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Entry
Database: EMDB / ID: EMD-5127
TitleThree-dimensional EM structure of an intact activator-dependent transcription initiation complex
Map dataE. coli Class I transcription activation complex
Sample
  • Sample: E. coli RNA polymerase holoenzyme (sigma70) and E. coli catabolite activator protein (CAP) bound to 98-mer DNA containing the lac promoter and engineered open transcription bubble
  • Protein or peptide: Catabolite Activator Protein
  • Protein or peptide: RNA polymerase holoenzyme (sigma70)
  • DNA: lac(ICAP)UP-UV5-bubble
Keywordstranscription / initiation / Class I / activator / RNA polymerase / holoenzyme / sigma70 / open complex / CAP / CRP / cAMP-dependent / DNA / prokaryotic
Function / homology
Function and homology information


carbon catabolite repression of transcription / sigma factor antagonist complex / DNA binding, bending / RNA polymerase complex / submerged biofilm formation / cellular response to cell envelope stress / regulation of DNA-templated transcription initiation / minor groove of adenine-thymine-rich DNA binding / sigma factor activity / bacterial-type flagellum assembly ...carbon catabolite repression of transcription / sigma factor antagonist complex / DNA binding, bending / RNA polymerase complex / submerged biofilm formation / cellular response to cell envelope stress / regulation of DNA-templated transcription initiation / minor groove of adenine-thymine-rich DNA binding / sigma factor activity / bacterial-type flagellum assembly / bacterial-type RNA polymerase core enzyme binding / cytosolic DNA-directed RNA polymerase complex / bacterial-type flagellum-dependent cell motility / nitrate assimilation / cAMP binding / regulation of DNA-templated transcription elongation / transcription elongation factor complex / transcription antitermination / cell motility / DNA-templated transcription initiation / protein-DNA complex / ribonucleoside binding / DNA-directed RNA polymerase / DNA-directed RNA polymerase activity / response to heat / protein-containing complex assembly / sequence-specific DNA binding / intracellular iron ion homeostasis / protein dimerization activity / DNA-binding transcription factor activity / response to antibiotic / negative regulation of DNA-templated transcription / DNA-templated transcription / regulation of DNA-templated transcription / positive regulation of DNA-templated transcription / magnesium ion binding / DNA binding / zinc ion binding / identical protein binding / membrane / cytoplasm / cytosol
Similarity search - Function
: / helix_turn_helix, cAMP Regulatory protein / Transcription regulator HTH, Crp-type, conserved site / Crp-type HTH domain signature. / : / Crp-like helix-turn-helix domain / Crp-type HTH domain profile. / Crp-type HTH domain / RNA polymerase sigma factor 70, non-essential domain / Sigma-70, non-essential region ...: / helix_turn_helix, cAMP Regulatory protein / Transcription regulator HTH, Crp-type, conserved site / Crp-type HTH domain signature. / : / Crp-like helix-turn-helix domain / Crp-type HTH domain profile. / Crp-type HTH domain / RNA polymerase sigma factor 70, non-essential domain / Sigma-70, non-essential region / RNA polymerase sigma factor 70, region 1.1 / Sigma-70 factor, region 1.1 superfamily / Sigma-70 factor, region 1.1 / Cyclic nucleotide-binding domain signature 2. / Cyclic nucleotide-binding domain signature 1. / Cyclic nucleotide-binding, conserved site / Sigma-70 factors family signature 1. / RNA polymerase sigma factor RpoD, C-terminal / RNA polymerase sigma factor RpoD / : / RNA polymerase sigma-70 region 1.2 / Sigma-70 factor, region 1.2 / RNA polymerase sigma-70 region 3 / Sigma-70 region 3 / Sigma-70 factors family signature 2. / RNA polymerase sigma-70 / RNA polymerase sigma-70 region 4 / Sigma-70, region 4 / RNA polymerase sigma-70 region 2 / RNA polymerase sigma-70 like domain / Sigma-70 region 2 / RNA polymerase sigma factor, region 2 / RNA polymerase sigma factor, region 3/4-like / Cyclic nucleotide-monophosphate binding domain / Cyclic nucleotide-binding domain / cAMP/cGMP binding motif profile. / Cyclic nucleotide-binding domain / Cyclic nucleotide-binding domain superfamily / DNA-directed RNA polymerase, omega subunit / DNA-directed RNA polymerase, subunit beta-prime, bacterial type / DNA-directed RNA polymerase, beta subunit, external 1 domain superfamily / DNA-directed RNA polymerase, beta