Discovery and Characterization of New Riboswitches

项目摘要 核糖开关是重要的和普遍存在的调节元件，它结合小分子效应器和 控制基因表达。许多已知的核糖开关效应部分是基于基因被鉴定出来的。 在核糖开关的控制下。然而，在某些情况下，这种信息流已经颠倒，其中 核糖开关类别的鉴定为了解其下基因的功能提供了有价值的见解 监管。 该建议描述了寻找新类别的核糖开关的配体，它们的结构 特异度改变的分子基础。我们将专注于突出的RNA基序 目前还没有确定其配基。我们将确定这些RNA的晶体结构。我们会 确定哪些核苷酸是改变几个核糖开关的特异性和功能所必需的 变异子类。 50多个“孤儿”核糖开关候选分子没有经过验证的配体。这些孤儿中的一些 核糖开关候选者提供了巨大的机会来扩大我们对重要新陈代谢和 来自生命各个领域的物种的信号传递过程。我们将继续进行配基的鉴定 最杰出的孤儿核糖开关候选人。为了增加我们的假设 关于孤儿的配基同一性，核糖开关对于最多的孤儿来说是强有力的 ，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 代理微生物中的额外基因。 我们将寻求多个核糖开关的结晶，因为它们被识别出来。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，便于发现以前被忽视的候选人。此外，每个结构 将提供深入的见解，为搜索核糖开关亚类提供信息。 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 改变配基的专一性。我们将继续对三个核糖开关家族进行研究 变异子类。我们已经开发了一种基于大规模并行测序的方法来生成 数千个个体突变体同时的定量核糖开关活性图谱。这项工作将 为RNA功能和底物专一性和能量格局建立进化途径 乱交。它还将有助于发现新的亚类，从而发现新的生物学。

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.