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主要原因的一个特征是其灵活的代谢，这使得能够在各种条件下存活。然而，很少有人知道E。粪便调节基因表达涉及采用替代能源。本申请的目的是阐明非典型B12核糖开关和参与调节乙醇胺利用的反应调节剂的作用，乙醇胺是宿主中潜在的有价值的碳和氮源。我们的中心假设是，核糖开关位于乙醇胺利用操纵子中的非翻译前导RNA中，结合腺苷钴胺素（腺苷钴胺素B12），促进反应调节剂的结合，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核糖开关，不同于任何以前描述的结构，调节结果（阳性与阴性），最重要的是，额外参与的调节蛋白。这种核糖开关的伴侣蛋白特征暗示了这些元素的机械多样性远远超出了目前的想法。目的#1将确定B12正调节乙醇胺利用基因表达的机制。工作假设是，THB12结合到前导RNA，诱导二级结构的变化，促进反应调节因子EutRR的结合。在建立前导RNA的转录起始位点后，我们将使用在线探测和SHAPE测定来证明B12结合引起RNA的结构变化，这增加了其对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，反应调节积极调节基因表达的终止子位于附近的编码区的开始破坏。我们将通过显示缺失突变体中基因表达的丧失来确定EutRR是一种正调控因子。然后，我们将通过单轮体外转录测定来证明EutRR具有抗终止子活性。由于拟议的调查，一个非典型的B12核糖开关的机制将被揭露。提出的研究是重要的，因为它将有助于努力集中在利用核糖开关作为可能的抗菌靶标在病原菌。公共卫生相关性：本申请中提出的研究将导致更好地理解人类细菌病原体中的调节RNA如何控制基因表达。这些知识与公共卫生有关，因为它将有助于集中利用这些调节机制作为潜在的抗菌靶标。

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.