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- PDB-6pb6: The E. coli class-II CAP-dependent transcription activation compl... -

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Basic information

Entry
Database: PDB / ID: 6pb6
TitleThe E. coli class-II CAP-dependent transcription activation complex at the state 2
Components
  • (DNA-directed RNA polymerase subunit ...Polymerase) x 4
  • RNA polymerase sigma factor RpoD
  • SYNTHETIC NONTEMPLATE STRAND DNA (78-MER)
  • SYNTHETIC TEMPLATE STRAND DNA (78-MER)
  • cAMP-activated global transcriptional regulator CRP
KeywordsTRANSCRIPTION/DNA / class-II / CAP-dependent / transcription activation complex / state 2 architecture / TRANSCRIPTION / TRANSCRIPTION-DNA complex
Function / homology
Function and homology information


sigma factor antagonist 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 / bacterial-type flagellum assembly / sigma factor activity / bacterial-type flagellum-dependent cell motility / nitrate assimilation ...sigma factor antagonist 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 / bacterial-type flagellum assembly / sigma factor activity / bacterial-type flagellum-dependent cell motility / nitrate assimilation / cAMP binding / transcription elongation factor complex / DNA-directed RNA polymerase complex / regulation of DNA-templated transcription elongation / transcription antitermination / cell motility / DNA-templated transcription initiation / 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 / DNA-binding transcription factor activity / negative regulation of DNA-templated transcription / DNA-templated transcription / magnesium ion binding / DNA binding / zinc ion binding / membrane / cytosol / cytoplasm
Similarity search - Function
Transcription regulator HTH, Crp-type, conserved site / Crp-type HTH domain signature. / helix_turn_helix, cAMP Regulatory protein / Crp-type HTH domain profile. / Crp-like helix-turn-helix domain / 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 ...Transcription regulator HTH, Crp-type, conserved site / Crp-type HTH domain signature. / helix_turn_helix, cAMP Regulatory protein / Crp-type HTH domain profile. / Crp-like helix-turn-helix domain / 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. / Sigma-70 factors family signature 1. / Cyclic nucleotide-binding, conserved site / 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, alpha subunit / DNA-directed RNA polymerase beta subunit, bacterial-type / RmlC-like jelly roll fold / RNA polymerase Rpb6 / RNA polymerase, subunit omega/Rpo6/RPB6 / RNA polymerase Rpb6 / RNA polymerase Rpb1, domain 3 superfamily / RNA polymerase Rpb1, clamp domain superfamily / RPB6/omega subunit-like superfamily / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb2, domain 2 superfamily / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 1 / RNA polymerase, alpha subunit / RNA polymerase Rpb1, domain 4 / RNA polymerase Rpb1, domain 2 / RNA polymerase Rpb1, domain 4 / RNA polymerase, N-terminal / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase I subunit A N-terminus / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 5 / RNA polymerase, beta subunit, protrusion / RNA polymerase beta subunit / 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 / DNA-directed RNA polymerase, insert domain superfamily / RNA polymerase, RBP11-like subunit / RNA polymerase Rpb2, domain 2 / RNA polymerase Rpb2, domain 2 / 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
ADENOSINE-3',5'-CYCLIC-MONOPHOSPHATE / DNA / DNA (> 10) / DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit beta / RNA polymerase sigma factor RpoD / DNA-directed RNA polymerase subunit alpha / DNA-directed RNA polymerase subunit alpha / DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit beta' / cAMP-activated global transcriptional regulator CRP
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.29 Å
AuthorsLiu, B. / Shi, W.
CitationJournal: PLoS Biol / Year: 2020
Title: Visualization of two architectures in class-II CAP-dependent transcription activation.
Authors: Wei Shi / Yanan Jiang / Yibin Deng / Zigang Dong / Bin Liu /
Abstract: Transcription activation by cyclic AMP (cAMP) receptor protein (CAP) is the classic paradigm of transcription regulation in bacteria. CAP was suggested to activate transcription on class-II promoters ...Transcription activation by cyclic AMP (cAMP) receptor protein (CAP) is the classic paradigm of transcription regulation in bacteria. CAP was suggested to activate transcription on class-II promoters via a recruitment and isomerization mechanism. However, whether and how it modifies RNA polymerase (RNAP) to initiate transcription remains unclear. Here, we report cryo-electron microscopy (cryo-EM) structures of an intact Escherichia coli class-II CAP-dependent transcription activation complex (CAP-TAC) with and without de novo RNA transcript. The structures reveal two distinct architectures of TAC and raise the possibility that CAP binding may induce substantial conformational changes in all the subunits of RNAP and transiently widen the main cleft of RNAP to facilitate DNA promoter entering and formation of the initiation open complex. These structural changes vanish during further RNA transcript synthesis. The observations in this study may reveal a possible on-pathway intermediate and suggest a possibility that CAP activates transcription by inducing intermediate state, in addition to the previously proposed stabilization mechanism.
History
DepositionJun 13, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 18, 2020Provider: repository / Type: Initial release
Revision 1.1Apr 29, 2020Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.pdbx_database_id_DOI
Revision 1.2Feb 10, 2021Group: Source and taxonomy / Category: entity_src_gen
Item: _entity_src_gen.gene_src_strain / _entity_src_gen.pdbx_gene_src_ncbi_taxonomy_id / _entity_src_gen.pdbx_gene_src_scientific_name
Revision 1.3Oct 20, 2021Group: Database references / Category: database_2 / pdbx_database_related
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_database_related.content_type

