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- PDB-9ei2: Cryo-EM structure of Human RNA polymerase II Elongation Complex b... -

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Basic information

Entry
Database: PDB / ID: 9ei2
TitleCryo-EM structure of Human RNA polymerase II Elongation Complex bound to an apo RECQL5 helicase (RECQL5 IRI Module focused-classified)
Components
  • ATP-dependent DNA helicase Q5
  • DNA-directed RNA polymerase II subunit RPB1
KeywordsTRANSCRIPTION / TRANSFERASE/DNA/RNA / translocation / Human RNA polymerase II / RECQL5 helicase / IRI Module / TRANSFERASE-DNA-RNA complex
Function / homology
Function and homology information


mitotic DNA-templated DNA replication / microfibril binding / chromosome separation / cellular response to camptothecin / four-way junction helicase activity / replication-born double-strand break repair via sister chromatid exchange / Abortive elongation of HIV-1 transcript in the absence of Tat / FGFR2 alternative splicing / transcription preinitiation complex / MicroRNA (miRNA) biogenesis ...mitotic DNA-templated DNA replication / microfibril binding / chromosome separation / cellular response to camptothecin / four-way junction helicase activity / replication-born double-strand break repair via sister chromatid exchange / Abortive elongation of HIV-1 transcript in the absence of Tat / FGFR2 alternative splicing / transcription preinitiation complex / MicroRNA (miRNA) biogenesis / Viral Messenger RNA Synthesis / Signaling by FGFR2 IIIa TM / DNA 3'-5' helicase / DNA metabolic process / 3'-5' DNA helicase activity / RNA Pol II CTD phosphorylation and interaction with CE during HIV infection / RNA Pol II CTD phosphorylation and interaction with CE / HIV Transcription Initiation / RNA Polymerase II HIV Promoter Escape / Transcription of the HIV genome / RNA Polymerase II Promoter Escape / RNA Polymerase II Transcription Pre-Initiation And Promoter Opening / RNA Polymerase II Transcription Initiation / RNA Polymerase II Transcription Initiation And Promoter Clearance / Formation of the Early Elongation Complex / Formation of the HIV-1 Early Elongation Complex / mRNA Capping / mRNA Splicing - Minor Pathway / PIWI-interacting RNA (piRNA) biogenesis / RNA polymerase II complex binding / Processing of Capped Intron-Containing Pre-mRNA / Pausing and recovery of Tat-mediated HIV elongation / Tat-mediated HIV elongation arrest and recovery / negative regulation of transcription elongation by RNA polymerase II / RNA polymerase II transcribes snRNA genes / HIV elongation arrest and recovery / Pausing and recovery of HIV elongation / negative regulation of double-strand break repair via homologous recombination / Tat-mediated elongation of the HIV-1 transcript / Formation of HIV-1 elongation complex containing HIV-1 Tat / Formation of HIV elongation complex in the absence of HIV Tat / RNA polymerase II, core complex / RNA Polymerase II Transcription Elongation / Formation of RNA Pol II elongation complex / RNA Polymerase II Pre-transcription Events / mRNA Splicing - Major Pathway / DNA helicase activity / Inhibition of DNA recombination at telomere / positive regulation of RNA splicing / replication fork / TP53 Regulates Transcription of DNA Repair Genes / promoter-specific chromatin binding / Transcriptional regulation by small RNAs / DNA-templated transcription termination / helicase activity / double-strand break repair via homologous recombination / Transcription-Coupled Nucleotide Excision Repair (TC-NER) / Formation of TC-NER Pre-Incision Complex / kinase binding / cellular response to xenobiotic stimulus / Activation of anterior HOX genes in hindbrain development during early embryogenesis / Dual incision in TC-NER / DNA-directed RNA polymerase / Gap-filling DNA repair synthesis and ligation in TC-NER / DNA-directed RNA polymerase activity / mitotic cell cycle / chromosome / Hydrolases; Acting on ester bonds; Exoribonucleases producing 5'-phosphomonoesters / Estrogen-dependent gene expression / transcription by RNA polymerase II / DNA replication / hydrolase activity / cell division / RNA-directed RNA polymerase / DNA repair / RNA-directed RNA polymerase activity / ubiquitin protein ligase binding / regulation of DNA-templated transcription / magnesium ion binding / ATP hydrolysis activity / mitochondrion / DNA binding / RNA binding / zinc ion binding / nucleoplasm / ATP binding / metal ion binding / identical protein binding / nucleus / cytoplasm / cytosol
Similarity search - Function
