Regulation of Ethanolamine Utilization in E. Faecalis by an Atypical Riboswitch

描述（由申请人提供）：粪肠球菌成功成为美国医院获得性感染的第二或第三大原因的一个特点是其灵活的代谢，使其能够在各种条件下生存。然而，关于粪肠杆菌如何调节与使用替代能源相关的基因表达，我们知之甚少。本应用的目的是阐明非典型B12核糖开关和反应调节因子在调节乙醇胺利用中的作用，乙醇胺是宿主体内潜在的有价值的碳和氮来源。我们的中心假设是，位于乙醇胺利用操纵子的未翻译先导RNA上的核糖体开关与腺苷钴胺素（AdoB12）结合，促进反应调节因子的结合，从而导致终止子的破坏，并导致下游基因的转录。这项研究的基本原理是，它阐明了一种新型的B12核糖体开关，不同于以往的任何结构，调节结果（阳性与阴性），最重要的是，调节蛋白的额外参与。这种核糖开关的伴侣蛋白特征暗示了这些元素的扩展机制多样性，远远超出了目前的想法。目的1将确定AdoB12正向调节乙醇胺利用基因表达的机制。工作假设是AdoB12与先导RNA结合，诱导二级结构的变化，从而促进反应调节因子EutRR的结合。在确定先导RNA的转录起始位点后，我们将使用在线探针和SHAPE分析来证明AdoB12结合导致RNA的结构变化，从而增加其对EutRR的亲和力。目的2将阐明EutRR调节eut基因表达的机制。我们假设反应调节因子通过破坏位于编码区开始附近的终止子来积极调节基因表达。我们将通过显示缺失突变体中基因表达的缺失来确定EutRR是一个积极的调节因子。然后，我们将通过单轮体外转录试验证明EutRR具有抗终止子活性。作为拟议的调查的结果，一个非典型的B12核糖开关的机制将被揭示。这项研究具有重要意义，因为它将有助于开发核蛋白开关作为致病菌中可能的抗菌靶点。公共卫生相关性：本申请中提出的研究将有助于更好地了解人类细菌病原体中的调控RNA如何控制基因表达。这些知识与公共卫生相关，因为它将有助于重点利用这些调节机制作为潜在的抗微生物靶点。

DESCRIPTION (provided by applicant): One characteristic that contributes to Enterococcus faecalis's success as the second or third leading cause of hospital-acquired infection in the U.S. is its flexible metabolism, which enables survival under a wide variety of conditions. However, little is known about how E. faecalis regulates gene expression involved in employing alternative energy sources. The objective of this application is to elucidate the roles of an atypical B12 riboswitch and a response regulator involved in regulating ethanolamine utilization, a potentially valuable source of carbon and nitrogen in the host. Our central hypothesis is that a riboswitch located in an untranslated leader RNA in the ethanolamine-utilization operon, binds adenosylcobalamin (AdoB12), facilitating binding of a response regulator that leads to disruption of a terminator, and results in transcription of the downstream genes. The rationale for this research is that it elucidates a novel type of B12 riboswitch unlike any previously described in terms of structure, regulatory outcome (positive vs. negative), and most importantly, the additional involvement of a regulatory protein. The partner protein feature of this riboswitch implicates an expanded mechanistic diversity for these elements well beyond the current thinking. Aim #1 will identify the mechanism by which AdoB12 positively regulates the expression of the ethanolamine-utilization genes. The working hypothesis is that AdoB12 binds to the leader RNA inducing a change in the secondary structure that facilitates binding of the response regulator, EutRR. After establishing the transcriptional start site of the leader RNA, we will use in-line probing and SHAPE assays to demonstrate that AdoB12 binding causes structural change in the RNA, which increases its affinity for EutRR. Aim #2 will elucidate the mechanism by which EutRR regulates the expression of the eut genes. We postulate that the response regulator positively regulates gene expression by disrupting a terminator located just proximal to the start of the coding region. We will establish that EutRR is a positive regulator by showing loss of gene expression in a deletion mutant. Then we will show that EutRR has anti-terminator activity by a single-round in vitro transcription assay. As a result of the proposed investigations, the mechanism of an atypical B12 riboswitch will be uncovered. The research proposed is significant because it will contribute to efforts focused on exploiting riboswitches as possible antimicrobial targets in pathogenic bacteria. PUBLIC HEALTH RELEVANCE: The research proposed in this application will lead to greater understanding of how regulatory RNA's in a human bacterial pathogen control gene expression. Such knowledge is relevant to public health because it will contribute to efforts focused on exploiting these regulatory mechanisms as potential antimicrobial targets.