Structural and Kinetic Basis of RNA-guided Adaptive Immunity in Bacteria

描述(申请人提供)：细菌和古生菌通过将外来DNA的短片段整合到成簇的规则间隔短回文重复(CRISPR)中获得抗药性病毒和质粒。这一过程导致了先前核酸入侵的遗传记录。CRISPR基因座被转录和加工成CRISPR衍生的短RNA(CrRNA)，其中包含来自以前的遗传挑战者的独特序列并与之互补。在大肠杆菌中，crRNA被组装成一个大的(405 KDa)多亚基监视复合体，称为Cascade(CRISPR相关的抗病毒防御复合体)。CASCADE在细胞内环境中巡逻，通过crRNA介导的碱基配对与入侵的DNA序列结合。靶结合导致级联亚单位和DNA靶的构象重排。我们假设这些重排揭示了增强Cas3的招募和激活的表面特征，Cas3是靶标降解所需的核酸酶/解旋酶。为了验证这一假设，我们使用结构和生化策略来确定Cascade如何与DNA靶标结合，以及核酸结合如何促进Cas3的招募。具体地说，这个建议的目的是(1)确定干扰每个阶段的级联结构，(2)使用表面等离子体共振来确定靶结合和Cas3募集的动力学参数。

DESCRIPTION (provided by applicant): Bacteria and archaea acquire resistance viruses and plasmids by integrating short fragments of foreign DNA into clustered regularly interspaced short palindromic repeat (CRISPR). This process results in a genetic record of previous nucleic acid invasions. CRISPR loci are transcribed and processed into short CRISPR- derived RNAs (crRNA) that contain unique sequences derived from and complementary to previous genetic challengers. In Escherichia coli, the crRNA is assembled into a large (405 kDa) multi-subunit surveillance complex called Cascade (CRISPR-associated complex for antiviral defense). Cascade patrols the intracellular environment and binds to invading DNA sequences through crRNA-mediated base pairing. Target binding causes a conformational rearrangement of Cascade subunits and the DNA target. We hypothesize that these rearrangements reveal surface features that enhance the recruitment and activation of Cas3, a nuclease/helicase that is required for target degradation. To test this hypothesis we use structural, and biochemical strategies to determine how Cascade binds DNA targets, and how nucleic acid binding promotes the recruitment of Cas3. Specifically, this proposal aims to (1) determine the structures of Cascade at each stage of interference and (2) use surface plasmon resonance to determine the kinetic parameters of target binding and Cas3 recruitment.