Molecular Basis for mRNA Decay in Bacteria

细菌中 mRNA 衰变的分子基础

• 批准号: 10250555
• 负责人: Alexander Serganov
• 金额: $ 35.6万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-21 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10250555
• 关键词: Address; Affect; Antibiotics; Applications Grants; Bacteria; Bacterial Infections; Binding Sites; Biochemical; Biochemistry; Biological; Catalytic RNA; Cell physiology; Cells; Complex; Cryoelectron Microscopy; DNA-Directed RNA Polymerase; Development; Electron Microscopy; Enzymes; Escherichia coli; Eukaryota; Excision; Foundations; Future; Genetic; Genetic Transcription; Goals; Growth; Health; Host Defense; Human; Hydrolase; Invaded; Kinetics; Knowledge; Life; Lysine; Mammalian Cell; Measures; Mediating; Messenger RNA; Metabolic; Metabolism; Modification; Molecular; Mutagenesis; Mutation Analysis; Nucleosides; Nucleotides; Organism; Pathway interactions; Peptidoglycan; Personal Satisfaction; Prokaryotic Cells; Property; Protein Biosynthesis; Proteins; RNA; RNA Binding; RNA Caps; RNA Decay; RNA Degradation; RNA Stability; Reaction; Research; Research Proposals; Roentgen Rays; Specificity; Stress; Structure; System; Time; Transcript; Transcription Initiation; Translating; Virulence; Work; X-Ray Crystallography; aminoacid biosynthesis; base; cryogenics; decapping enzyme; design; diadenosine tetraphosphate; environmental change; enzyme structure; experimental study; inhibitor/antagonist; insight; mRNA Decay; mRNA Stability; mRNA Transcript Degradation; mRNA capping; mRNA decapping; novel; nucleoside triphosphate; pathogenic bacteria; response; three dimensional structure; transcriptome sequencing; virtual

Summary mRNA degradation affects virtually all cellular activities by limiting the number of times each mRNA can be translated into protein molecules. By affecting protein synthesis, mRNA degradation allows organisms to adapt to changing environmental conditions and is therefore particularly important in enabling pathogenic bacteria to invade and survive in host cells. mRNA degradation in E. coli and many other bacteria involves a decay pathway triggered by modification of the 5¢ end of the mRNA transcript by RppH and other enzymes. A better understanding of this pathway could enable the development of new strategies to impede bacterial invasion and survival in hosts. A recently discovered 5¢-end modification of E. coli mRNAs is nucleoside tetraphosphate (Np4) caps, originated from stress-induced “alarmones”, such as Ap4A, present in all domains of life. Despite the identification of capping and decapping enzymes in E. coli, practically nothing is known about mechanisms of cap addition and removal. This proposal details a research plan to elucidate the mechanisms of Np4 capping and decapping and the connection between cellular metabolism and RNA degradation in E. coli. Aim 1 addresses how the RNA polymerase adds Np4A cap precursor to mRNA molecules by using cryogenic electron microscopy, X-ray crystallography, and biochemical experiments to reveal the mechanism and specificity of incorporation. Aim 2 will uncover how the Np4 cap is removed by RppH, using X-ray crystallography and biochemistry to understand the specificity and the catalytic mechanisms of decapping. Aim 3 elucidates the molecular basis of Np4 cap removal by ApaH, using X-ray crystallography and biochemistry to understand the catalytic mechanism and RNA binding rules of this enzyme. Aim 4 reveals a relationship between cellular metabolism and mRNA degradation. This aim uses structure-based genetic uncoupling to identify how the metabolic enzyme DapF affects RNA degradation under various growth conditions. The results of these studies will significantly advance our knowledge of the steps leading to 5¢-end-dependent mRNA degradation and how modulation of this pathway affects the viability of bacteria.

概括 mRNA 降解通过限制每个 mRNA 降解的次数，影响几乎所有细胞活动。 翻译成蛋白质分子。通过影响蛋白质合成，mRNA 降解使生物体能够适应 改变环境条件，因此对于使病原菌能够 侵入宿主细胞并在其中存活。大肠杆菌和许多其他细菌中的 mRNA 降解涉及衰变 RppH 和其他酶对 mRNA 转录物 5￠ 末端的修饰触发的途径。更好的 了解这一途径可以帮助开发阻止细菌入侵的新策略 和在宿主体内的生存。最近发现的大肠杆菌 mRNA 5 末端修饰是四磷酸核苷 (Np4) 帽源自压力诱导的“警报素”，例如 Ap4A，存在于生命的各个领域。尽管 大肠杆菌中加帽和脱帽酶的鉴定，实际上对其机制一无所知 盖的添加和移除。该提案详细阐述了阐明 Np4 封端机制的研究计划 大肠杆菌中的脱帽以及细胞代谢和 RNA 降解之间的联系。目标1 解决了 RNA 聚合酶如何使用低温将 Np4A 帽前体添加到 mRNA 分子中 电子显微镜、X射线晶体学和生化实验揭示了其机制和 合并的特殊性。目标 2 将揭示 RppH 如何使用 X 射线去除 Np4 帽 晶体学和生物化学，以了解脱盖的特异性和催化机制。目的 图 3 使用 X 射线晶体学和生物化学阐明了 ApaH 去除 Np4 帽的分子基础 了解该酶的催化机制和RNA结合规则。目标 4 揭示关系 细胞代谢和 mRNA 降解之间的关系。该目标使用基于结构的遗传解偶联来 确定代谢酶 DapF 在不同生长条件下如何影响 RNA 降解。结果 这些研究将显着提高我们对 5′ 端依赖性 mRNA 步骤的了解 降解以及该途径的调节如何影响细菌的活力。