subunit, external 1 domain / RNA polymerase beta subunit external 1 domain / RNA polymerase, alpha subunit, C-terminal / Bacterial RNA polymerase, alpha chain C terminal domain / DNA-directed RNA polymerase beta subunit, bacterial-type / DNA-directed RNA polymerase, alpha subunit / RmlC-like jelly roll fold / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase Rpb6 / RNA polymerase, subunit omega/Rpo6/RPB6 / RNA polymerase Rpb6 / RNA polymerase Rpb2, domain 2 superfamily / RNA polymerase Rpb1, domain 3 superfamily / RPB6/omega subunit-like superfamily / DNA-directed RNA polymerase, insert domain / DNA-directed RNA polymerase, RpoA/D/Rpb3-type / RNA polymerase Rpb3/RpoA insert domain / RNA polymerase Rpb3/Rpb11 dimerisation domain / RNA polymerases D / RNA polymerase Rpb1, clamp domain superfamily / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 1 / RNA polymerase, beta subunit, protrusion / RNA polymerase beta subunit / RNA polymerase, alpha subunit / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 4 / RNA polymerase Rpb1, domain 2 / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 4 / DNA-directed RNA polymerase, insert domain superfamily / RNA polymerase, N-terminal / RNA polymerase Rpb2, domain 2 / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase Rpb2, domain 2 / RNA polymerase I subunit A N-terminus / RNA polymerase, RBP11-like subunit / RNA polymerase, beta subunit, conserved site / RNA polymerase Rpb2, domain 7 / RNA polymerase Rpb2, domain 3 / RNA polymerase Rpb2, OB-fold / RNA polymerase Rpb2, domain 7 / RNA polymerase Rpb2, domain 3 / RNA polymerases beta chain signature. / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain / DNA-directed RNA polymerase, subunit 2 / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain superfamily / RNA polymerase Rpb2, domain 6 / Winged helix DNA-binding domain superfamily / Winged helix-like DNA-binding domain superfamily
Similarity search - Domain/homology
RNA polymerase sigma factor RpoD / DNA-directed RNA polymerase subunit alpha / DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit beta / DNA-binding transcriptional dual regulator CRP
Similarity search - Component
Biological speciesEscherichia coli (E. coli) / unidentified (others)
Methodsingle particle reconstruction / negative staining / Resolution: 19.8 Å
AuthorsHudson BP / Quispe J / Lara S / Kim Y / Berman HM / Arnold E / Ebright RH / Lawson CL
CitationJournal: Proc Natl Acad Sci U S A / Year: 2009
Title: Three-dimensional EM structure of an intact activator-dependent transcription initiation complex.
Authors: Brian P Hudson / Joel Quispe / Samuel Lara-González / Younggyu Kim / Helen M Berman / Eddy Arnold / Richard H Ebright / Catherine L Lawson /
Abstract: We present the experimentally determined 3D structure of an intact activator-dependent transcription initiation complex comprising the Escherichia coli catabolite activator protein (CAP), RNA ...We present the experimentally determined 3D structure of an intact activator-dependent transcription initiation complex comprising the Escherichia coli catabolite activator protein (CAP), RNA polymerase holoenzyme (RNAP), and a DNA fragment containing positions -78 to +20 of a Class I CAP-dependent promoter with a CAP site at position -61.5 and a premelted transcription bubble. A 20-A electron microscopy reconstruction was obtained by iterative projection-based matching of single particles visualized in carbon-sandwich negative stain and was fitted using atomic coordinate sets for CAP, RNAP, and DNA. The structure defines the organization of a Class I CAP-RNAP-promoter complex and supports previously proposed interactions of CAP with RNAP alpha subunit C-terminal domain (alphaCTD), interactions of alphaCTD with sigma(70) region 4, interactions of CAP and RNAP with promoter DNA, and phased-DNA-bend-dependent partial wrapping of DNA around the complex. The structure also reveals the positions and shapes of species-specific domains within the RNAP beta', beta, and sigma(70) subunits.
History
DepositionJul 30, 2009-
Header (metadata) releaseAug 24, 2009-
Map releaseNov 4, 2009-
UpdateMay 21, 2012-
Current statusMay 21, 2012Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 2.8
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 2.8
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3iyd
  • Surface level: 2.8
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5127_1.tif

