Administrative Supplement to Existing NIH Grant and Cooperative Agreement

描述（由申请人提供）：生物学中出现了一种新的范式，其中RNA分子是调节、催化和控制基本细胞过程的积极参与者-直到最近才为蛋白质保留的角色。有两个新兴主题特别吸引人，也是我实验室的研究重点。第一个主题涉及RNA作为一种向导，一种信息载体，指导蛋白质对核酸目标的作用。以RNAi和CRISPR-Cas为例，这种系统的力量可以用于治疗和基因组工程应用。到目前为止，已有88%的古细菌基因组和39%的细菌基因组中发现了CRISPR-Cas防御系统，包括重要的人类病原体，如人类空肠弯曲杆菌、肉毒梭菌、大肠杆菌、单核增生李斯特菌、结核分枝杆菌和鼠疫杆菌。它已被证明可以调节水平基因转移和生物膜的形成。我们提出的项目1是基于成功地确定几个重要的Cas蛋白的结构，并成功地重建了B. halodurans的I-C型级联复合体。在本提案中，我们提出了在I-C型CRISPR- Cas系统中了解CRISPR干扰机制的实验。我们建立在强大的初步数据基础上：(1)表征I- c型系统中目标搜索级联复合体的结构-功能，(2)表征级联相互作用蛋白Cas3的结构-功能，Cas3是所有I型CRISPR-Cas系统中的重要因素。(3)获取Cascade-dsDNA和Cascade-Cas3复合物的结构快照。我们的发现将有助于揭示不同CRISPR-Cas系统之间的共同主题和机制多样性。RNA生物学的第二个中心主题涉及在顺式中执行基因调控功能的结构化RNA。被称为核糖开关的短顺式作用RNA元件的发现导致了基因调控概念的范式转变。核开关在原核生物中广泛存在，据估计它们控制着厚壁菌门中多达2-4%的基因。它们几乎完全顺式发挥作用，通常位于宿主mRNA的5‘非翻译区（5’- UTRs），主要通过过早终止转录或抑制翻译起始来调节基因表达，尽管已经证明了其他调节机制，包括控制mRNA的切割、稳定性和选择性剪接。我们确定了核糖开关研究的以下前沿，并相应地调整了我们的努力：1。利用核糖开关的新型配体传感策略，其研究可能揭示细菌生理学的新方面（项目2中对T盒核糖开关的研究）；2. 更深入地了解构象开关机制（项目3中的yybP-ykoY孤儿核开关）；3. 孤儿核开关家族的结构-功能表征（项目3）；和4。合成生物学在工业、医学、制药或环境保护方面的应用（项目3中的荧光Mn2+传感器应用）。

DESCRIPTION (provided by applicant): A new paradigm has emerged in biology in which RNA molecules are active participants in regulating, catalyzing and controlling fundamental cellular processes - roles that were reserved for proteins until recently. Two emerging themes are particularly fascinating and have been the research focus in my lab. The first theme involves RNA serving as a guide, an information carrier, to direct the action of proteins on nucleic acid targets. The power in such systems, exemplified by RNAi and CRISPR-Cas, can be harnessed for therapeutics as well as genome engineering applications. CRISPR-Cas defense systems have been identified in 88% of archaeal genomes and 39% of bacterial genomes thus far sequenced, including important human pathogens such as Campylobacter human jejuni, Clostridium botulinum, Escherichia coli, Listeria monocytogenes, Mycobacterium tuberculosis and Yersinia pestis. It has been shown to modulate the horizontal gene transfer and biofilm formation. Our proposed Project 1 is based on the successful structure determination of several important Cas proteins and the successful reconstitution of the Type I-C Cascade complex from B. halodurans. In this proposal, we propose experiments to understand the CRISPR interference mechanism in Type I-C CRISPR- Cas system. We build upon strong preliminary data to (1) characterize the structure-function of the target searching Cascade complex in Type I-C system, (2) characterize the structure-function of the Cascade- interacting protein Cas3, an essential factor in all Type I CRISPR-Cas systems. (3) capture structure snapshots of the Cascade-dsDNA and the Cascade-Cas3 complexes. Our finding will serve to reveal the common theme and mechanistic diversity among different CRISPR-Cas systems. The second central theme in RNA biology involves structured RNAs performing gene regulatory function in cis. The discovery of short cis- acting RNA elements termed riboswitches led to a paradigm shift in the concept of gene regulation. Riboswitches are widespread in prokaryotes, where they are estimated to control as many as 2-4% of all genes in Firmicutes. They almost exclusively function in cis, usually reside in the 5' untranslated regions (5'- UTRs) of the host mRNAs, and regulate gene expression mainly through the means of premature transcription termination or inhibition of translation initiation, although other regulatory mechanisms including the control of mRNA cleavage, stability, and alternative splicing have been demonstrated. We identify the following frontiers in the riboswitch research and align our efforts accordingly: 1. novel ligand sensing strategy utilized riboswitches, the study of which may reveal novel aspects of bacterial physiology (the study of T box riboswitches in Project 2); 2. deeper understanding of the conformational switching mechanism (the yybP-ykoY orphan riboswitches in Project 3); 3. structure-function characterization of orphan riboswitch families (Project 3); and 4. synthetic biology applications in industry, medicine, pharmacy or environmental protection (fluorescent Mn2+ sensor applications in Project 3).