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- PDB-6xl9: Cryo-EM structure of EcmrR-RNAP-promoter initial transcribing com... -

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Basic information

Entry
Database: PDB / ID: 6xl9
TitleCryo-EM structure of EcmrR-RNAP-promoter initial transcribing complex with 3-nt RNA transcript (EcmrR-RPitc-3nt)
Components
  • (DNA-directed RNA polymerase subunit ...) x 3
  • MerR family transcriptional regulator EcmrR
  • RNA (5'-D(*(GTP))-R(P*AP*G)-3')
  • RNA polymerase sigma factor RpoD
  • synthetic non-template strand DNA (54-MER)
  • synthetic template strand DNA (54-MER)
KeywordsTRANSCRIPTION / TRANSFERASE/DNA / Transcriptional factor / TRANSCRIPTION TRANSFERASE-DNA complex / promoter / multidrug recognition / TRANSFERASE-DNA complex
Function / homology
Function and homology information


sigma factor antagonist complex / cytosolic DNA-directed RNA polymerase complex / regulation of DNA-templated transcription initiation / sigma factor activity / DNA-directed RNA polymerase complex / DNA-templated transcription initiation / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / response to heat ...sigma factor antagonist complex / cytosolic DNA-directed RNA polymerase complex / regulation of DNA-templated transcription initiation / sigma factor activity / DNA-directed RNA polymerase complex / DNA-templated transcription initiation / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / response to heat / protein dimerization activity / negative regulation of DNA-templated transcription / DNA-templated transcription / magnesium ion binding / DNA binding / zinc ion binding / cytosol / cytoplasm
Similarity search - Function
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 / 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 ...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 / 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 / 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 / RNA polymerase Rpb1, domain 3 superfamily / RNA polymerase Rpb1, clamp domain superfamily / RNA polymerase Rpb2, domain 2 superfamily / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 3 / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 1 / 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 / RNA polymerase, beta subunit, protrusion / RNA polymerase beta subunit / RNA polymerase, N-terminal / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase I subunit A N-terminus / 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-like DNA-binding domain superfamily
Similarity search - Domain/homology
TETRAPHENYLANTIMONIUM ION / DNA / DNA (> 10) / RNA / DNA-directed RNA polymerase subunit beta / RNA polymerase sigma factor RpoD / DNA-directed RNA polymerase subunit alpha / DNA-directed RNA polymerase subunit beta'
Similarity search - Component
Biological speciesEscherichia coli O157:H7 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.5 Å
AuthorsYang, Y. / Liu, C. / Shi, W. / Liu, B.
CitationJournal: Nat Commun / Year: 2021
Title: Structural visualization of transcription activated by a multidrug-sensing MerR family regulator.
Authors: Yang Yang / Chang Liu / Wei Zhou / Wei Shi / Ming Chen / Baoyue Zhang / David G Schatz / Yangbo Hu / Bin Liu /
Abstract: Bacterial RNA polymerase (RNAP) holoenzyme initiates transcription by recognizing the conserved -35 and -10 promoter elements that are optimally separated by a 17-bp spacer. The MerR family of ...Bacterial RNA polymerase (RNAP) holoenzyme initiates transcription by recognizing the conserved -35 and -10 promoter elements that are optimally separated by a 17-bp spacer. The MerR family of transcriptional regulators activate suboptimal 19-20 bp spacer promoters in response to myriad cellular signals, ranging from heavy metals to drug-like compounds. The regulation of transcription by MerR family regulators is not fully understood. Here we report one crystal structure of a multidrug-sensing MerR family regulator EcmrR and nine cryo-electron microscopy structures that capture the EcmrR-dependent transcription process from promoter opening to initial transcription to RNA elongation. These structures reveal that EcmrR is a dual ligand-binding factor that reshapes the suboptimal 19-bp spacer DNA to enable optimal promoter recognition, sustains promoter remodeling to stabilize initial transcribing complexes, and finally dissociates from the promoter to reverse DNA remodeling and facilitate the transition to elongation. Our findings yield a comprehensive model for transcription regulation by MerR family factors and provide insights into the transition from transcription initiation to elongation.
History
DepositionJun 28, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 7, 2021Provider: repository / Type: Initial release
Revision 1.1Apr 21, 2021Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.title / _citation.year
Revision 1.2May 26, 2021Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.3Mar 6, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.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'
F: RNA polymerase sigma factor RpoD
N: synthetic non-template strand DNA (54-MER)
R: RNA (5'-D(*(GTP))-R(P*AP*G)-3')
T: synthetic template strand DNA (54-MER)
G: MerR family transcriptional regulator EcmrR
H: MerR family transcriptional regulator EcmrR
hetero molecules


