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- PDB-7mki: Cryo-EM structure of Escherichia coli RNA polymerase bound to lam... -
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Basic information
Entry | Database: PDB / ID: 7mki | ||||||
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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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![]() | TRANSCRIPTION/DNA / 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 | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å | ||||||
![]() | Saecker, R.M. / Darst, S.A. / Chen, J. | ||||||
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
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Structure viewer | Molecule: ![]() ![]() |
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PDBx/mmCIF format | ![]() | 705.1 KB | Display | ![]() |
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PDB format | ![]() | 558.5 KB | Display | ![]() |
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-Validation report
Arichive directory | ![]() ![]() | HTTPS FTP |
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-Related structure data
Related structure data | ![]() 23895MC ![]() 7mkdC ![]() 7mkeC ![]() 7mkjC C: citing same article ( M: map data used to model this data |
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Similar structure data |
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Links
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Assembly
Deposited unit | ![]()
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Components
-DNA chain , 2 types, 2 molecules PQ
#1: DNA chain | Mass: 27841.791 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) ![]() |
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#7: DNA chain | Mass: 27673.764 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) ![]() |
-DNA-directed RNA polymerase subunit ... , 4 types, 5 molecules GHIJK
#2: Protein | Mass: 36558.680 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() ![]() References: UniProt: A0A073G207, DNA-directed RNA polymerase #3: Protein | | Mass: 150820.875 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() ![]() #4: Protein | | Mass: 155366.781 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() ![]() References: UniProt: A0A4S1NBU2, DNA-directed RNA polymerase #5: Protein | | Mass: 10249.547 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() ![]() |
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-Protein , 1 types, 1 molecules L
#6: Protein | Mass: 70352.242 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() ![]() |
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-Non-polymers , 3 types, 6 molecules ![](data/chem/img/1N7.gif)
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#8: Chemical | #9: Chemical | ChemComp-MG / | #10: Chemical | |
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-Details
Has ligand of interest | N |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
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Sample preparation
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Source (recombinant) | Organism: ![]() ![]() | ||||||||||||||||||||||||
Buffer solution | pH: 8 | ||||||||||||||||||||||||
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | ||||||||||||||||||||||||
Vitrification | Cryogen name: ETHANE |
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Electron microscopy imaging
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: ![]() |
Electron lens | Mode: BRIGHT FIELD |
Image recording | Electron dose: 75 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k) |
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Processing
Software | Name: PHENIX / Version: 1.18.2_3874: / Classification: refinement | ||||||||||||||||||||||||
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
3D reconstruction | Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 353304 / Symmetry type: POINT | ||||||||||||||||||||||||
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