NsrR regulation of the small noncoding RNA RybB in Escherichia coli

NsrR 对大肠杆菌中小非编码 RNA RybB 的调控

• 批准号: 8701310
• 负责人: Karl M Thompson
• 金额: $ 15.1万
• 依托单位: HOWARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-07-15 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8701310
• 关键词: Affect; Animal Model; Bacteria; Bacterial Infections; Binding; Biochemical; Biological; Biological Models; Cell physiology; Cells; Cellular biology; ChIP-on-chip; Computer Simulation; Critical Pathways; DNA; Data; Down-Regulation; Drug Metabolic Detoxication; E protein; Elements; Endoplasmic Reticulum; Enzymes; Escherichia coli; Escherichia coli K12; Functional RNA; Future; Genes; Genetic; Immune response; Individual; Investigation; Kinetics; Knowledge; Left; Link; Mediating; Mediator of activation protein; Medicine; Membrane Proteins; Metabolic Pathway; Modeling; Molecular; Nitric Oxide; Pathway interactions; Physiological; Physiological Adaptation; Play; Post-Transcriptional Regulation; Promoter Regions; Proteins; Public Health; RNA; Regulation; Regulon; Repression; Role; Sigma Factor; Signal Transduction; Small RNA; Stress; Systems Biology; Testing; Transcription Repressor/Corepressor; Untranslated RNA; base; biological adaptation to stress; biological systems; environmental change; in vivo; metabolomics; network models; nitrosative stress; novel therapeutics; prevent; promoter; protein expression; public health relevance; research study; response; sigma-E factor; synthetic biology

DESCRIPTION (provided by applicant): Systems biology knowledge is crucial for our understanding of the cellular and molecular basis of medicine and synthetic biology applications. It also provides a framework for metabolomic studies of various biological systems. Systems biology knowledge will advance through investigations that confirm computational models of cellular physiology or discover new pathways not previously described. Using E. coli K12 as a model system for cellular physiology studies is optimal due to its biological simplicity and genetic tractability. One crucial element missing from bacterial cell physiology studies is precise mechanisms defining cross-talk between two different stress responses. Stress responses are rapid physiological adaptations to environmental changes. Bacterial small regulatory RNAs are noncoding RNA molecules with a post- transcriptional regulatory function. Small RNAs frequently act as genetic switches, by affecting specific metabolic pathways in major ways, via post-transcriptional regulation of a central regulator or enzyme involved in a pathway. For this reason, the study of small RNAs is uniquely suited for studies aimed at defining physiological circuits and cross-talk. The small regulatory RNA RybB is regulated by the Extracytoplasmic function (ECF) sigma factor, RpoE, in E. coli. RpoE is a central regulator of the envelope stress response in E. coli and is analogous to the unfolded protein response in eukaryotic endoplasmic reticulum. NsrR responds to nitric oxide (NO) exposure by inducing the expression of HmpA, a NO-detoxification enzyme. Hence, NsrR is a central mediator of the nitrosative stress response in E. coli. We have preliminary evidence to suggest that a nitric oxide sensing transcriptional regulator, NsrR, has a regulatory effect on RybB expression as well. The implications for this observation is that there may be physiological cross-talk between the nitrosative stress and envelope stress responses in E. coli; and, it can be studied by characterizing the role NsrR plays on RybB. Our main hypothesis is that NsrR is a direct regulator of RybB expression. We have two models we propose as a mechanism for our hypothesis that NsrR directly regulates RybB. Our first model is that NsrR exerts its regulatory effect on RybB via an interaction with DNA, specifically the promoter of the rybB gene. Our second model is that NsrR exerts its regulatory effect on RybB via a direct interaction with the RpoE protein, acting as an anti-sigma factor and preventing it from regulating is transcriptional targets (including rybB). To test our hypothesis, we will first determine the regulatory level at which NsrR acts on RybB expression (Specific Aim #1). Then, we will identify biochemical interactions necessary for the NsrR effect on RybB expression (Specific Aim #2). Finally, we will determine the role that exogenous NO exposure plays on the expression of RybB (Specific Aim #3). Taken together, these experiments will characterize NsrR's regulatory effect on RybB and contribute to knowledge of physiological cross-talk in bacterial cells. The models resulting from these studies will contribute to refined physiological models for systems biology applications.

描述（由申请人提供）：系统生物学知识对于我们对医学和合成生物学应用的细胞和分子基础的理解至关重要。它还为各种生物系统的代谢组学研究提供了一个框架。系统生物学知识将通过确认细胞生理的计算模型或发现先前未描述的新途径的研究来推进。将大肠杆菌K12用作细胞生理研究的模型系统，由于其生物学简单性和遗传性障碍性是最佳的。细菌细胞生理学研究中缺少的一个关键元素是定义两个不同应力反应之间串扰的精确机制。压力反应是对环境变化的快速生理适应。细菌小调节性RNA是具有转录后调节功能的非编码RNA分子。小型RNA经常用作遗传转换，通过以主要方式影响特定的代谢途径，通过对途径中的中央调节剂或酶进行的转录后调节。因此，对小RNA的研究非常适合旨在定义生理回路和串扰的研究。小的调节RNA RYBB受大肠杆菌中的RPOE（ECF）Sigma因子（ECF）的调节。 RPOE是大肠杆菌中包膜应力反应的中心调节剂，类似于真核生物内质网中展开的蛋白质反应。 NSRR通过诱导NO氧化酶HMPA的表达来应对一氧化氮（NO）暴露。因此，NSRR是大肠杆菌中亚硝化应激反应的中心介体。我们有初步的证据表明，一氧化氮传感转录调节剂NSRR也对RYBB表达具有调节作用。这一观察结果的含义是，大肠杆菌中的亚硝化应激和包膜应力反应之间可能存在生理串扰。而且，可以通过表征NSRR在RYBB上的作用来研究它。我们的主要假设是NSRR是RYBB表达的直接调节剂。我们提出了两个模型，作为我们的假设，即NSRR直接调节RYBB。我们的第一个模型是NSRR通过与DNA的相互作用，特别是RYBB基因的启动子对RYBB发挥调节作用。我们的第二个模型是，NSRR通过与RPOE蛋白的直接相互作用对RYBB发挥调节作用，充当抗sigma因子，并防止其调节它是转录靶标（包括RYBB）。为了检验我们的假设，我们将首先确定NSRR对RYBB表达的调节水平（特定目标＃1）。然后，我们将确定NSRR对RYBB表达的影响所需的生化相互作用（特定目标＃2）。最后，我们将确定外源不暴露在RYBB表达中的作用（特定的目标＃3）。综上所述，这些实验将表征NSRR对RYBB的调节作用，并有助于细菌细胞中的生理串扰知识。这些研究产生的模型将有助于系统生物学应用的精致生理模型。