Molecular Basis for mRNA Decay in Bacteria

细菌中 mRNA 衰变的分子基础

基本信息

批准号：
10058513
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
项目类别：
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/10058513
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

项目摘要

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。通过影响蛋白质合成，mRNA 降解可以使生物体适应。改变环境条件，因此对于使病原菌能够大肠杆菌和许多其他细菌中的 mRNA 侵入并在宿主细胞中存活，从而导致衰变。 RppH 和其他酶对 mRNA 转录物 5￠末端的修饰触发了更好的途径。了解这一途径可以帮助开发阻止细菌入侵的新策略最近发现的大肠杆菌 mRNA 的 5￠末端修饰是四磷酸核苷。 (Np4)帽起源于压力诱导的“警报素”，例如Ap4A，存在于生活的各个领域。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该提案详细说明了阐明 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。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。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 揭示了一种关系。该目标利用基于结构的遗传解偶联来研究细胞代谢和 mRNA 降解之间的关系。确定代谢酶 DapF 在不同生长条件下如何影响 RNA 降解。这些研究将显着提高我们对 5￠末端依赖性 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。