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：
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
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/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

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降解几乎通过限制每个mRNA的次数来影响所有细胞活性翻译成蛋白质分子。通过影响蛋白质合成，mRNA降解使生物可以适应改变环境条件，因此对于使致病细菌的能力尤其重要入侵并在宿主细胞中生存。大肠杆菌和许多其他细菌中的mRNA降解涉及衰减通过RPPH和其他酶对mRNA转录的5¢末端修饰触发的途径。更好了解这一途径可以使新策略的发展阻碍细菌入侵和宿主的生存。最近发现的大肠杆菌mRNA的5¢末端修饰是核苷四磷酸盐（NP4）CAPS起源于应力引起的“警报器”，例如AP4A，存在于生命的所有领域中。尽管在大肠杆菌中识别封盖和解替酶，几乎没有任何知识加上盖的添加和去除。该建议详细介绍了一项研究计划，以阐明NP4封盖的机制大肠杆菌中的细胞代谢和RNA降解之间的连接。目标1 解决RNA聚合酶如何通过使用低温为mRNA分子添加NP4A帽前体电子显微镜，X射线晶体学和生化实验，以揭示机制和保险的特异性。 AIM 2将使用X射线删除RPPH的NP4盖晶体学和生物化学，以理解脱皮的特异性和催化机制。目的 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揭示了一段关系在细胞代谢和mRNA降解之间。此目的使用基于结构的遗传解偶联到确定代谢酶DAPF如何在各种生长条件下影响RNA降解。结果这些研究将大大提高我们对导致5¢端子依赖mRNA的步骤的了解降解以及该途径的调节如何影响细菌的活力。