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- EMDB-23895: Cryo-EM structure of Escherichia coli RNA polymerase bound to lam... -
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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-23895 | |||||||||
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Title | Cryo-EM structure of Escherichia coli RNA polymerase bound to lambda PR (-5G to C) promoter DNA | |||||||||
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![]() | DNA-dependent RNA polymerase / transcription / DNA promoter / open complex / TRANSCRIPTION-DNA complex | |||||||||
Function / homology | ![]() sigma factor activity / DNA-directed RNA polymerase complex / DNA-templated transcription initiation / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / protein dimerization activity / DNA-templated transcription / magnesium ion binding / DNA binding ...sigma factor activity / DNA-directed RNA polymerase complex / DNA-templated transcription initiation / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / protein dimerization activity / DNA-templated transcription / magnesium ion binding / DNA binding / zinc ion binding / metal ion binding / cytoplasm Similarity search - Function | |||||||||
Biological species | ![]() ![]() ![]() | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.5 Å | |||||||||
![]() | Saecker RM / Darst SA | |||||||||
Funding support | ![]()
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![]() | ![]() Title: Structural origins of RNA polymerase open promoter complex stability. Authors: Ruth M Saecker / James Chen / Courtney E Chiu / Brandon Malone / Johanna Sotiris / Mark Ebrahim / Laura Y Yen / Edward T Eng / Seth A Darst / ![]() Abstract: The first step in gene expression in all organisms requires opening the DNA duplex to expose one strand for templated RNA synthesis. In , promoter DNA sequence fundamentally determines how fast the ...The first step in gene expression in all organisms requires opening the DNA duplex to expose one strand for templated RNA synthesis. In , promoter DNA sequence fundamentally determines how fast the RNA polymerase (RNAP) forms "open" complexes (RPo), whether RPo persists for seconds or hours, and how quickly RNAP transitions from initiation to elongation. These rates control promoter strength in vivo, but their structural origins remain largely unknown. Here, we use cryoelectron microscopy to determine the structures of RPo formed de novo at three promoters with widely differing lifetimes at 37 °C: λP (t ∼10 h), T7A1 (t ∼4 min), and a point mutant in λP (λP) (t ∼2 h). Two distinct RPo conformers are populated at λP, likely representing productive and unproductive forms of RPo observed in solution studies. We find that changes in the sequence and length of DNA in the transcription bubble just upstream of the start site (+1) globally alter the network of DNA-RNAP interactions, base stacking, and strand order in the single-stranded DNA of the transcription bubble; these differences propagate beyond the bubble to upstream and downstream DNA. After expanding the transcription bubble by one base (T7A1), the nontemplate strand "scrunches" inside the active site cleft; the template strand bulges outside the cleft at the upstream edge of the bubble. The structures illustrate how limited sequence changes trigger global alterations in the transcription bubble that modulate the RPo lifetime and affect the subsequent steps of the transcription cycle. | |||||||||
History |
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Structure visualization
Movie |
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Structure viewer | EM map: ![]() ![]() ![]() |
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 59.3 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 28.3 KB 28.3 KB | Display Display | ![]() |
Images | ![]() | 142 KB | ||
Filedesc metadata | ![]() | 8.7 KB | ||
Others | ![]() ![]() ![]() | 49.3 MB 16.7 MB 16.7 MB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 7mkiMC ![]() 7mkdC ![]() 7mkeC ![]() 7mkjC C: citing same article ( M: atomic model generated by this map |
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Similar structure data |
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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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Voxel size | X=Y=Z: 1.03 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Additional map: #1
File | emd_23895_additional_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: #2
File | emd_23895_half_map_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: #1
File | emd_23895_half_map_2.map | ||||||||||||
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Density Histograms |
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Sample components
+Entire : Escherichia coli RNA polymerase bound to lambda PR promoter DNA (...
+Supramolecule #1: Escherichia coli RNA polymerase bound to lambda PR promoter DNA (...
+Supramolecule #2: RNA polymerase
+Supramolecule #3: lambda PR (-5G to C) promoter DNA
+Macromolecule #1: Nontemplate strand of lambda PR DNA promoter (-5G to C)
+Macromolecule #7: Template strand of lambda PR promoter DNA (-5C to G)
+Macromolecule #2: DNA-directed RNA polymerase subunit alpha
+Macromolecule #3: DNA-directed RNA polymerase subunit beta
+Macromolecule #4: DNA-directed RNA polymerase subunit beta'
+Macromolecule #5: DNA-directed RNA polymerase subunit omega
+Macromolecule #6: RNA polymerase sigma factor RpoD
+Macromolecule #8: CHAPSO
+Macromolecule #9: MAGNESIUM ION
+Macromolecule #10: ZINC 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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Vitrification | Cryogen name: ETHANE |
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Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 75.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD |
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
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Image processing
Startup model | Type of model: INSILICO MODEL |
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Final reconstruction | Resolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 353304 |
Initial angle assignment | Type: MAXIMUM LIKELIHOOD |
Final angle assignment | Type: MAXIMUM LIKELIHOOD |