Mechanisms for Stress-Induced Transcriptional Reprogramming via Anti-Adaptors

通过反适配器进行应激诱导转录重编程的机制

• 批准号: 9229317
• 负责人: Alexandra M. Deaconescu
• 金额: $ 31.5万
• 依托单位: BROWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-04-01 至 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9229317
• 关键词: Animals; Anti-Bacterial Agents; Antibiotics; Bacteria; Bacteria sigma factor KatF protein; Behavior; Binding; Binding Sites; Biochemistry; Biocide; Biological Assay; Cells; Communities; Complement; Complex; Cryoelectron Microscopy; Cues; DNA Damage; DNA-Directed RNA Polymerase; Desiccation; Development; Environment; Escherichia coli; Gammaproteobacteria; Genes; Genetic Transcription; Genome; Human; In Vitro; Individual; Infection; Knowledge; Laboratories; Magnesium; Mediating; Metals; Methods; Microbial Biofilms; Microscopy; Molecular Conformation; Molecular Genetics; Molecular Structure; Molecular Weight; Nutrient; Oxidative Stress; Pathway interactions; Phagocytosis; Phase; Phosphorylation; Plants; Play; Process; Protein Family; Proteins; Proteolysis; Regulation; Regulon; Reporting; Research Personnel; Resolution; Role; Sequence Homology; Signal Transduction; Starvation; Stress; Structural Models; Structure; System; Testing; Ubiquitin; Up-Regulation; Virulence; Virulence Factors; Work; X-Ray Crystallography; antimicrobial; biological adaptation to stress; biological systems; biophysical techniques; combat; in vivo; inhibitor/antagonist; inorganic phosphate; insight; logarithm; multicatalytic endopeptidase complex; novel; particle; pathogen; promoter; protein protein interaction; response; structural biology; toxic metal

PROJECT SUMMARY The dissociable promoter recognition subunit RpoS (also known as σs) is the master transcriptional regulator of the general stress response in γ-proteobacteria, and plays key roles in the virulence of many pathogens, including human, plant and animal pathogens. Under certain conditions, such as at the transition from the logarithmic to the stationary phase or in the presence of stress signals, RpoS redirects the core RNA polymerase machinery to a subset of promoters to reprogram transcription. However, intracellular RpoS levels are not steady – they are low in actively dividing cells, and substantially increased upon entering the stationary phase or upon encountering stress. To achieve proper regulation, there is tight control over RpoS levels, with the major point of regulation occurring at the level of RpoS proteolysis by the ATP-dependent ClpXP machine. Our central focus is to understand the mechanisms of RpoS proteolysis by ClpXP as well as its regulation by an emerging family of proteins collectively called anti-adaptors. In order to be degraded, RpoS is presented to ClpXP by a unique, highly specific adaptor called RssB, which acts catalytically, without being degraded. In turn, RssB itself is regulated by interactions with stress-specific anti-adaptors. Our work will focus on the structure and function of three-anti-adaptors: IraD (induced by oxidative stress and DNA damage), IraM (induced by magnesium starvation) and IraP (induced by phosphate starvation). These anti-adaptors share no sequence homology, and only weak homology with protein of known structure, warranting a structural biology effort aimed at deciphering the underlying mechanisms of RssB recognition. We will determine the structures of IraD, IraM and IraP, both in isolation and bound to RssB using X-ray crystallography. This will allow us to pinpoint, at atomic resolution, residues important for anti-adaptor/RssB interactions, and also regulation of the anti-adaptors themselves by oligomerization. We will complement these structural studies with molecular genetics, microscopy and functional assays for protein-protein interactions and RpoS degradation, which will allow us to correlate in vitro behavior with in vivo observations. We will also determine structures of a RpoS- RssB-ClpXP assembly using cutting-edge methods in electron cryo-microscopy, which will allow us to understand the RpoS and RssB conformational dynamics at the core of this paradigmatic mode of regulated proteolysis in bacteria. Overall, this work will not only bring fundamental, mechanistic insights, but will also open the way to the development of novel antibacterials that could target ClpXP directly, or, adaptor/anti- adaptor interfaces. The RpoS regulon has been reported to comprise up to 10% of the Escherichia coli genome, and RpoS itself plays important roles in bacterial persistence, host-pathogen interactions and biofilm formation, which underlie 80% of all infections.

项目总结