Theoretical massNumber of molelcules
Total (without water)548,40417
Polymers546,93410
Non-polymers1,4707
Water37821
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area58490 Å2
ΔGint-329 kcal/mol
Surface area208580 Å2

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Components

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DNA-directed RNA polymerase subunit ... , 3 types, 4 molecules ABCD

#1: Protein DNA-directed RNA polymerase subunit alpha / 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 O157:H7 (bacteria) / Gene: rpoA, Z4665, ECs4160 / Production host: Escherichia coli O157:H7 (bacteria) / References: UniProt: P0A7Z6, DNA-directed RNA polymerase
#2: Protein DNA-directed RNA polymerase subunit beta / 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 O157:H7 (bacteria) / Gene: rpoB, ECS88_4448 / Production host: Escherichia coli O157:H7 (bacteria) / References: UniProt: B7MIX3, DNA-directed RNA polymerase
#3: Protein DNA-directed RNA polymerase subunit beta' / 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 O157:H7 (bacteria) / Gene: rpoC, Z5561, ECs4911 / Production host: Escherichia coli O157:H7 (bacteria) / References: UniProt: P0A8T8, DNA-directed RNA polymerase

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Protein , 2 types, 3 molecules FGH

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


Mass: 70352.242 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli O157:H7 (bacteria) / Gene: rpoD, alt, b3067, JW3039 / Production host: Escherichia coli O157:H7 (bacteria) / References: UniProt: P00579
#8: Protein MerR family transcriptional regulator EcmrR


Mass: 31394.191 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli O157:H7 (bacteria) / Production host: Escherichia coli O157:H7 (bacteria)

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DNA chain , 2 types, 2 molecules NT

#5: DNA chain synthetic non-template strand DNA (54-MER)


Mass: 16718.658 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli O157:H7 (bacteria)
#7: DNA chain synthetic template strand DNA (54-MER)


Mass: 16634.643 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli O157:H7 (bacteria)

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RNA chain , 1 types, 1 molecules R

#6: RNA chain RNA (5'-D(*(GTP))-R(P*AP*G)-3')


Mass: 1134.619 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli O157:H7 (bacteria)

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Non-polymers , 4 types, 28 molecules

#9: Chemical ChemComp-ZN / ZINC ION


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


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
#11: Chemical ChemComp-118 / TETRAPHENYLANTIMONIUM ION


Mass: 430.176 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C24H20Sb / Feature type: SUBJECT OF INVESTIGATION
#12: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 21 / Source method: isolated from a natural source / Formula: H2O

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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

ComponentName: EcmrR-RNAP-promoter initial transcribing complex with 3-nt RNA transcript (EcmrR-RPitc-3nt)
Type: COMPLEX / Entity ID: #1-#8 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Escherichia coli O157:H7 (bacteria)
Source (recombinant)Organism: Escherichia coli O157:H7 (bacteria)
Buffer solutionpH: 7.5
SpecimenConc.: 0.7 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid 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: 295 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 96000 X / Nominal defocus max: 2600 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 30 sec. / Electron dose: 30 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k)
Image scansMovie frames/image: 40 / Used frames/image: 1-40

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Processing

SoftwareName: PHENIX / Version: 1.16_3549: / Classification: refinement
EM software
IDNameVersionCategory
1Gautomatch0.56particle selection
2EPUimage acquisition
4RELION3.1CTF correction
7ISOLDEmodel fitting
9PHENIX1.16model refinement
13cryoSPARC2.143D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 446920
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 110796 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: Correlation coefficient
Atomic model buildingPDB-ID: 6OUL

6oul


Accession code: 6OUL / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00835899
ELECTRON MICROSCOPYf_angle_d0.88748967
ELECTRON MICROSCOPYf_dihedral_angle_d12.1921614
ELECTRON MICROSCOPYf_chiral_restr0.0575529
ELECTRON MICROSCOPYf_plane_restr0.0065981

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