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- PDB-8ree: Cryo-EM structure of bacterial RNA polymerase-sigma54 initial tra... -

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Basic information

Entry
Database: PDB / ID: 8ree
TitleCryo-EM structure of bacterial RNA polymerase-sigma54 initial transcribing complex - 9nt complex
Components
  • (DNA-directed RNA polymerase subunit ...Polymerase) x 4
  • DNA (45-MER)
  • DNA (49-MER)
  • RNA (5'-R(P*GP*CP*CP*GP*CP*GP*AP*UP*C)-3')
  • RNA polymerase sigma-54 factor
KeywordsTRANSCRIPTION / initiation / rna polymerase / sigma54
Function / homology
Function and homology information


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 / bacterial-type flagellum-dependent cell motility / nitrate assimilation / transcription elongation factor complex / regulation of DNA-templated transcription elongation ...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 / bacterial-type flagellum-dependent cell motility / nitrate assimilation / transcription elongation factor 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 / response to antibiotic / magnesium ion binding / DNA binding / zinc ion binding / membrane / cytosol / cytoplasm
Similarity search - Function
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 / RNA polymerase Rpb6 ...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 / 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
Similarity search - Domain/homology
DNA / DNA (> 10) / RNA / DNA-directed RNA polymerase subunit alpha / DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit beta
Similarity search - Component
Biological speciesEscherichia coli K-12 (bacteria)
Klebsiella oxytoca (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsGao, F. / Zhang, X.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
UK Research and Innovation (UKRI)BB/M011178/1 United Kingdom
CitationJournal: Proc Natl Acad Sci U S A / Year: 2024
Title: Structural basis of σ displacement and promoter escape in bacterial transcription.
Authors: Forson Gao / Fuzhou Ye / Bowen Zhang / Nora Cronin / Martin Buck / Xiaodong Zhang /
Abstract: Gene transcription is a fundamental cellular process carried out by RNA polymerase (RNAP). Transcription initiation is highly regulated, and in bacteria, transcription initiation is mediated by sigma ...Gene transcription is a fundamental cellular process carried out by RNA polymerase (RNAP). Transcription initiation is highly regulated, and in bacteria, transcription initiation is mediated by sigma (σ) factors. σ recruits RNAP to the promoter DNA region, located upstream of the transcription start site (TSS) and facilitates open complex formation, where double-stranded DNA is opened up into a transcription bubble and template strand DNA is positioned inside RNAP for initial RNA synthesis. During initial transcription, RNAP remains bound to σ and upstream DNA, presumably with an enlarging transcription bubble. The release of RNAP from upstream DNA is required for promoter escape and processive transcription elongation. Bacteria sigma factors can be broadly separated into two classes with the majority belonging to the σ class, represented by the σ that regulates housekeeping genes. σ forms a class on its own and regulates stress response genes. Extensive studies on σ have revealed the molecular mechanisms of the σ dependent process while how σ transitions from initial transcription to elongation is currently unknown. Here, we present a series of cryo-electron microscopy structures of the RNAP-σ initial transcribing complexes with progressively longer RNA, which reveal structural changes that lead to promoter escape. Our data show that initially, the transcription bubble enlarges, DNA strands scrunch, reducing the interactions between σ and DNA strands in the transcription bubble. RNA extension and further DNA scrunching help to release RNAP from σ and upstream DNA, enabling the transition to elongation.
History
DepositionDec 10, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 17, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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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
M: RNA polymerase sigma-54 factor
N: DNA (45-MER)
R: RNA (5'-R(P*GP*CP*CP*GP*CP*GP*AP*UP*C)-3')
T: DNA (49-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)451,32312
Polymers451,1689
Non-polymers1553
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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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: 35726.789 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Gene: rpoA, pez, phs, sez, b3295, JW3257 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P0A7Z4, DNA-directed RNA polymerase
#2: Protein DNA-directed RNA polymerase subunit beta / Polymerase


Mass: 150691.750 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Gene: rpoB / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P0A8V2
#3: Protein DNA-directed RNA polymerase subunit beta' / Polymerase


Mass: 152040.266 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Gene: rpoC / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P0A8T7
#4: Protein DNA-directed RNA polymerase subunit omega / Polymerase / RNAP omega subunit / RNA polymerase omega subunit / Transcriptase subunit omega


Mass: 8449.504 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Gene: rpoZ, b3649, JW3624 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P0A800, DNA-directed RNA polymerase

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DNA chain , 2 types, 2 molecules NT

#6: DNA chain DNA (45-MER)


Mass: 13794.841 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Klebsiella oxytoca (bacteria) / Production host: Klebsiella oxytoca (bacteria)
#8: DNA chain DNA (49-MER)


Mass: 15165.748 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Klebsiella oxytoca (bacteria) / Production host: Klebsiella oxytoca (bacteria)

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Protein / RNA chain , 2 types, 2 molecules MR

#5: Protein RNA polymerase sigma-54 factor


Mass: 36725.473 Da / Num. of mol.: 1 / Mutation: R336A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Klebsiella oxytoca (bacteria) / Production host: Klebsiella oxytoca (bacteria)
#7: RNA chain RNA (5'-R(P*GP*CP*CP*GP*CP*GP*AP*UP*C)-3')


Mass: 2846.759 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Klebsiella oxytoca (bacteria) / Production host: Klebsiella oxytoca (bacteria)

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Non-polymers , 2 types, 3 molecules

#9: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#10: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn

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Details

Has ligand of interestN

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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: RNA polymerase-sigma54 initial transcribing complex - 9nt complex
Type: COMPLEX / Entity ID: #1-#8 / Source: RECOMBINANT
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Source (recombinant)Organism: Escherichia coli K-12 (bacteria)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 3000 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 30 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 13704 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00329224
ELECTRON MICROSCOPYf_angle_d0.61440154
ELECTRON MICROSCOPYf_dihedral_angle_d15.6524914
ELECTRON MICROSCOPYf_chiral_restr0.0414788
ELECTRON MICROSCOPYf_plane_restr0.0054906

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