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- EMDB-41433: Escherichia coli RNA polymerase unwinding intermediate (I1a) at t... -
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Open data
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Basic information
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Title | Escherichia coli RNA polymerase unwinding intermediate (I1a) at the lambda PR promoter | |||||||||
![]() | melting intermediate I1a map filtered by local resolution | |||||||||
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![]() | 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 / cytoplasm / cytosol Similarity search - Function | |||||||||
Biological species | ![]() ![]() ![]() ![]() ![]() | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 2.8 Å | |||||||||
![]() | Darst SA / Saecker RM / Mueller AU | |||||||||
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 SA / Saecker RM / Mueller AU / Malone B / Chen J / Budell WC / Dandey VP / Maruthi K / Mendez JH / Molina N / Eng ET / Yen LY / Potter CS / Carragher B | |||||||||
History |
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Structure visualization
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 8.8 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 34.4 KB 34.4 KB | Display Display | ![]() |
Images | ![]() | 127.5 KB | ||
Filedesc metadata | ![]() | 9.8 KB | ||
Others | ![]() ![]() ![]() ![]() | 204.1 MB 108.2 MB 200.3 MB 200.2 MB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Validation report
Summary document | ![]() | 819.1 KB | Display | ![]() |
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Full document | ![]() | 818.7 KB | Display | |
Data in XML | ![]() | 15.7 KB | Display | |
Data in CIF | ![]() | 18.6 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 8to1MC ![]() 8to6C ![]() 8to8C ![]() 8toeC ![]() 8tomC M: atomic model generated by this map C: citing same article ( |
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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 | melting intermediate I1a map filtered by local resolution | ||||||||||||||||||||
Voxel size | X=Y=Z: 0.844 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Additional map: melting intermediate I1a sharpened map
File | emd_41433_additional_1.map | ||||||||||||
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Annotation | melting intermediate I1a sharpened map | ||||||||||||
Projections & Slices |
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Density Histograms |
-Additional map: melting intermediate I1a unsharpened map
File | emd_41433_additional_2.map | ||||||||||||
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Annotation | melting intermediate I1a unsharpened map | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: melting intermediate I1a half map B
File | emd_41433_half_map_1.map | ||||||||||||
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Annotation | melting intermediate I1a half map B | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: melting intermediate I1a half map A
File | emd_41433_half_map_2.map | ||||||||||||
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Annotation | melting intermediate I1a half map A | ||||||||||||
Projections & Slices |
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Density Histograms |
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Sample components
+Entire : Escherichia coli RNA polymerase unwinding intermediate (I1a) at t...
+Supramolecule #1: Escherichia coli RNA polymerase unwinding intermediate (I1a) at t...
+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 RpoD
+Macromolecule #6: Nontemplate strand of lamdba PR promoter DNA
+Macromolecule #7: Template strand of lamdba PR promoter DNA
+Macromolecule #8: (3R,5S,7R,8R,9S,10S,12S,13R,14S,17R)-10,13-dimethyl-17-[(2R)-pent...
+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 Component:
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Grid | Pretreatment - Type: PLASMA CLEANING Details: Nanowire grids were plasma-treated (Gatan Solarus) at 5 W in H2 (g) and O2 (g) for 1 to 2 minutes. | |||||||||||||||
Vitrification | Cryogen name: ETHANE / Chamber temperature: 296 K / Instrument: SPOTITON Details: CHAPSO was added (from 80 mM stock) to 8 mM final in each sample just prior to spray mixing.. | |||||||||||||||
Details | tip 1: 26 or 30 micromolar Es70 RNAP tip 2: 52 or 60 micromolar LPR DNA |
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Electron microscopy
Microscope | FEI TITAN KRIOS |
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Specialist optics | Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV |
Details | Please see publication for details. |
Image recording | Film or detector model: GATAN K3 (6k x 4k) / Average electron dose: 60.0 e/Å2 Details: Cryo-EM map from images from multiple data collections, please see publication. |
Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.8 µm |
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |