[English] 日本語
Yorodumi- EMDB-40864: E. coli DNA-directed RNA polymerase transcription elongation comp... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-40864 | |||||||||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Title | E. coli DNA-directed RNA polymerase transcription elongation complex bound the unnatural dB-STP base pair in the active site | |||||||||||||||||||||||||||
Map data | The full map of E. coli DNA-directed RNA polymerase transcription elongation complex bound the unnatural dB-STP base pair in the active site | |||||||||||||||||||||||||||
Sample |
| |||||||||||||||||||||||||||
Keywords | complex / unnatural base pairs / synthetic biology / transcription / TRANSCRIPTION-DNA-RNA complex | |||||||||||||||||||||||||||
Function / homology | Function and homology information submerged biofilm formation / cellular response to cell envelope stress / cytosolic DNA-directed RNA polymerase complex / regulation of DNA-templated transcription initiation / bacterial-type flagellum assembly / bacterial-type flagellum-dependent cell motility / nitrate assimilation / DNA-directed RNA polymerase complex / transcription elongation factor complex / regulation of DNA-templated transcription elongation ...submerged biofilm formation / cellular response to cell envelope stress / cytosolic DNA-directed RNA polymerase complex / regulation of DNA-templated transcription initiation / bacterial-type flagellum assembly / bacterial-type flagellum-dependent cell motility / nitrate assimilation / DNA-directed RNA polymerase complex / transcription elongation factor complex / regulation of DNA-templated transcription elongation / DNA-templated transcription initiation / transcription antitermination / cell motility / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / response to heat / intracellular iron ion homeostasis / protein dimerization activity / response to antibiotic / DNA-templated transcription / magnesium ion binding / DNA binding / zinc ion binding / membrane / cytosol / cytoplasm Similarity search - Function | |||||||||||||||||||||||||||
Biological species | Escherichia coli (E. coli) / synthetic construct (others) | |||||||||||||||||||||||||||
Method | single particle reconstruction / cryo EM / Resolution: 2.65 Å | |||||||||||||||||||||||||||
Authors | Shan Z / Lyumkis D / Oh J / Wang D | |||||||||||||||||||||||||||
Funding support | United States, 8 items
| |||||||||||||||||||||||||||
Citation | Journal: Nat Commun / Year: 2023 Title: A unified Watson-Crick geometry drives transcription of six-letter expanded DNA alphabets by E. coli RNA polymerase. Authors: Juntaek Oh / Zelin Shan / Shuichi Hoshika / Jun Xu / Jenny Chong / Steven A Benner / Dmitry Lyumkis / Dong Wang / Abstract: Artificially Expanded Genetic Information Systems (AEGIS) add independently replicable unnatural nucleotide pairs to the natural G:C and A:T/U pairs found in native DNA, joining the unnatural pairs ...Artificially Expanded Genetic Information Systems (AEGIS) add independently replicable unnatural nucleotide pairs to the natural G:C and A:T/U pairs found in native DNA, joining the unnatural pairs through alternative modes of hydrogen bonding. Whether and how AEGIS pairs are recognized and processed by multi-subunit cellular RNA polymerases (RNAPs) remains unknown. Here, we show that E. coli RNAP selectively recognizes unnatural nucleobases in a six-letter expanded genetic system. High-resolution cryo-EM structures of three RNAP elongation complexes containing template-substrate UBPs reveal the shared principles behind the recognition of AEGIS and natural base pairs. In these structures, RNAPs are captured in an active state, poised to perform the chemistry step. At this point, the unnatural base pair adopts a Watson-Crick geometry, and the trigger loop is folded into an active conformation, indicating that the mechanistic principles underlying recognition and incorporation of natural base pairs also apply to AEGIS unnatural base pairs. These data validate the design philosophy of AEGIS unnatural basepairs. Further, we provide structural evidence supporting a long-standing hypothesis that pair mismatch during transcription occurs via tautomerization. Together, our work highlights the importance of Watson-Crick complementarity underlying the design principles of AEGIS base pair recognition. | |||||||||||||||||||||||||||
History |
|
-Structure visualization
Supplemental images |
---|
-Downloads & links
-EMDB archive
