title: Mechanism of CRISPR-RNA guided recognition of DNA targets in Escherichia coli.
abstract: In 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 Å 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.
authors.affiliation: Department of Microbiology and Immunology, Montana State University, Bozeman, MT 59717, USA.
authors.forename: Paul B G
authors.initials: PB
authors.lastname: van Erp
authors.affiliation: Department of Microbiology and Immunology, Montana State University, Bozeman, MT 59717, USA.
authors.forename: Ryan N
authors.initials: RN
authors.lastname: Jackson
authors.affiliation: Department of Microbiology and Immunology, Montana State University, Bozeman, MT 59717, USA.
authors.forename: Joshua
authors.initials: J
authors.lastname: Carter
authors.affiliation: Department of Microbiology and Immunology, Montana State University, Bozeman, MT 59717, USA.
authors.forename: Sarah M
authors.initials: SM
authors.lastname: Golden
authors.affiliation: Department of Biochemistry and Molecular Biology, Johns Hopkins School of Public Health, Baltimore, MD 21205, USA.
authors.forename: Scott
authors.initials: S
authors.lastname: Bailey
authors.affiliation: Department of Microbiology and Immunology, Montana State University, Bozeman, MT 59717, USA bwiedenheft@gmail.com.
authors.forename: Blake
authors.initials: B
authors.lastname: Wiedenheft
mesh_terms: D001120:Arginine / Q000737:chemistry; D064130:CRISPR-Associated Proteins / Q000737:chemistry* / Q000378:metabolism; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D008239:Lysine / Q000737:chemistry; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D059372:Nucleotide Motifs; D011485:Protein Binding; D011487:Protein Conformation; D012329:RNA, Bacterial / Q000378:metabolism
chemical_list: D064130:CRISPR-Associated Proteins; D029968:Escherichia coli Proteins; D012329:RNA, Bacterial; D004247:DNA; D001120:Arginine; D008239:Lysine