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Molecular Basis for mRNA Decay in Bacteria - summer supplement

细菌 mRNA 衰变的分子基础 - 夏季补充品

基本信息

批准号：
10805871
负责人：
Alexander Serganov
金额：
$ 1.5万
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：
2023
资助国家：
美国
起止时间：
2023-06-01 至 2024-05-31
项目状态：
已结题

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.

摘要信使核糖核酸的降解通过限制每一个信使核糖核酸的作用次数来影响几乎所有的细胞活动。转化成蛋白质分子。通过影响蛋白质合成，信使核糖核酸的降解使生物体能够适应环境条件的变化，因此在使病原菌能够侵入并存活于宿主细胞内。在大肠杆菌和许多其他细菌中，mRNA的降解涉及一种腐烂由RppH和其他酶对转录产物5‘端进行修饰而触发的通路。更好的对这一途径的了解可能有助于开发新的策略来阻止细菌入侵以及在宿主体内存活。最近发现的一种对大肠杆菌mRNAs的5‘端修饰是四磷酸核苷 (NP4)帽，起源于应激诱导的“杏仁醇”，如Ap4A，存在于生活的各个领域。尽管大肠杆菌中封盖和去封盖酶的鉴定，对其作用机制几乎一无所知盖子的添加和移除。这项建议详细说明了一项研究计划，以阐明NP4封顶的机制以及在大肠杆菌中细胞新陈代谢和RNA降解之间的联系。目标1 阐述了RNA聚合酶如何通过低温将Np4A帽前体添加到mRNA分子中电子显微镜、X射线结晶学和生化实验以揭示其作用机制和公司成立的特殊性。Aim 2将使用X射线揭示NP4帽是如何被RppH移除的结晶学和生物化学，以了解脱壳的特异性和催化机制。目标 3用X射线结晶学和生化方法阐明了APAH去除NP4帽的分子基础。了解该酶的催化机理和RNA结合规律。《目标4》揭示了一种关系细胞代谢和信使核糖核酸降解之间的关系。这一目标使用基于结构的基因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。结果是这些研究将极大地促进我们对导致5‘端依赖信使核糖核酸的步骤的了解降解以及这一途径的调节如何影响细菌的生存能力。