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- PDB-8to6: Escherichia coli RNA polymerase unwinding intermediate (I1d) at t... -
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Open data
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Basic information
Entry | Database: PDB / ID: 8to6 | ||||||
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Title | Escherichia coli RNA polymerase unwinding intermediate (I1d) at the lambda PR promoter | ||||||
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![]() | transcription/DNA / DNA-dependent RNA polymerase / transcription / intermediate / DNA promoter / DNA unwinding / transcription-DNA complex | ||||||
Function / homology | ![]() RNA polymerase complex / submerged biofilm formation / cellular response to cell envelope stress / cytosolic DNA-directed RNA polymerase complex / regulation of DNA-templated transcription initiation / sigma factor activity / bacterial-type flagellum assembly / bacterial-type flagellum-dependent cell motility / nitrate assimilation / transcription elongation factor complex ...RNA polymerase complex / submerged biofilm formation / cellular response to cell envelope stress / cytosolic DNA-directed RNA polymerase complex / regulation of DNA-templated transcription initiation / sigma factor activity / bacterial-type flagellum assembly / bacterial-type flagellum-dependent cell motility / nitrate assimilation / transcription elongation factor complex / regulation of DNA-templated transcription elongation / transcription antitermination / DNA-templated transcription initiation / cell motility / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / response to heat / protein-containing complex assembly / intracellular iron ion homeostasis / protein dimerization activity / response to antibiotic / magnesium ion binding / DNA binding / zinc ion binding / membrane / metal ion binding / cytosol / cytoplasm Similarity search - Function | ||||||
Biological species | ![]() ![]() ![]() | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å | ||||||
![]() | Darst, S.A. / Saecker, R.M. / Mueller, A.U. | ||||||
Funding support | ![]()
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![]() | ![]() Title: Early intermediates in bacterial RNA polymerase promoter melting visualized by time-resolved cryo-electron microscopy. Authors: Ruth M Saecker / Andreas U Mueller / Brandon Malone / James Chen / William C Budell / Venkata P Dandey / Kashyap Maruthi / Joshua H Mendez / Nina Molina / Edward T Eng / Laura Y Yen / ...Authors: Ruth M Saecker / Andreas U Mueller / Brandon Malone / James Chen / William C Budell / Venkata P Dandey / Kashyap Maruthi / Joshua H Mendez / Nina Molina / Edward T Eng / Laura Y Yen / Clinton S Potter / Bridget Carragher / Seth A Darst / ![]() Abstract: During formation of the transcription-competent open complex (RPo) by bacterial RNA polymerases (RNAPs), transient intermediates pile up before overcoming a rate-limiting step. Structural ...During formation of the transcription-competent open complex (RPo) by bacterial RNA polymerases (RNAPs), transient intermediates pile up before overcoming a rate-limiting step. Structural descriptions of these interconversions in real time are unavailable. To address this gap, here we use time-resolved cryogenic electron microscopy (cryo-EM) to capture four intermediates populated 120 ms or 500 ms after mixing Escherichia coli σ-RNAP and the λP promoter. Cryo-EM snapshots revealed that the upstream edge of the transcription bubble unpairs rapidly, followed by stepwise insertion of two conserved nontemplate strand (nt-strand) bases into RNAP pockets. As the nt-strand 'read-out' extends, the RNAP clamp closes, expelling an inhibitory σ domain from the active-site cleft. The template strand is fully unpaired by 120 ms but remains dynamic, indicating that yet unknown conformational changes complete RPo formation in subsequent steps. Given that these events likely describe DNA opening at many bacterial promoters, this study provides insights into how DNA sequence regulates steps of RPo formation. #1: ![]() Title: Early intermediates in bacterial RNA polymerase promoter melting visualized by time-resolved cryo-electron microscopy Authors: Darst, S.A. / Saecker, R.M. / Mueller, A.U. / Malone, B. / Chen, J. / Budell, W.C. / Dandey, V.P. / Maruthi, K. / Mendez, J.H. / Molina, N. / Eng, E.T. / Yen, L.Y. / Potter, C.S. / Carragher, B. | ||||||
History |
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Structure visualization
Structure viewer | Molecule: ![]() ![]() |
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Downloads & links
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Download
PDBx/mmCIF format | ![]() | 802.7 KB | Display | ![]() |
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PDB format | ![]() | 631.2 KB | Display | ![]() |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
Others | ![]() |
-Validation report
Arichive directory | ![]() ![]() | HTTPS FTP |
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-Related structure data
Related structure data | ![]() 41437MC ![]() 8to1C ![]() 8to8C ![]() 8toeC ![]() 8tomC M: map data used to model this data C: citing same article ( |
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Similar structure data | Similarity search - Function & homology ![]() |
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Links
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Assembly
Deposited unit | ![]()
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Components
-DNA-directed RNA polymerase subunit ... , 4 types, 6 molecules GHMIJK
#1: Protein | Mass: 36558.680 Da / Num. of mol.: 3 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() Gene: rpoA, pez, phs, sez, b3295, JW3257 Production host: ![]() ![]() References: UniProt: P0A7Z4, DNA-directed RNA polymerase #2: Protein | | Mass: 150820.875 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() Gene: rpoB, groN, nitB, rif, ron, stl, stv, tabD, b3987, JW3950 Production host: ![]() ![]() References: UniProt: P0A8V2, DNA-directed RNA polymerase #3: Protein | | Mass: 155366.781 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() Gene: rpoC, tabB, b3988, JW3951 Production host: ![]() ![]() References: UniProt: P0A8T7, DNA-directed RNA polymerase #4: Protein | | Mass: 10249.547 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() Gene: rpoZ, b3649, JW3624 Production host: ![]() ![]() References: UniProt: P0A800, DNA-directed RNA polymerase |
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-Protein , 1 types, 1 molecules L
#5: Protein | Mass: 70352.242 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() Gene: rpoD Production host: ![]() ![]() References: UniProt: Q0P6L9 |
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-DNA chain , 2 types, 2 molecules OP
#6: DNA chain | Mass: 32600.814 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) ![]() |
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#7: DNA chain | Mass: 32183.631 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) ![]() |
-Non-polymers , 3 types, 6 molecules ![](data/chem/img/4QM.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
Component | Name: Escherichia coli RNA polymerase unwinding intermediate (I1d) at the lambda PR promoter Type: COMPLEX / Entity ID: #1-#7 / Source: RECOMBINANT | |||||||||||||||||||||||||
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Molecular weight | Experimental value: NO | |||||||||||||||||||||||||
Source (natural) | Organism: ![]() ![]() | |||||||||||||||||||||||||
Source (recombinant) | Organism: ![]() ![]() | |||||||||||||||||||||||||
Buffer solution | pH: 8 | |||||||||||||||||||||||||
Buffer component |
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Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES Details: tip 1: 26 or 30 micromolar Es70 RNAP tip 2: 52 or 60 micromolar LPR DNA | |||||||||||||||||||||||||
Vitrification | Instrument: SPOTITON / Cryogen name: ETHANE / Chamber temperature: 296 K Details: CHAPSO was added (from 80 mM stock) to 8 mM final in each sample just prior to spray mixing. |
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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 / Details: Please see publication for details. |
Electron gun | Electron source: ![]() |
Electron lens | Mode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 800 nm |
Image recording | Electron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) Details: Cryo-EM map from images from multiple data collections, please see publication. |
EM imaging optics | Energyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV |
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Processing
EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | |||||||||||||||||||||||||
3D reconstruction | Resolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 111510 / Symmetry type: POINT | |||||||||||||||||||||||||
Refine LS restraints |
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