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（由应用程序提供）：发火机制，例如在大肠杆菌中控制乙醇胺利用率（EUT）基因的机制，这是不完全理解代表知识中关键差距的。这项研究的长期目标是确定乙醇胺（EA）如何在粪肠球菌中调节利用。此应用的目的是阐明 控制基因表达的定位调节机制。中心假设是，AMIR和NASR转录抗固定器调节剂（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来控制基因表达。中心假设将以三个目标进行检验。 AIM 1将阐明EUT系统中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具有具有特定特征的双发夹RNA结构。为了进一步理解这种复合物的结构，将进行生物物理方法，包括通过X射线晶体学通过X射线RNA复合物的三维结构解决。在AIM 2中，将发现Adocbl核糖开关调节基因表达的机制。小说的工作假设是，核糖开关下游的双发夹底物结合并隔离活性EUTV，可防止仅EA诱导。 ADOCBL与核糖开关的结合会导致会议变化，从而阻止EUTV会话，从而在存在EA和ADOCBL时诱导。该模型将通过量化蛋白质和RNA的体内水平，测量结合常数以及对SRNA的突变分析来评估该模型。最后，在计算机分析中，将研究这种机制在微生物系统中的广泛应用。 AIM 3将确定如何关闭EUT基因表达。我们的工作假设是细菌微室（BMC）的形成隔离了基因表达的一种或多种至关重要的含量-EA，ADOCBL和/或EUTV/EUTW。通过荧光标记和透射电子显微镜（TEM）表征基因表达和BMC形成的动力学，将测试该假设。该提案中的研究将进一步了解如何调节EUT基因，从而为原核基因调节领域和潜在抗菌靶标的鉴定提供知识。具体而言，这种贡献的重要性将是揭示新机制，而新机制，核糖开关和BMC控制基因表达的新机制。拟议的研究具有创新性，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。