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- EMDB-50591: Composite map of the mycobacterial PafBC-bound transcription init... -
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Open data
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Basic information
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Title | Composite map of the mycobacterial PafBC-bound transcription initiation complex | |||||||||
![]() | Sharpened map. | |||||||||
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![]() | RNAP / PafBC / sigma adaptation / WYL domain / TRANSCRIPTION | |||||||||
Function / homology | ![]() sigma factor activity / bacterial-type RNA polymerase core enzyme binding / DNA-directed RNA polymerase complex / DNA-templated transcription initiation / ribonucleoside binding / : / : / : / : / : ...sigma factor activity / bacterial-type RNA polymerase core enzyme binding / DNA-directed RNA polymerase complex / DNA-templated transcription initiation / ribonucleoside binding / : / : / : / : / : / : / DNA-directed RNA polymerase / protein dimerization activity / response to antibiotic / positive regulation of DNA-templated transcription / magnesium ion binding / DNA binding / zinc ion binding / cytoplasm Similarity search - Function | |||||||||
Biological species | ![]() | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 4.0 Å | |||||||||
![]() | Zdanowicz R / Schilling CM / Rabl J / Mueller AU / Boehringer D / Glockshuber R / Weber-Ban E | |||||||||
Funding support | ![]()
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![]() | ![]() Title: Single-stranded DNA binding to the transcription factor PafBC triggers the mycobacterial DNA damage response. Authors: Charlotte M Schilling / Rafal Zdanowicz / Julius Rabl / Andreas U Müller / Daniel Boehringer / Rudi Glockshuber / Eilika Weber-Ban / ![]() Abstract: The DNA damage response in mycobacteria is controlled by the heterodimeric transcription factor PafBC, a member of the WYL domain-containing protein family. It has been shown that PafBC induces ...The DNA damage response in mycobacteria is controlled by the heterodimeric transcription factor PafBC, a member of the WYL domain-containing protein family. It has been shown that PafBC induces transcription of its regulon by reprogramming the housekeeping RNA polymerase holoenzyme to recognize PafBC-dependent promoters through sigma adaptation. However, the mechanism by which DNA damage is sensed and translated into PafBC activation has remained unclear. Here, we demonstrate that the binding of single-stranded DNA (ssDNA) to the WYL domains of PafBC activates the transcription factor. Our cryo-electron microscopy structure of full-length PafBC in its active conformation, bound to the transcription initiation complex, reveals a previously unknown mode of interaction between the ssDNA and the WYL domains. Using biochemical experiments, we show that short ssDNA fragments bind to PafBC dynamically, resulting in deactivation as ssDNA levels decrease postrepair. Our findings shed light on the mechanism linking DNA damage to PafBC activation and expand our understanding of WYL domain-containing proteins. | |||||||||
History |
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Structure visualization
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 161.8 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 27.4 KB 27.4 KB | Display Display | ![]() |
Images | ![]() | 45.9 KB | ||
Filedesc metadata | ![]() | 9.6 KB | ||
Others | ![]() | 164.4 MB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 9fneMC ![]() 9fndC C: citing same article ( M: atomic model generated by this map |
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Similar structure data | Similarity search - Function & homology ![]() |
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Links
EMDB pages | ![]() ![]() |
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Related items in Molecule of the Month |
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Map
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Annotation | Sharpened map. | ||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.12 Å | ||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Additional map: Unsharpened map.
File | emd_50591_additional_1.map | ||||||||||||
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Annotation | Unsharpened map. | ||||||||||||
Projections & Slices |
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Density Histograms |
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Sample components
+Entire : DNA-directed RNA polymerase with transcriptional activator PafBC
+Supramolecule #1: DNA-directed RNA polymerase with transcriptional activator PafBC
+Macromolecule #1: DNA-directed RNA polymerase subunit alpha
+Macromolecule #2: DNA-directed RNA polymerase subunit beta
+Macromolecule #3: DNA-directed RNA polymerase subunit beta'
+Macromolecule #4: DNA-directed RNA polymerase subunit omega
+Macromolecule #5: RNA polymerase sigma factor SigA
+Macromolecule #6: RNA polymerase-binding protein RbpA
+Macromolecule #9: PafC
+Macromolecule #10: Transcriptional regulator-like protein
+Macromolecule #7: recA-op non-template strand
+Macromolecule #8: recA-op template strand
+Macromolecule #11: ZINC ION
+Macromolecule #12: MAGNESIUM ION
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Buffer | pH: 8 |
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Grid | Model: Quantifoil R2/2 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 15 sec. |
Vitrification | Cryogen name: ETHANE-PROPANE / Chamber humidity: 95 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV |
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Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 78.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm |
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
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Image processing
Startup model | Type of model: OTHER / Details: cryoSPARC Ab-Initio model |
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Final reconstruction | Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 470245 |
Initial angle assignment | Type: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC |
Final angle assignment | Type: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC |