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- EMDB-40178: Cryo-EM composited map of the E. coli transcription-translation c... -

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Entry
Database: EMDB / ID: EMD-40178
TitleCryo-EM composited map of the E. coli transcription-translation complex (RNAP in an anti-swiveled conformation)
Map dataCryo-EM composited map of the E. coli Transcription-Translation Complex where RNAP adopts an anti-swiveled conformation
Sample
  • Complex: Cryo-EM composited map of the E. coli transcription-translation complex (RNAP adopts an anti-swiveled conformation)
    • Complex: Cryo-EM structure of E. coli RNA polymerase Elongation complex in the Transcription-Translation Complex (RNAP in an anti-swiveled conformation)
    • Complex: Cryo-EM structure of E. coli 70S Ribosome containing mRNA and tRNA (in the transcription-translation complex)
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsFlorez Ariza A / Wee L / Tong A / Canari C / Grob P / Nogales E / Bustamante C
Funding support United States, 2 items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
National Institutes of Health/National Center for Research Resources (NIH/NCRR) United States
CitationJournal: Cell / Year: 2023
Title: A trailing ribosome speeds up RNA polymerase at the expense of transcript fidelity via force and allostery.
Authors: Liang Meng Wee / Alexander B Tong / Alfredo Jose Florez Ariza / Cristhian Cañari-Chumpitaz / Patricia Grob / Eva Nogales / Carlos J Bustamante /
Abstract: In prokaryotes, translation can occur on mRNA that is being transcribed in a process called coupling. How the ribosome affects the RNA polymerase (RNAP) during coupling is not well understood. Here, ...In prokaryotes, translation can occur on mRNA that is being transcribed in a process called coupling. How the ribosome affects the RNA polymerase (RNAP) during coupling is not well understood. Here, we reconstituted the E. coli coupling system and demonstrated that the ribosome can prevent pausing and termination of RNAP and double the overall transcription rate at the expense of fidelity. Moreover, we monitored single RNAPs coupled to ribosomes and show that coupling increases the pause-free velocity of the polymerase and that a mechanical assisting force is sufficient to explain the majority of the effects of coupling. Also, by cryo-EM, we observed that RNAPs with a terminal mismatch adopt a backtracked conformation, while a coupled ribosome allosterically induces these polymerases toward a catalytically active anti-swiveled state. Finally, we demonstrate that prolonged RNAP pausing is detrimental to cell viability, which could be prevented by polymerase reactivation through a coupled ribosome.
History
DepositionMar 17, 2023-
Header (metadata) releaseApr 12, 2023-
Map releaseApr 12, 2023-
UpdateApr 12, 2023-
Current statusApr 12, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_40178.png

Downloads & links

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Map

FileDownload / File: emd_40178.map.gz / Format: CCP4 / Size: 325 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM composited map of the E. coli Transcription-Translation Complex where RNAP adopts an anti-swiveled conformation
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
1.45 Å/pix.
x 440 pix.
= 636.68 Å		1.45 Å/pix.
x 440 pix.
= 636.68 Å		1.45 Å/pix.
x 440 pix.
= 636.68 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.447 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 4.5
Minimum - Maximum-32.1911 - 75.14211
Average (Standard dev.)0.060853213 (±1.3078992)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions440440440
Spacing440440440
CellA=B=C: 636.68 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Cryo-EM composited map of the E. coli transcription-translation c...

EntireName: Cryo-EM composited map of the E. coli transcription-translation complex (RNAP adopts an anti-swiveled conformation)
Components
  • Complex: Cryo-EM composited map of the E. coli transcription-translation complex (RNAP adopts an anti-swiveled conformation)
    • Complex: Cryo-EM structure of E. coli RNA polymerase Elongation complex in the Transcription-Translation Complex (RNAP in an anti-swiveled conformation)
    • Complex: Cryo-EM structure of E. coli 70S Ribosome containing mRNA and tRNA (in the transcription-translation complex)

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Supramolecule #1: Cryo-EM composited map of the E. coli transcription-translation c...

SupramoleculeName: Cryo-EM composited map of the E. coli transcription-translation complex (RNAP adopts an anti-swiveled conformation)
type: complex / ID: 1 / Chimera: Yes / Parent: 0
Molecular weightTheoretical: 2.9 MDa

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Supramolecule #2: Cryo-EM structure of E. coli RNA polymerase Elongation complex in...

SupramoleculeName: Cryo-EM structure of E. coli RNA polymerase Elongation complex in the Transcription-Translation Complex (RNAP in an anti-swiveled conformation)
type: complex / ID: 2 / Chimera: Yes / Parent: 1
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 400 KDa

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Supramolecule #3: Cryo-EM structure of E. coli 70S Ribosome containing mRNA and tRN...

SupramoleculeName: Cryo-EM structure of E. coli 70S Ribosome containing mRNA and tRNA (in the transcription-translation complex)
type: complex / ID: 3 / Chimera: Yes / Parent: 1
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 2.5 MDa

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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: 7.5
GridModel: C-flat-1.2/1.3 / Support film - Material: CARBON / Support film - topology: CONTINUOUS
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6ri9

Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF
Details: This is a composited map. The resolution reported here is the one obtained only for one of the components of the map that was focus refined (see EMD-29214).
Number images used: 18000
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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