Structural and functional characterization of the Thermotoga maritima Cmr complex

海栖热袍菌 Cmr 复合体的结构和功能表征

• 批准号: 8821632
• 负责人: Michael Anthony Estrella
• 金额: $ 4.31万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-04-01 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8821632
• 关键词: Address; Antimicrobial Resistance; Architecture; Bacteria; Binding; Biochemical; Biochemistry; Biological Assay; Catalytic Domain; Cleaved cell; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Complex; Crystallography; DNA; Data; Data Set; Development; Genetic Materials; Genome; Goals; Guide RNA; Health; Horizontal Gene Transfer; Human; Immune; Immune system; Immunity; In Vitro; Incubated; Individual; Kinetics; Knowledge; Length; Maps; Methods; Modeling; Molecular; Molecular Sieve Chromatography; Mutation Analysis; Names; Nucleic Acids; Nucleotides; Organism; Pathway interactions; Plasmids; Prokaryotic Cells; Property; Protein Subunits; Proteins; Public Health Applications Research; Publishing; Pyrococcus furiosus; RNA; RNA Interference; RNA-Induced Silencing Complex; RNA-Protein Interaction; Reaction; Reading; Recombinants; Resistance; Resolution; Role; Route; Set protein; Small RNA; Structural Models; Structure; System; Techniques; Testing; Thermotoga maritima; Thermus thermophilus; Time; Transcript; United States National Institutes of Health; Virus; bacterial resistance; base; biological systems; disorder control; electron density; expectation; genetic element; improved; in vivo; interest; new technology; novel; nuclease; protein complex; reconstitution; resistance factors; structural biology

DESCRIPTION (provided by applicant): In prokaryotes, clustered regularly interspaced short palindromic repeat (CRISPR) loci encode small CRISPR RNAs (crRNA) that protect against invasive genetic elements. These crRNAs are used as guides by various CRISPR associated (Cas) proteins to target foreign genetic elements and are the bases of a prokaryotic defense system that preserves the host genome. The Cas module RAMP (Cmr) proteins form a complex that is unique amongst the CRISPR/Cas systems for its ability to target and cleave RNA, whereas the other systems target DNA. The Cmr complex is composed of 6 protein subunits, named Cmr1-6, and a crRNA. Our goal is to understand the roles of the individual Cmr subunits and their interactions in the complex using biochemical and structural approaches. It is important to understand these fundamental properties of CRISPR/Cas complexes because of their central role in prokaryotic immunity and our lack of knowledge in this important pathway. Upon better understanding, we hope that manipulation of this natural pathway may be used to impair the spread of antimicrobial resistance factors. It has been demonstrated that the CRISPR/Cas system is able to limit horizontal gene transfer, which is the main route for bacteria to acquire resistance, by targeting the degradation of conjugative plasmids. A role can be conceived whereby specifically Cmr can be used silence RNA transcripts that may code for proteins that facilitate HGT. Such developments may have considerable public health applications and would be consistent with the NIH goals of advancing our understanding of biological systems, controlling disease and improving human health.

描述（由申请人提供）：在原核生物中，成簇的规则间隔短回文重复序列（CRISPR）基因座编码小CRISPR RNA（crRNA），其保护免受入侵遗传元件的侵害。这些crRNA被各种CRISPR相关（Cas）蛋白用作靶向外源遗传元件的向导，并且是保留宿主基因组的原核防御系统的基础。Cas模块RAMP（Cmr）蛋白形成复合物，该复合物在CRISPR/Cas系统中是独特的，因为其靶向和切割RNA的能力，而其他系统靶向DNA。Cmr复合物由6个蛋白质亚基（命名为Cmr 1 -6）和一个crRNA组成。我们的目标是了解的作用，个别CMR亚基和它们的相互作用，在复杂的使用生化和结构的方法。理解CRISPR/Cas复合物的这些基本特性很重要，因为它们在原核免疫中起着核心作用，而我们对这一重要途径缺乏了解。在更好的理解之后，我们希望操纵这种天然途径可以用来削弱抗菌素耐药因子的传播。已经证明，CRISPR/Cas系统能够通过靶向接合质粒的降解来限制水平基因转移，这是细菌获得抗性的主要途径。可以设想一种作用，由此可以特异性地使用Cmr沉默RNA转录物，其可以编码促进HGT的蛋白质。这些发展可能具有相当大的公共卫生应用，并与NIH的目标相一致，即促进我们对生物系统的理解，控制疾病和改善人类健康。