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:
-
- Montana State Univ., Bozeman, MT (United States)
- Johns Hopkins School of Public Health, Baltimore, MD (United States)
- Publication Date:
- Research Org.:
- Argonne National Laboratory (ANL), Argonne, IL (United States)
- Sponsoring Org.:
- USDOE; National Science Foundation (NSF); National Inst. of Health
- OSTI Identifier:
- 1252758
- Grant/Contract Number:
- AC02-06CH11357; AC02-76SF00515; P41GM103393; F32 GM108436; P20GM10347; R01GM097330; P20GM103500; R01GM108888; EPS-110134
- Resource Type:
- Accepted Manuscript
- Journal Name:
- Nucleic Acids Research
- Additional Journal Information:
- Journal Volume: 43; Journal Issue: 17; Journal ID: ISSN 0305-1048
- Publisher:
- Oxford University Press
- Country of Publication:
- United States
- Language:
- ENGLISH
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES
Citation Formats
van Erp, Paul B. G., Jackson, Ryan N., Carter, Joshua, Golden, Sarah M., Bailey, Scott, and Wiedenheft, Blake. Mechanism of CRISPR-RNA guided recognition of DNA targets in Escherichia coli. United States: N. p., 2015.
Web. doi:10.1093/nar/gkv793.
van Erp, Paul B. G., Jackson, Ryan N., Carter, Joshua, Golden, Sarah M., Bailey, Scott, & Wiedenheft, Blake. Mechanism of CRISPR-RNA guided recognition of DNA targets in Escherichia coli. United States. https://doi.org/10.1093/nar/gkv793
van Erp, Paul B. G., Jackson, Ryan N., Carter, Joshua, Golden, Sarah M., Bailey, Scott, and Wiedenheft, Blake. Mon .
"Mechanism of CRISPR-RNA guided recognition of DNA targets in Escherichia coli". United States. https://doi.org/10.1093/nar/gkv793. https://www.osti.gov/servlets/purl/1252758.
@article{osti_1252758,
title = {Mechanism of CRISPR-RNA guided recognition of DNA targets in Escherichia coli},
author = {van Erp, Paul B. G. and Jackson, Ryan N. and Carter, Joshua and Golden, Sarah M. and Bailey, Scott and Wiedenheft, Blake},
abstractNote = {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.},
doi = {10.1093/nar/gkv793},
journal = {Nucleic Acids Research},
number = 17,
volume = 43,
place = {United States},
year = {Mon Aug 03 00:00:00 EDT 2015},
month = {Mon Aug 03 00:00:00 EDT 2015}
}
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