Administrative Supplement to Existing NIH Grant and Cooperative Agreement

描述(申请人提供)：生物学中出现了一种新的范式，在这种范式中，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，这些作用还被保留给蛋白质。有两个新出现的主题特别吸引人，一直是我实验室的研究重点。第一个主题涉及RNA作为向导，一个信息载体，指导蛋白质对核酸靶标的作用。以RNAi和CRISPR-Cas为例的这种系统中的力量，可以被用于治疗学和基因组工程应用。到目前为止，已在88%的古菌基因组和39%的细菌基因组中发现了CRISPR-Cas防御系统，其中包括重要的人类病原体，如人类空肠弯曲菌、肉毒梭菌、大肠杆菌、单核细胞增生性李斯特菌、结核分枝杆菌和鼠疫耶尔森氏菌。它已被证明可以调节水平的基因转移和生物膜的形成。我们提出的项目1是基于几个重要的CaS蛋白的成功结构确定和从盐生双歧杆菌中成功地重组I-C型级联复合体。在该方案中，我们建议通过实验来理解I-C CRISPR-CAS系统中的CRISPR干扰机制。我们基于强大的初步数据来(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系统中的一个必不可少的因子。(3)捕获Cascade-dsDNA和Casade-Cas3复合体的结构快照。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结构RNA在顺式结构中执行基因调控功能。被称为核糖开关的顺式作用RNA短元件的发现导致了基因调控概念的范式转变。核糖开关广泛存在于原核生物中，据估计，它们控制着原核生物中多达2%-4%的基因。它们几乎只在顺式结构中起作用，通常位于宿主mRNAs的5‘非翻译区(5’-UTRs)，主要通过提前终止转录或抑制翻译起始来调节基因的表达，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，并相应地协调了我们的努力：1.利用核糖开关的新的配体传感策略，其研究可能揭示细菌生理的新方面(在项目2中对T盒核糖开关的研究)；2.对构象转换机制的更深层次的理解(在项目3中的yplop-ykoY孤儿核糖开关)；3.孤儿核糖开关家族的结构-功能表征(项目3)；以及4.合成生物学在工业、医学、制药或环境保护中的应用(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).