5cd4
The Type IE CRISPR Cascade complex from E. coli, with two assemblies in the asymmetric unit arranged back-to-back
Structural highlights
FunctionCAS5_ECOLI CRISPR (clustered regularly interspaced short palindromic repeat), is an adaptive immune system that provides protection against mobile genetic elements (viruses, transposable elements and conjugative plasmids). CRISPR clusters contain sequences complementary to antecedent mobile elements and target invading nucleic acids. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA).[1] [2] [3] A component of Cascade, which participates in CRISPR interference, the third stage of CRISPR immunity. Cascade binds both crRNA and in a sequence-specific manner negatively supercoiled dsDNA target. This leads to the formation of an R-loop in which the crRNA binds the target DNA, displacing the noncomplementary strand. Cas3 is recruited to Cascade, nicks target DNA and then unwinds and cleaves the target, leading to DNA degradation and invader neutralization. CasCDE alone is also able to form R-loops.[4] [5] [6] Publication Abstract from PubMedIn bacteria and archaea, short fragments of foreign DNA are integrated into Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) loci, providing a molecular memory of previous encounters with foreign genetic elements. In Escherichia coli, short CRISPR-derived RNAs are incorporated into a multi-subunit surveillance complex called Cascade (CRISPR-associated complex for antiviral defense). Recent structures of Cascade capture snapshots of this seahorse-shaped RNA-guided surveillance complex before and after binding to a DNA target. Here we determine a 3.2 A x-ray crystal structure of Cascade in a new crystal form that provides insight into the mechanism of double-stranded DNA binding. Molecular dynamic simulations performed using available structures reveal functional roles for residues in the tail, backbone and belly subunits of Cascade that are critical for binding double-stranded DNA. Structural comparisons are used to make functional predictions and these predictions are tested in vivo and in vitro. Collectively, the results in this study reveal underlying mechanisms involved in target-induced conformational changes and highlight residues important in DNA binding and protospacer adjacent motif recognition. Mechanism of CRISPR-RNA guided recognition of DNA targets in Escherichia coli.,van Erp PB, Jackson RN, Carter J, Golden SM, Bailey S, Wiedenheft B Nucleic Acids Res. 2015 Aug 3. pii: gkv793. PMID:26243775[7] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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