Discovery and Characterization of New Riboswitches

新核糖开关的发现和表征

基本信息

批准号：
10580082
负责人：
SCOTT A STROBEL
金额：
$ 39.43万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-04-01 至 2024-03-31
项目状态：
已结题

项目摘要

Project Summary Riboswitches are important and ubiquitous regulatory elements that bind small-molecule effectors and control gene expression. Many of the known riboswitch effectors have been identified based in part on the genes under riboswitch control. However, in some cases this flow of information has been reversed, where the identification of a riboswitch class has provided valuable insight about the function of the genes under its regulation. This proposal describes the search for the ligands for new classes of riboswitches, their structural characterization, and the molecular basis of altered specificity. We will focus on prominent RNA motifs for which no ligand has been identified. We will determine the crystal structures of these RNAs. We will determine which nucleotides are necessary for altered specificity and function for several riboswitches that have variant subclasses. More than 50 “orphan” riboswitch candidates do not have validated ligands. Some of these orphan riboswitch candidates present enormous opportunities to expand our knowledge of important metabolic and signaling processes in species from various domains of life. We will pursue the identification of the ligands for the most prominent orphan riboswitch candidates. To increase the probability that our hypotheses regarding the ligand identity for an orphan riboswitch is strong for the largest number of orphan candidates, we will employ a new genetic screening/selection approach to link the function of a riboswitch to additional genes in a surrogate microorganism. We will pursue crystallization of multiple riboswitches as they are identified. The structures will establish the mechanism of ligand binding and further reveal the complexity of RNA tertiary folds. Structural analysis of ligand binding and investigation of helical switching will allow for feedback into riboswitch search methods, facilitating discovery of previously overlooked candidates. Furthermore, each structure will provide insights that will inform the search for riboswitch subclasses. There are several examples where small changes in riboswitch sequence result in dramatic changes to the specificity of the ligand. We will pursue studies of three riboswitch families that have variant subclasses. We have developed a massively parallel sequencing-based approach to generate quantitative riboswitch activity profiles for thousands of individual mutants simultaneously. This work will establish evolutionary pathways for RNA function and the energetic landscape of substrate specificity and promiscuity. It will also aid in discovery of new subclasses and therefore new biology.

项目概要核糖开关是重要且普遍存在的调节元件，可结合小分子效应子和许多已知的核糖开关效应子已部分基于这些基因被识别。然而，在某些情况下，这种信息流是相反的，其中核糖开关类别的鉴定为了解其下的基因功能提供了宝贵的见解规定。该提案描述了寻找新型核糖开关的配体、其结构我们将重点关注重要的 RNA 基序。我们将确定这些 RNA 的晶体结构。确定哪些核苷酸对于具有某些核糖开关的特异性和功能是必需的变体子类。超过 50 个“孤儿”核糖开关候选物没有经过验证的配体，其中一些孤儿核糖开关候选者没有经过验证的配体。核糖开关候选者为扩展我们对重要代谢和代谢的知识提供了巨大的机会我们将致力于鉴定来自生命各个领域的物种的信号传导过程。为最突出的孤儿核糖开关候选者增加我们的假设的可能性。关于孤儿核糖开关的配体身份对于最大数量的孤儿来说是很强的对于候选者，我们将采用一种新的基因筛选/选择方法将核糖开关的功能与替代微生物中的额外基因。当结构被确定时，我们将追求多个核糖开关的结晶。建立配体结合机制并进一步揭示RNA三级折叠的复杂性。配体结合的分析和螺旋开关的研究将允许反馈到核糖开关搜索方法，有助于发现以前被忽视的候选人。将提供有助于寻找核糖开关子类的见解。有几个例子表明核糖开关序列的微小变化会导致戏剧性的结果我们将继续研究具有配体特异性的三个核糖开关家族。我们开发了一种基于大规模并行测序的方法来生成变体子类。这项工作将同时对数千个个体突变体进行定量核糖开关活性分析。建立 RNA 功能的进化途径和底物特异性的能量景观它还将有助于发现新的亚类，从而发现新的生物学。