Map data | emd_40864.map.gz | 62.7 MB | EMDB map data format | |
---|---|---|---|---|
Header (meta data) | emd-40864-v30.xml emd-40864.xml | 31.3 KB 31.3 KB | Display Display | EMDB header |
Images | emd_40864.png | 91.6 KB | ||
Masks | emd_40864_msk_1.map | 125 MB | Mask map | |
Filedesc metadata | emd-40864.cif.gz | 9.4 KB | ||
Others | emd_40864_additional_1.map.gz emd_40864_half_map_1.map.gz emd_40864_half_map_2.map.gz | 32.4 MB 116.2 MB 116.2 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-40864 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-40864 | HTTPS FTP |
-Validation report
Summary document | emd_40864_validation.pdf.gz | 971.4 KB | Display | EMDB validaton report |
---|---|---|---|---|
Full document | emd_40864_full_validation.pdf.gz | 971 KB | Display | |
Data in XML | emd_40864_validation.xml.gz | 13.9 KB | Display | |
Data in CIF | emd_40864_validation.cif.gz | 16.4 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-40864 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-40864 | HTTPS FTP |
-Related structure data
Related structure data | 8sy7MC 8sy5C 8sy6C M: atomic model generated by this map C: citing same article (ref.) |
---|---|
Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
---|---|
Related items in Molecule of the Month |
-Map
File | Download / File: emd_40864.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Annotation | The full map of E. coli DNA-directed RNA polymerase transcription elongation complex bound the unnatural dB-STP base pair in the active site | ||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 0.86 Å | ||||||||||||||||||||||||||||||||||||
Density |
| ||||||||||||||||||||||||||||||||||||
Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
|
-Supplemental data
-Mask #1
File | emd_40864_msk_1.map | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Projections & Slices |
| ||||||||||||
Density Histograms |
-Additional map: The density modified map of E. coli DNA-directed...
File | emd_40864_additional_1.map | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Annotation | The density modified map of E. coli DNA-directed RNA polymerase transcription elongation complex bound the unnatural dB-STP base pair in the active site that used for atomic modeling | ||||||||||||
Projections & Slices |
| ||||||||||||
Density Histograms |
-Half map: The half map1 of E. coli DNA-directed RNA...
File | emd_40864_half_map_1.map | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Annotation | The half map1 of E. coli DNA-directed RNA polymerase transcription elongation complex bound the unnatural dB-STP base pair in the active site | ||||||||||||
Projections & Slices |
| ||||||||||||
Density Histograms |
-Half map: The half map2 of E. coli DNA-directed RNA...
File | emd_40864_half_map_2.map | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Annotation | The half map2 of E. coli DNA-directed RNA polymerase transcription elongation complex bound the unnatural dB-STP base pair in the active site | ||||||||||||
Projections & Slices |
| ||||||||||||
Density Histograms |
-Sample components
+Entire : E. coli RNAP transcription elongation complex bound the unnatural...
+Supramolecule #1: E. coli RNAP transcription elongation complex bound the unnatural...
+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: Non-template single stranded DNA
+Macromolecule #7: Template single stranded DNA
+Macromolecule #6: RNA oligomer
+Macromolecule #8: ZINC ION
+Macromolecule #9: MAGNESIUM ION
+Macromolecule #10: [[(2~{R},3~{S},4~{R},5~{S})-5-(4-azanyl-1-methyl-2-oxidanylidene-...
+Macromolecule #11: water
-Experimental details
-Structure determination
Method | cryo EM |
---|---|
Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 15 mg/mL |
---|---|
Buffer | pH: 8 Details: 20mM Tris (pH 8.0), 40mM KCl, 5mM DTT and 5mM MgCl2 |
Grid | Model: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 90 % |
Details | The sample was concentrated to 15 mg/ml with 4 mM CHAPSO |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
---|---|
Specialist optics | Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV |
Image recording | Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number real images: 11268 / Average electron dose: 35.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | C2 aperture diameter: 70.0 µm / Calibrated magnification: 58140 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.4 µm / Nominal defocus min: 1.0 µm |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |