Structural and functional characterization of the Thermotoga maritima Cmr complex

描述(申请人提供)：在原核生物中，成簇的规则间隔短回文重复(CRISPR)基因座编码小CRISPR RNAs(CrRNA)，保护免受入侵的遗传因素。这些crRNAs被各种CRISPR相关(CAS)蛋白用作靶向外源遗传元件的向导，是保护宿主基因组的原核防御系统的基础。Cas模块RAMP(Cmr)蛋白形成的复合体在CRISPR/Cas系统中是独一无二的，因为它具有靶向和切割RNA的能力，而其他系统则靶向DNA。CmR复合体由6个蛋白质亚基和一个crRNA组成，命名为CMR1-6。我们的目标是利用生化和结构方法了解单个CmR亚单位的作用及其在复合体中的相互作用。了解CRISPR/Cas复合体的这些基本性质是很重要的，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。在更好地了解后，我们希望这一自然途径的操纵可能被用来阻止抗菌素耐药因素的传播。已有研究表明，CRISPR/Cas系统能够通过靶向降解结合质粒来限制水平基因转移，这是细菌获得耐药性的主要途径。可以设想这样一种作用，即Cmr可以特别地被用来沉默RNA转录本，它可能编码促进HGT的蛋白质。这些发展可能会有相当大的公共卫生应用，并将与NIH的目标保持一致，即促进我们对生物系统的了解，控制疾病和改善人类健康。

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.