is outpacing the development of new antibiotics and posing a serious challenge to infectious disease treatment. Understanding how bacteria adapt to stress during infection will spur the search for novel therapeutic approaches. To become therapeutic targets, these mechanisms have to play key functional roles in pathogens but not in mammalian organisms. Bacterial “stringent response”—the master regulator of bacterial stress adaptation—drives bacterial pathogenesis and antibiotic resistance. Stringent response depends on RelA-like proteins that make and regulate the second messenger molecules pppGpp and ppGpp, which reformat cellular transcription to adapt to stresses. RelA-like proteins, such as RelA and SpoT exist in most pathogenic bacteria. Despite many cellular and biochemical studies, the mechanisms of activation of RelA-like proteins and stringent response remain poorly understood. How RelA-like proteins sense stress and catalyze pppGpp synthesis and hydrolysis is unclear. RelA is generally thought to be activated by deacyl-tRNAs, which accumulate in response to amino acid deprivation and bind specific mRNA codons. Paradoxically, many other types of stress activate stringent response, and our preliminary biochemical and structural studies suggest that RelA and SpoT can be activated by ribosomes in the absence of deacyl-tRNA. The proposed studies will test the paradigm-shifting hypothesis that RelA-like proteins sense many stresses via ribosome stalling, and that ribosomes and other activators induce distinct conformational changes in RelA and SpoT to regulate their activities. This project will take advantage of recent methodological innovations and advances in biochemistry and cryogenic electron microscopy (cryo-EM) to dissect the molecular mechanisms of two RelA-like proteins. Aim 1 will dissect the biochemical basis of the stringent response activation by RelA by identifying the functions of regulatory domains and by kinetic characterization of RelA autoinhibition and activation by stalled ribosomes. Aim 2 will elucidate the structural mechanisms of RelA activation by stressed ribosomes primarily using cryo- EM. Aim 3 will dissect mechanisms of the dual enzyme SpoT, which possesses the hydrolase and weak synthetase activities and interacts with many molecular partners via unknown mechanisms. How the ribosome and other partners control opposing SpoT activities will be determined using biochemical and structural approaches. Completion of this project will expand the understanding of stringent response in bacteria, and may inform the development of new drugs to treat severe bacterial infections.