Post-initiation regulatory mechanisms controlling ethanolamine utilization

控制乙醇胺利用的引发后调节机制

• 批准号: 9193056
• 负责人: Danielle A Garsin
• 金额: $ 45.6万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-01-15 至 2019-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9193056
• 关键词: 5' Untranslated Regions; Affect; Bacteria; Binding; Binding Proteins; Carbon; Complex; Computer Simulation; Containment; Data; Down-Regulation; Drug Targeting; Enterococcus faecalis; Ethanolamines; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Goals; Human; Intestines; Knowledge; Measures; Metabolic Pathway; Metabolism; Modeling; Molecular; Molecular Conformation; Mutation Analysis; Nitrogen; Nosocomial Infections; Operon; Organelles; Pathogenesis; Phosphotransferases; Proteins; Public Health; RNA; Regulation; Research; Resolution; Source; Structure; System; Terminator Regions; Testing; Thinking; Translations; Transmission Electron Microscopy; Untranslated RNA; Work; X-Ray Crystallography; antimicrobial; antitermination; base; biophysical techniques; cobamamide; falls; in vivo; innovation; microbial; novel; pathogen; prevent; protein structure; public health relevance; response; sensor; small molecule; stem; therapeutic development; therapeutic target; three dimensional structure

 DESCRIPTION (provided by applicant): Post-initiation mechanisms, like those regulating the ethanolamine utilization (eut) genes in E. faecalis, are incompletely understood representing a critical gap in knowledge. The long-term goal of this research is to determine how ethanolamine (EA) utilization is regulated in E. faecalis. The objective of this application is to elucidate the post-initiation regulatory mechanisms that control gene expression. The central hypothesis is that the AmiR and NasR Transcriptional Antiterminator Regulators' (ANTARs) RNA substrates are the central regulatory feature of the system and control gene expression by three interrelated mechanisms. The central hypothesis will be tested in three aims. Aim 1 will elucidate the molecular details of how EutV, the ANTAR in the eut system, interacts with its RNA substrates to control gene expression. There is evidence that EutV binds a dual hairpin RNA structure with specific features. To further understand the structure of this complex, biophysical approaches will be employed, including resolution of three-dimensional structures of the protein-RNA complex by X-ray crystallography. In Aim 2, the mechanism by which the AdoCbl riboswitch regulates gene expression will be uncovered. The novel, working hypothesis is that a dual hairpin substrate just downstream of the riboswitch binds and sequesters active EutV, preventing induction by EA alone. AdoCbl binding to the riboswitch causes a conformational change that prevents EutV sequestration, allowing for induction when both EA and AdoCbl are present. The model will be assessed by quantifying in vivo levels of protein and RNA, measuring binding constants, and by mutational analysis of the sRNA. Finally, in silico analysis will investigate how broadly this mechanism applies to microbial systems. Aim 3 will identify how eut gene expression is turned off. Our working hypothesis is that bacterial microcompartment (BMC) formation sequesters one or more of the crucial ingredients for gene expression - EA, AdoCbl and/or EutV/EutW. By characterizing the dynamics of gene expression and BMC formation with fluorescent markers and transmission electron microscopy (TEM), the hypothesis will be tested. The research in this proposal will further the understanding of how the eut genes are regulated, contributing knowledge to the field of prokaryotic gene regulation and to the identification of potential antimicrobial targets. Specifically, the significance of this contribution will be the uncovering of novel mechanisms by which ANTARs, riboswitches, and BMCs control gene expression. The proposed research is innovative because these new mechanisms will challenge the status quo and expand the field's thinking on how RNA structural features and BMCs can operate.

 描述（申请人提供）：启动后机制，如调节大肠杆菌中乙醇胺利用（eut）基因的机制。对粪便细菌的认识不完全，这是一个重大的知识空白。本研究的长期目标是确定乙醇胺（EA）的利用是如何在E。粪便本申请的目的是阐明 启动后调控机制，控制基因表达。核心假设是AmiR和NasR转录抗终止子调节因子（ANTAR）RNA底物是系统的中心调节特征，并通过三种相互关联的机制控制基因表达。中心假设将在三个目标进行测试。目的1将阐明Eut系统中的ANTAR EutV如何与其RNA底物相互作用以控制基因表达的分子细节。有证据表明EutV结合具有特定特征的双发夹RNA结构。为了进一步了解这种复合物的结构，将采用生物物理方法，包括通过X射线晶体学解析蛋白质-RNA复合物的三维结构。在目标2中，将揭示BBCbl核糖开关调节基因表达的机制。新的工作假设是，双发夹底物下游的核糖开关结合和隔离活性EutV，防止诱导EA单独。与核糖开关结合的EutCbl引起构象变化，防止EutV螯合，允许在EA和EutCbl都存在时进行诱导。将通过定量体内蛋白质和RNA水平、测量结合常数和sRNA突变分析来评估模型。最后，计算机分析将研究这种机制在微生物系统中的应用范围。目标3将确定eut基因表达是如何被关闭的。我们的工作假设是，细菌微区室（BMC）的形成螯合一个或多个基因表达的关键成分- EA，EUTV/EutW和/或EUTV/EutW。通过用荧光标记物和透射电子显微镜（TEM）表征基因表达和BMC形成的动态，将检验该假设。这项研究将进一步了解eut基因是如何调控的，为原核基因调控领域和潜在抗菌靶标的鉴定提供知识。具体而言，这一贡献的意义将是揭示ANTAR，核糖开关和BMC控制基因表达的新机制。拟议的研究是创新的，因为这些新机制将挑战现状，并扩大该领域对RNA结构特征和BMC如何运作的思考。