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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.

总结 mRNA降解通过限制每个mRNA可以降解的次数而影响几乎所有的细胞活性。转化成蛋白质分子。通过影响蛋白质合成，mRNA降解使生物体能够适应对环境条件的变化，因此在使病原菌侵入并在宿主细胞中存活。E.大肠杆菌和许多其他细菌的腐烂通过RppH和其他酶修饰mRNA转录物的5 ′末端而触发的途径。更好的对这一途径的了解可以帮助开发阻止细菌入侵的新策略在宿主体内存活最近发现的E.大肠杆菌mRNA是核苷四磷酸 (Np4)帽，起源于压力诱导的“alarmones”，如Ap 4A，目前在所有领域的生活。尽管 E.大肠杆菌，几乎没有什么是已知的机制添加和删除的上限。该提案详细说明了阐明Np 4加帽机制的研究计划以及E.杆菌要求1 解决了RNA聚合酶如何添加Np 4A帽前体的mRNA分子通过使用低温电子显微镜，X射线晶体学和生化实验，以揭示机制，结合的特异性。目的2将揭示如何Np 4帽被RppH去除，使用X射线晶体学和生物化学，以了解特异性和催化机制的脱帽。目的 3阐明了ApaH去除Np 4帽的分子基础，使用X射线晶体学和生物化学，了解这种酶的催化机制和RNA结合规则。Aim 4揭示关系细胞代谢和mRNA降解之间的关系。这一目标使用基于结构的遗传解偶联，确定代谢酶DapF在各种生长条件下如何影响RNA降解。结果这些研究将大大提高我们对5 ′-末端依赖性mRNA形成步骤的认识，降解以及该途径的调节如何影响细菌的生存力。