Downloads & links

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Map

FileDownload / File: emd_5127.map.gz / Format: CCP4 / Size: 1.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationE. coli Class I transcription activation complex
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
4.64 Å/pix.
x 80 pix.
= 371.2 Å		4.64 Å/pix.
x 80 pix.
= 371.2 Å		4.64 Å/pix.
x 80 pix.
= 371.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 4.64 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 2.8 / Movie #1: 2.8
Minimum - Maximum-3.34078479 - 14.395241739999999
Average (Standard dev.)0.0 (±0.90271008)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-40-40-40
Dimensions808080
Spacing808080
CellA=B=C: 371.19998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.644.644.64
M x/y/z808080
origin x/y/z0.0000.0000.000
length x/y/z371.200371.200371.200
α/β/γ90.00090.00090.000
start NX/NY/NZ-34-26-72
NX/NY/NZ6953145
MAP C/R/S123
start NC/NR/NS-40-40-40
NC/NR/NS808080
D min/max/mean-3.34114.395-0.000

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Supplemental data

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Sample components

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Entire : E. coli RNA polymerase holoenzyme (sigma70) and E. coli catabolit...

EntireName: E. coli RNA polymerase holoenzyme (sigma70) and E. coli catabolite activator protein (CAP) bound to 98-mer DNA containing the lac promoter and engineered open transcription bubble
Components
  • Sample: E. coli RNA polymerase holoenzyme (sigma70) and E. coli catabolite activator protein (CAP) bound to 98-mer DNA containing the lac promoter and engineered open transcription bubble
  • Protein or peptide: Catabolite Activator Protein
  • Protein or peptide: RNA polymerase holoenzyme (sigma70)
  • DNA: lac(ICAP)UP-UV5-bubble

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Supramolecule #1000: E. coli RNA polymerase holoenzyme (sigma70) and E. coli catabolit...

SupramoleculeName: E. coli RNA polymerase holoenzyme (sigma70) and E. coli catabolite activator protein (CAP) bound to 98-mer DNA containing the lac promoter and engineered open transcription bubble
type: sample / ID: 1000 / Details: Complex formation was verified by gel shift
Oligomeric state: One molecule of RNAP (containing six subunits) and one CAP homodimer bound to a DNA duplex)
Number unique components: 3
Molecular weightTheoretical: 570 KDa

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Macromolecule #1: Catabolite Activator Protein

MacromoleculeName: Catabolite Activator Protein / type: protein_or_peptide / ID: 1 / Name.synonym: CAP / Number of copies: 1 / Oligomeric state: homodimer / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli (E. coli) / Strain: K12
Molecular weightTheoretical: 50 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant plasmid: pET21(a)
SequenceGO: regulation of DNA-templated transcription / InterPro: INTERPRO: IPR001808

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Macromolecule #3: RNA polymerase holoenzyme (sigma70)

MacromoleculeName: RNA polymerase holoenzyme (sigma70) / type: protein_or_peptide / ID: 3 / Name.synonym: RNAP
Details: Six subunits include alphaI (RpoA), alphaII (RpoA), beta (RpoB), beta prime (RpoC) with C-terminal 6His-tag, omega (RpoZ), and sigma70.
Number of copies: 1 / Oligomeric state: heterohexamer / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli (E. coli) / Strain: K12 / synonym: Escherichia coli
Molecular weightTheoretical: 460 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant plasmid: pEcABC-H6, pRSFduet-sigma, pCDF-omega
SequenceGO: DNA-templated transcription

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Macromolecule #2: lac(ICAP)UP-UV5-bubble

MacromoleculeName: lac(ICAP)UP-UV5-bubble / type: dna / ID: 2 / Name.synonym: lac(ICAP)UP-UV5-bubble
Details: An engineered 98mer duplex based on positions -78 to 20 of the Class I CAP-dependent promoter lac but containing consensus -10 sequence and consensus binding sites for CAP and RNAP alpha-CTD. ...Details: An engineered 98mer duplex based on positions -78 to 20 of the Class I CAP-dependent promoter lac but containing consensus -10 sequence and consensus binding sites for CAP and RNAP alpha-CTD. Positions -11 to 2 are non-complementary to create an artificial transcription bubble. Top strand 5'-CGCAATAAATGTGATCTAGATCACATTTTAGGCAAAAAAGGCTTTACACTTTATGCTTCCGGCTCGTATAATCGCACCTTATGTGAGCGGATAACAAG-3' Bottom strand 5'-CTTGTTATCCGCTCACAATTCCACACTAATAACGAGCCGGAAGCATAAAGTGTAAAGCCTTTTTTGCCTAAAATGTGATCTAGATCACATTTATTGCG-3'
Classification: DNA / Structure: OTHER / Synthetic?: Yes
Source (natural)Organism: unidentified (others)
Molecular weightExperimental: 60 KDa / Theoretical: 60 KDa

