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

Montana State University - Bozeman, Bozeman MT

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Abstract

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.