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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.

项目摘要核糖开关是重要且普遍存在的调节元件，其结合小分子效应物，控制基因表达。许多已知的核糖开关效应子已经部分地基于以下基因被鉴定：在核糖开关控制下。然而，在某些情况下，这种信息流动发生了逆转，核糖开关类的鉴定提供了关于其下基因功能的有价值的见解。调控该提案描述了寻找新类型的核糖开关的配体，其结构特征，以及改变特异性的分子基础。我们将集中在突出的RNA基序，没有发现配体。我们将确定这些RNA的晶体结构。我们将确定哪些核苷酸是改变特异性和功能所必需的几个核糖开关，变体子类。超过50种“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人提供了巨大的机会，以扩大我们的知识，重要的代谢和在不同生命领域的物种中的信号传递过程。我们会继续鉴定配体最突出的孤儿核糖开关候选者。为了增加我们的假设关于孤儿核糖开关的配体身份，对于最大数量的孤儿核糖开关，候选人，我们将采用一种新的遗传筛选/选择方法来连接核糖开关的功能，替代微生物中的额外基因。我们将追求多个核糖开关的结晶，因为他们被确定。这些结构将建立配体结合机制，进一步揭示RNA三级折叠的复杂性。结构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中搜索方法，促进发现以前被忽视的候选人。此外，每个结构将提供见解，将通知核糖开关子类的搜索。有几个例子，核糖开关序列的微小变化导致了戏剧性的改变配体的特异性。我们将继续研究三个核糖开关家族，变体子类。我们开发了一种基于大规模并行测序的方法来生成同时对数千个个体突变体的定量核糖开关活性谱进行分析。这项工作将建立RNA功能的进化途径和底物特异性的能量景观，滥交它还将有助于发现新的子类，从而发现新的生物学。