Discovery and Characterization of New Riboswitches

新核糖开关的发现和表征

• 批准号: 10378525
• 负责人: SCOTT A STROBEL
• 金额: $ 41.64万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 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
• 来源: https://reporter.nih.gov/project-details/10378525
• 关键词: Algorithms; Architecture; Binding; Biochemical; Biology; Collaborations; Crystallization; Dinucleoside Phosphates; Diphosphates; Elements; Family; Feedback; Fluorides; Gene Expression; Genes; Genetic Screening; Glycine; Guanidines; Individual; Investigation; Knowledge; Laboratories; Life; Ligand Binding; Ligands; Link; Massive Parallel Sequencing; Metabolic; Metabolism; Methods; Modeling; Molecular; Nucleotides; Organism; Orphan; Pathway interactions; Pattern; Periodicity; Probability; Process; RNA; Regulation; Regulatory Element; Resources; Second Messenger Systems; Serc; Signal Transduction; Specificity; Structure; Substrate Specificity; System; Validation; Variant; Work; aptamer; base; biophysical properties; chemical binding; gene function; insight; microorganism; mutant; novel; response; small molecule

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 核糖开关是重要的，无处不在的调节元素，结合小分子效应和 控制基因表达。许多已知的核糖开关效应器已被部分鉴定在基因上 在Riboswitch控制下。但是，在某些情况下，这种信息流已经逆转， 核糖开关类别的识别提供了有关基因功能的宝贵见解 规定。 该建议描述了寻找新的核糖开关的配体的搜索 表征和特异性改变的分子基础。我们将专注于突出的RNA图案 尚未确定配体。我们将确定这些RNA的晶体结构。我们将 确定哪种核苷酸对于几种具有的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”核糖开关候选者没有经过验证的配体。其中一些孤儿 Riboswitch候选人提供了巨大的机会，以扩大我们对重要代谢和 来自生命的各个领域的物种中的信号传导过程。我们将追求配体的识别 对于最突出的孤儿核糖开关候选人。增加我们的假设的可能性 有关孤儿的孤儿的配体身份对于孤儿数量最多 候选人，我们将采用一种新的基因筛查/选择方法将核糖开关的功能联系起来 替代微生物中的其他基因。 我们将在确定的多个核糖开关的结晶中进行结晶。结构将 建立配体结合的机理，并进一步揭示RNA三级褶皱的复杂性。结构 配体结合和对螺旋开关的研究将允许反馈到Riboswitch 搜索方法，支持发现先前被忽视的候选人。此外，每个结构 将提供洞察力，以告知搜索Riboswitch子类。 有几个示例，核糖开关序列的微小变化导致戏剧性 变化配体的特异性。我们将研究三个核糖开关家庭 变体子类。我们已经开发了一种基于平行的测序方法来生成 简单的数千个单个突变体的定量核糖开关活性谱。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和 滥交。它还将有助于发现新的子类，从而发现新的生物学。