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PDBsum entry 3ps0
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RNA binding protein
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PDB id
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3ps0
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References listed in PDB file
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Key reference
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Title
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Structural and functional characterization of an archaeal clustered regularly interspaced short palindromic repeat (crispr)-Associated complex for antiviral defense (cascade).
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Authors
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N.G.Lintner,
M.Kerou,
S.K.Brumfield,
S.Graham,
H.Liu,
J.H.Naismith,
M.Sdano,
N.Peng,
Q.She,
V.Copié,
M.J.Young,
M.F.White,
C.M.Lawrence.
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Ref.
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J Biol Chem, 2011,
286,
21643-21656.
[DOI no: ]
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PubMed id
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Abstract
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In response to viral infection, many prokaryotes incorporate fragments of
virus-derived DNA into loci called clustered regularly interspaced short
palindromic repeats (CRISPRs). The loci are then transcribed, and the processed
CRISPR transcripts are used to target invading viral DNA and RNA. The
Escherichia coli "CRISPR-associated complex for antiviral defense"
(CASCADE) is central in targeting invading DNA. Here we report the structural
and functional characterization of an archaeal CASCADE (aCASCADE) from
Sulfolobus solfataricus. Tagged Csa2 (Cas7) expressed in S. solfataricus
co-purifies with Cas5a-, Cas6-, Csa5-, and Cas6-processed CRISPR-RNA (crRNA).
Csa2, the dominant protein in aCASCADE, forms a stable complex with Cas5a.
Transmission electron microscopy reveals a helical complex of variable length,
perhaps due to substoichiometric amounts of other CASCADE components. A
recombinant Csa2-Cas5a complex is sufficient to bind crRNA and complementary
ssDNA. The structure of Csa2 reveals a crescent-shaped structure unexpectedly
composed of a modified RNA-recognition motif and two additional domains present
as insertions in the RNA-recognition motif. Conserved residues indicate
potential crRNA- and target DNA-binding sites, and the H160A variant shows
significantly reduced affinity for crRNA. We propose a general subunit
architecture for CASCADE in other bacteria and Archaea.
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Figure 2.
S. solfataricus Cas6 generates crRNA. A, a two-repeat spacer
unit CRISPR transcript was cleaved by Cas6 at a single site in
each repeat, yielding fragments of 109 and 43 nt for cleavage at
repeat 1, 106 and 46 nt for cleavage at repeat 2, and the 63-nt
mature crRNA for cleavage at both repeats. B, a synthetic RNA
corresponding to a single CRISPR repeat with a 15-unit 5′
extension is cleaved by Cas6 at a single site, generating an
8-nt repeat-derived 5′ extension (“psi-tag”). C, schematic
illustrating the 2-repeat transcript and the expected cleavage
products. D, schematic illustrating the synthetic substrate.
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Figure 5.
The structural similarity between Csa2 and Cas6 is limited to
the RNA-recognition motif. A, Csa2 and Cas6 are shown in
equivalent orientations based on an SSM Structural alignment.
The RRM-like subdomains are colored violet in both structures.
The Csa2 1–3 domain is colored red, the 2–4 domain is
colored orange, and the C-terminal subdomain is colored yellow.
The portions of the Cas6 N-terminal domain that do not exhibit
similarity to the Csa2 RRM subdomain are depicted in green and
the C-terminal domain in light cyan. The conserved clusters on
Csa2 and the putative active site residues on Cas6 are shown as
“sticks” colored in dark cyan.
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The above figures are
reprinted
from an Open Access publication published by the ASBMB:
J Biol Chem
(2011,
286,
21643-21656)
copyright 2011.
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