National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
P01GM051487
United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
F32GM122334
United States
Life Sciences Research Foundation
United States
Citation
Journal: Nat Commun / Year: 2019 Title: Target preference of Type III-A CRISPR-Cas complexes at the transcription bubble. Authors: Tina Y Liu / Jun-Jie Liu / Abhishek J Aditham / Eva Nogales / Jennifer A Doudna / Abstract: Type III-A CRISPR-Cas systems are prokaryotic RNA-guided adaptive immune systems that use a protein-RNA complex, Csm, for transcription-dependent immunity against foreign DNA. Csm can cleave RNA and ...Type III-A CRISPR-Cas systems are prokaryotic RNA-guided adaptive immune systems that use a protein-RNA complex, Csm, for transcription-dependent immunity against foreign DNA. Csm can cleave RNA and single-stranded DNA (ssDNA), but whether it targets one or both nucleic acids during transcription elongation is unknown. Here, we show that binding of a Thermus thermophilus (T. thermophilus) Csm (TthCsm) to a nascent transcript in a transcription elongation complex (TEC) promotes tethering but not direct contact of TthCsm with RNA polymerase (RNAP). Biochemical experiments show that both TthCsm and Staphylococcus epidermidis (S. epidermidis) Csm (SepCsm) cleave RNA transcripts, but not ssDNA, at the transcription bubble. Taken together, these results suggest that Type III systems primarily target transcripts, instead of unwound ssDNA in TECs, for immunity against double-stranded DNA (dsDNA) phages and plasmids. This reveals similarities between Csm and eukaryotic RNA interference, which also uses RNA-guided RNA targeting to silence actively transcribed genes.
History
Deposition
Jan 12, 2019
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Header (metadata) release
Feb 20, 2019
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Map release
Jul 10, 2019
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Update
Sep 6, 2023
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Current status
Sep 6, 2023
Processing site: RCSB / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
Download / File: emd_0455.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation
Csm-RNAP
Voxel size
X=Y=Z: 4.36 Å
Density
Contour Level
By AUTHOR: 0.03 / Movie #1: 0.03
Minimum - Maximum
-0.03912864 - 0.112419434
Average (Standard dev.)
0.0001628129 (±0.006225997)
Symmetry
Space group: 1
Details
EMDB XML:
Map geometry
Axis order
X
Y
Z
Origin
-64
-64
-64
Dimensions
128
128
128
Spacing
128
128
128
Cell
A=B=C: 558.08 Å α=β=γ: 90.0 °
CCP4 map header:
mode
Image stored as Reals
Å/pix. X/Y/Z
4.36
4.36
4.36
M x/y/z
128
128
128
origin x/y/z
0.000
0.000
0.000
length x/y/z
558.080
558.080
558.080
α/β/γ
90.000
90.000
90.000
MAP C/R/S
1
2
3
start NC/NR/NS
-64
-64
-64
NC/NR/NS
128
128
128
D min/max/mean
-0.039
0.112
0.000
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Supplemental data
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Sample components
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Entire : Complex of Thermus thermophilus Csm with the Thermus thermophilus...
Entire
Name: Complex of Thermus thermophilus Csm with the Thermus thermophilus transcription elongation complex
Components
Complex: Complex of Thermus thermophilus Csm with the Thermus thermophilus transcription elongation complex
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Supramolecule #1: Complex of Thermus thermophilus Csm with the Thermus thermophilus...
Supramolecule
Name: Complex of Thermus thermophilus Csm with the Thermus thermophilus transcription elongation complex type: complex / ID: 1 / Parent: 0 Details: T. thermophilus Csm was recombinantly expressed in E. coli, and T. thermophilus RNA polymerase was isolated from the natural source.
Source (natural)
Organism: Thermus thermophilus (bacteria)
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Experimental details
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Structure determination
Method
negative staining, cryo EM
Processing
single particle reconstruction
Aggregation state
particle
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Sample preparation
Buffer
pH: 7.9
Staining
Type: NEGATIVE / Material: UA
Vitrification
Cryogen name: ETHANE
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Electron microscopy
Microscope
FEI TECNAI 12
Image recording
Film or detector model: FEI CETA (4k x 4k) / Average electron dose: 58.0 e/Å2
Electron beam
Acceleration voltage: 120 kV / Electron source: LAB6
Electron optics
Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
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Image processing
Startup model
Type of model: NONE
Final reconstruction
Resolution.type: BY AUTHOR / Resolution: 17.0 Å / Resolution method: FSC 0.5 CUT-OFF / Number images used: 8351
Initial angle assignment
Type: ANGULAR RECONSTITUTION
Final angle assignment
Type: MAXIMUM LIKELIHOOD
+
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