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- EMDB-73127: De novo initial transcribing RNA polymerase on T7A1 promoter (wea... -

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Basic information

Entry
Database: EMDB / ID: EMD-73127
TitleDe novo initial transcribing RNA polymerase on T7A1 promoter (weak downstream)
Map dataUnsharpened map
Sample
  • Complex: Bacterial RNA polymerase open complex on T7A1 promoter with NTP substrates
KeywordsRNA polymerase / initial transcribing complex / nucleotide addition / translocation / TRANSCRIPTION
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.0 Å
AuthorsMueller AU / Darst SA
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM118130 United States
CitationJournal: Mol Cell / Year: 2026
Title: Structural basis for multi-subunit DNA-dependent RNA polymerase catalytic activity.
Authors: Andreas U Mueller / Seth A Darst /
Abstract: Multi-subunit DNA-dependent RNA polymerase (RNAP) is the central enzyme of transcription, yet its catalytic mechanism remains obscure because high-resolution structures of intermediates with native ...Multi-subunit DNA-dependent RNA polymerase (RNAP) is the central enzyme of transcription, yet its catalytic mechanism remains obscure because high-resolution structures of intermediates with native substrates are not available. We visualized E. coli RNAP on a promoter DNA template with nucleoside triphosphate substrates engaged in active RNA synthesis by cryo-electron microscopy. From this heterogeneous mixture, we determined five high-resolution structures of initial transcribing complexes, including a true Michaelis complex (MC) and a post-catalytic product complex (PC). The MC reveals key conformational transitions during catalysis. Waters in the MC and PC structures show striking overlap with those in corresponding S. cerevisiae RNA polymerase II (RNAPII) structures (see related paper by Li et al.), pointing to functional importance. Together, these results establish that RNAP catalyzes nucleotidyl transfer through a positional (entropic) mechanism, revealing structural determinants of the first step of gene expression.
History
DepositionOct 10, 2025-
Header (metadata) releaseApr 29, 2026-
Map releaseApr 29, 2026-
UpdateMay 13, 2026-
Current statusMay 13, 2026Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_73127.png

Downloads & links

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Map

FileDownload / File: emd_73127.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationUnsharpened map
Projections & slices

Image control

Size
Brightness
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.86 Å/pix.
x 400 pix.
= 344. Å		0.86 Å/pix.
x 400 pix.
= 344. Å		0.86 Å/pix.
x 400 pix.
= 344. Å

Surface

Projections

Slices (1/3)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.86 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.1
Minimum - Maximum-0.23148267 - 0.61913747
Average (Standard dev.)0.0003551955 (±0.01779442)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 344.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Sharpened map (b-factor -65.0)

Fileemd_73127_additional_1.map
AnnotationSharpened map (b-factor -65.0)
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Additional map: Locally filtered map

Fileemd_73127_additional_2.map
AnnotationLocally filtered map
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Additional map: Local resolution map

Fileemd_73127_additional_3.map
AnnotationLocal resolution map
Projections & Slices
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Half map: Half map B

Fileemd_73127_half_map_1.map
AnnotationHalf map B
Projections & Slices
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Histogram

Histogram (log scale)

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Half map: Half map A

Fileemd_73127_half_map_2.map
AnnotationHalf map A
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Sample components

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Entire : Bacterial RNA polymerase open complex on T7A1 promoter with NTP s...

EntireName: Bacterial RNA polymerase open complex on T7A1 promoter with NTP substrates
Components
  • Complex: Bacterial RNA polymerase open complex on T7A1 promoter with NTP substrates

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Supramolecule #1: Bacterial RNA polymerase open complex on T7A1 promoter with NTP s...

SupramoleculeName: Bacterial RNA polymerase open complex on T7A1 promoter with NTP substrates
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#8
Details: Bacterial RNA polymerase open complexes on the T7A1 promoter were prepared in vitro. Addition of ATP, UTP, and CTP started the reaction. Sample was vitrified to halt reaction.
Source (natural)Organism: Escherichia coli (E. coli)

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 8
Component:
ConcentrationFormulaName
40.0 mMC4H11NO3Tris
120.0 NaClNaClsodium chloride
10.0 mMMgCl2magnesium chloride
1.0 mMC4H10O2S2dithiothreitol
1.5 mMC13H17F13NO4Pfluorinated fos-choline-8
GridModel: C-flat-1.2/1.3 / Material: GOLD / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 5 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 30.0 kPa / Details: 25mA; Pelco easiGlow
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 283 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 1.343 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.2 µm / Nominal defocus min: 0.8 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

7mkj

Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.3.1) / Number images used: 63226
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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