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Structure visualization

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Assembly

Deposited unit
A: DNA-directed RNA polymerase subunit alpha
B: DNA-directed RNA polymerase subunit alpha
C: DNA-directed RNA polymerase subunit beta
D: DNA-directed RNA polymerase subunit beta'
E: DNA-directed RNA polymerase subunit omega
F: RNA polymerase sigma factor RpoD
G: cAMP-activated global transcriptional regulator CRP
H: cAMP-activated global transcriptional regulator CRP
1: SYNTHETIC NONTEMPLATE STRAND DNA (78-MER)
2: SYNTHETIC TEMPLATE STRAND DNA (78-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)558,08715
Polymers557,27410
Non-polymers8145
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area54750 Å2
ΔGint-311 kcal/mol
Surface area223430 Å2

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Components

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DNA-directed RNA polymerase subunit ... , 4 types, 5 molecules ABCDE

#1: Protein DNA-directed RNA polymerase subunit alpha / Polymerase / RNAP subunit alpha / RNA polymerase subunit alpha / Transcriptase subunit alpha


Mass: 36558.680 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: rpoA, Z4665, ECs4160 / Production host: Escherichia coli (E. coli)
References: UniProt: P0A7Z6, UniProt: P0A7Z4*PLUS, DNA-directed RNA polymerase
#2: Protein DNA-directed RNA polymerase subunit beta / Polymerase / RNAP subunit beta / RNA polymerase subunit beta / Transcriptase subunit beta


Mass: 150820.875 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: rpoB, ECS88_4448 / Production host: Escherichia coli (E. coli) / References: UniProt: B7MIX3, DNA-directed RNA polymerase
#3: Protein DNA-directed RNA polymerase subunit beta' / Polymerase / RNAP subunit beta' / RNA polymerase subunit beta' / Transcriptase subunit beta'


Mass: 155366.781 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: rpoC, Z5561, ECs4911 / Production host: Escherichia coli (E. coli) / References: UniProt: P0A8T8, DNA-directed RNA polymerase
#4: Protein DNA-directed RNA polymerase subunit omega / Polymerase / RNAP omega subunit / RNA polymerase omega subunit / Transcriptase subunit omega


Mass: 10249.547 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: rpoZ, ECS88_4064 / Production host: Escherichia coli (E. coli)
References: UniProt: B7MFL0, UniProt: P0A800*PLUS, DNA-directed RNA polymerase

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Protein , 2 types, 3 molecules FGH

#5: Protein RNA polymerase sigma factor RpoD / Sigma-70


Mass: 72206.266 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: rpoD, alt, b3067, JW3039 / Production host: Escherichia coli (E. coli) / References: UniProt: P00579
#6: Protein cAMP-activated global transcriptional regulator CRP / Catabolite activator protein / CAP / Catabolite gene activator / cAMP receptor protein / CRP / cAMP ...Catabolite activator protein / CAP / Catabolite gene activator / cAMP receptor protein / CRP / cAMP regulatory protein


Mass: 23672.439 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: crp, Z4718, ECs4208 / Production host: Escherichia coli (E. coli) / References: UniProt: P0ACK0

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DNA chain , 2 types, 2 molecules 12

#7: DNA chain SYNTHETIC NONTEMPLATE STRAND DNA (78-MER)


Mass: 23976.332 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)
#8: DNA chain SYNTHETIC TEMPLATE STRAND DNA (78-MER)


Mass: 24191.590 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)

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Non-polymers , 3 types, 5 molecules

#9: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
#10: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#11: Chemical ChemComp-CMP / ADENOSINE-3',5'-CYCLIC-MONOPHOSPHATE / CYCLIC AMP / CAMP / Cyclic adenosine monophosphate


Mass: 329.206 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H12N5O6P / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1The E. coli class-II CAP-dependent transcription activation complex at the state 2COMPLEX#1-#80RECOMBINANT
2DNA-directed RNA polymerase subunit alpha, DNA-directed RNA polymerase subunit beta, DNA-directed RNA polymerase subunit beta', DNA-directed RNA polymerase subunit omega, RNA polymerase sigma factor RpoD, cAMP-activated global transcriptional regulator CRPPolymeraseCOMPLEX#1-#61RECOMBINANT
3DNACOMPLEX#7-#81RECOMBINANT
Molecular weightValue: 0.55 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
11Escherichia coli (E. coli)562
22Escherichia coli (E. coli)562
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
11Escherichia coli (E. coli)562
22synthetic construct (others)32630
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mM2-Amino-2-hydroxymethyl-1,3-propanediolTris1
250 mMsodium chlorideNaClSodium chloride1
35 mMmagnesium chlorideMgCl21
SpecimenConc.: 0.55 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 15 mA / Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K / Details: blot for 3 seconds before plunging

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Details: Direct alignments: Beam tilt pivot points, Beam shift, Comma Free. C2 aperture centering, C2 lens astigmatism correction. Objective aperture centering and objective lens astigmatism correction.
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 96000 X / Nominal defocus max: 2600 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 77 K / Temperature (min): 77 K
Image recordingAverage exposure time: 45 sec. / Electron dose: 45 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k)

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Processing

EM software
IDNameCategory
2EPUimage acquisition
12cisTEMclassification
13cisTEM3D reconstruction
CTF correctionType: NONE
3D reconstructionResolution: 4.29 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 37456 / Symmetry type: POINT

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