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration6.18 mg/mL
BufferpH: 8
Details: 25mM HEPES, 100mM KCl, 10mM MgCl2, 1mM DTT, 0.2mM cAMP
StainingType: NEGATIVE
Details: Sample and 2% uranyl formate stain were applied to the grid in rapid succession, with the last drop of stain remaining on the sample for 1 minute. The grid was then submerged in stain and ...Details: Sample and 2% uranyl formate stain were applied to the grid in rapid succession, with the last drop of stain remaining on the sample for 1 minute. The grid was then submerged in stain and brought up under thin carbon to form an upper sandwich layer. The grid was then blotted and dried for 10 minutes.
GridDetails: 400-mesh copper 2.0x0.5 hole pattern C-flat grid covered with thin layer of continuous carbon
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI F20
TemperatureAverage: 293 K
Details15 um pixel size on detector
DateNov 4, 2008
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F415 (4k x 4k) / Number real images: 349 / Average electron dose: 16 e/Å2
Electron beamAcceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 50000
Sample stageSpecimen holder: standard side-entry room-temperature stage / Specimen holder model: SIDE ENTRY, EUCENTRIC
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

Details32816 particles were automatically selected by the Appion DoGpicker initially
CTF correctionDetails: ACE
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 19.8 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN, SPIDER
Details: EMAN interleaved with SPIDER correspondence analysis
Number images used: 14097
Final two d classificationNumber classes: 280

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Atomic model buiding 1

Initial modelPDB ID:

1lb2

SoftwareName: Chimera, Yup.scx
DetailsProtocol: rigid body, Yup.scx simulated annealing. A complete ternary complex model was generated using multiple PDB entries, with a homology modelling step for RNAP. The model was regularized with PHENIX and the refined against the EM map with Yup.scx using default parameters.
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: map-derived potential energy
Output model

PDB-3iyd:
Three-dimensional EM structure of an intact activator-dependent transcription initiation complex

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Atomic model buiding 2

Initial modelPDB ID:

1bdf

SoftwareName: Chimera, Yup.scx
DetailsProtocol: rigid body fit followed by Yup.scx simulated annealing. A complete ternary complex model was generated using multiple PDB entries, with a homology modelling step for RNAP. The model was regularized with PHENIX and the refined against the EM map with Yup.scx using default parameters.
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: map-derived potential energy
Output model

PDB-3iyd:
Three-dimensional EM structure of an intact activator-dependent transcription initiation complex

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Atomic model buiding 3

Initial modelPDB ID:

2auk


Chain - Chain ID: A
SoftwareName: Chimera, Yup.scx
DetailsProtocol: manual fit followed by Yup.scx simulated annealing. A complete ternary complex model was generated using multiple PDB entries, with a homology modelling step for RNAP. The model was regularized with PHENIX and the refined against the EM map with Yup.scx using default parameters.
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: map-derived potential energy
Output model

PDB-3iyd:
Three-dimensional EM structure of an intact activator-dependent transcription initiation complex

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Atomic model buiding 4

Initial modelPDB ID:

1sig


Chain - Chain ID: A
SoftwareName: Chimera, Yup.scx
DetailsProtocol: rigid body fit followed by Yup.scx simulated annealing. A complete ternary complex model was generated using multiple PDB entries, with a homology modelling step for RNAP. The model was regularized with PHENIX and the refined against the EM map with Yup.scx using default parameters.
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: map-derived potential energy
Output model

PDB-3iyd:
Three-dimensional EM structure of an intact activator-dependent transcription initiation complex

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Atomic model buiding 5

Initial modelPDB ID:

3dxj

SoftwareName: Chimera, Modeller, Yup.scx
DetailsProtocol: rigid body fit followed by Yup.scx simulated annealing. A complete ternary complex model was generated using multiple PDB entries, with a homology modelling step for RNAP. The model was regularized with PHENIX and the refined against the EM map with Yup.scx using default parameters.
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: map-derived potential energy
Output model

PDB-3iyd:
Three-dimensional EM structure of an intact activator-dependent transcription initiation complex

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