Post-initiation regulatory mechanisms controlling ethanolamine utilization

 描述(由申请人提供)：启动后的机制，如调节粪肠球菌中乙醇胺利用(EUT)基因的机制，尚不完全了解，这是知识中的一个关键差距。这项研究的长期目标是确定粪肠球菌对乙醇胺(EA)的利用是如何调节的。本申请的目的是阐明 控制基因表达的启动后调控机制。中心假说是，Amir和Nasr转录抗终止调节剂(ANTARs)RNA底物是该系统的中央调控功能，并通过三种相互关联的机制控制基因表达。核心假设将在三个目标上得到检验。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与具有特定功能的双发夹状RNA结构结合。为了进一步了解该复合体的结构，将采用生物物理方法，包括通过X射线结晶学解析蛋白质-RNA复合体的三维结构。在目标2中，我们将揭示ADOCbl核糖开关调节基因表达的机制。新颖的工作假说是，核糖开关下游的双发夹状底物结合并隔离活性EutV，防止单独电针诱导。ADOCbl与核糖开关的结合会导致构象变化，从而阻止EutV的隔离，从而在EA和ADOCbl同时存在的情况下进行诱导。该模型将通过对体内蛋白质和RNA水平的量化、结合常数的测量以及sRNA的突变分析来评估。最后，在硅胶分析中，将研究这一机制在多大程度上适用于微生物系统。目标3将确定ut基因表达是如何关闭的。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个或多个关键成分-EA、ADOCbl和/或EutV/EutW。通过用荧光标记和透射电子显微镜(TEM)表征基因表达和BMC形成的动态，这一假说将得到验证。这一建议的研究将进一步了解EUT基因是如何调控的，为原核基因调控领域和确定潜在的抗菌靶点贡献知识。具体地说，这项贡献的意义将是揭示ANTARs、核糖开关和BMC控制基因表达的新机制。这项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。

 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.