RecQ helicase-like 5 / RecQ helicase protein-like 5 (RecQ5) / Set2 Rpb1 interacting domain / SRI (Set2 Rpb1 interacting) domain / ATP-dependent DNA helicase RecQ, zinc-binding domain / RecQ zinc-binding / DNA helicase, ATP-dependent, RecQ type / DNA/RNA helicase, ATP-dependent, DEAH-box type, conserved site / DEAH-box subfamily ATP-dependent helicases signature. / RNA polymerase Rpb1 C-terminal repeat ...RecQ helicase-like 5 / RecQ helicase protein-like 5 (RecQ5) / Set2 Rpb1 interacting domain / SRI (Set2 Rpb1 interacting) domain / ATP-dependent DNA helicase RecQ, zinc-binding domain / RecQ zinc-binding / DNA helicase, ATP-dependent, RecQ type / DNA/RNA helicase, ATP-dependent, DEAH-box type, conserved site / DEAH-box subfamily ATP-dependent helicases signature. / RNA polymerase Rpb1 C-terminal repeat / RNA polymerase II, heptapeptide repeat, eukaryotic / Eukaryotic RNA polymerase II heptapeptide repeat. / RNA polymerase Rpb1, domain 6 / RNA polymerase Rpb1, domain 6 / RNA polymerase Rpb1, domain 7 / RNA polymerase Rpb1, domain 7 superfamily / RNA polymerase Rpb1, domain 7 / DEAD/DEAH box helicase domain / DEAD/DEAH box helicase / DNA-directed RNA polymerase, subunit beta-prime / Helicase conserved C-terminal domain / RNA polymerase Rpb1, domain 3 superfamily / 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, 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, N-terminal / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase I subunit A N-terminus / helicase superfamily c-terminal domain / Superfamilies 1 and 2 helicase C-terminal domain profile. / Superfamilies 1 and 2 helicase ATP-binding type-1 domain profile. / DEAD-like helicases superfamily / Helicase, C-terminal / Helicase superfamily 1/2, ATP-binding domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ATP-dependent DNA helicase Q5 / DNA-directed RNA polymerase II subunit RPB1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsFlorez Ariza, A. / Lue, N. / Nogales, E.
Funding support United States, 2items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
National Institutes of Health/National Institute of Biomedical Imaging and Bioengineering (NIH/NIBIB)R35 GM127018 United States
Citation
Journal: Nat Struct Mol Biol / Year: 2025
Title: Structural insights into transcriptional regulation by the helicase RECQL5.
Authors: Alfredo Jose Florez Ariza / Nicholas Z Lue / Patricia Grob / Benjamin Kaeser / Jie Fang / Susanne A Kassube / Eva Nogales /
Abstract: Transcription poses a major challenge for genome stability. The RECQL5 helicase helps safeguard genome integrity and is the only member of the human RecQ helicase family that directly binds to RNA ...Transcription poses a major challenge for genome stability. The RECQL5 helicase helps safeguard genome integrity and is the only member of the human RecQ helicase family that directly binds to RNA polymerase II (Pol II) and affects its progression. While RECQL5 mitigates transcription stress in cells, the molecular mechanism underlying this phenomenon is unclear. Here, we use cryo-electron microscopy to determine the structures of stalled human Pol II elongation complexes (ECs) bound to RECQL5. Our structures reveal the molecular interactions stabilizing RECQL5 binding to the Pol II EC and highlight its role as a transcriptional roadblock. Additionally, we find that, in its nucleotide-free state, RECQL5 twists the downstream DNA in the EC and, upon nucleotide binding, undergoes a conformational change that allosterically induces Pol II toward a post-translocation state. We propose that this mechanism may help restart Pol II elongation and, therefore, contribute to reducing transcription stress.
#1: Journal: bioRxiv / Year: 2025
Title: Structural insights into transcriptional regulation by the helicase RECQL5.
Authors: Alfredo Jose Florez Ariza / Nicholas Z Lue / Patricia Grob / Benjamin Kaeser / Jie Fang / Susanne A Kassube / Eva Nogales /
Abstract: Transcription and its regulation pose a major challenge for genome stability. The helicase RECQL5 has been proposed as an important factor to help safeguard the genome, and is the only member of the ...Transcription and its regulation pose a major challenge for genome stability. The helicase RECQL5 has been proposed as an important factor to help safeguard the genome, and is the only member of the human RecQ helicase family that directly binds to RNA Polymerase II (Pol II) and affects its progression. RECQL5 mitigates transcription stress and genome instability in cells, yet the molecular mechanism underlying this phenomenon is unclear. Here, we employ cryo-electron microscopy (cryo-EM) to determine the structures of stalled Pol II elongation complexes (ECs) bound to RECQL5. Our structures reveal the molecular interactions stabilizing RECQL5 binding to the Pol II EC and highlight its role as a transcriptional roadblock. Additionally, we find that RECQL5 can modulate the Pol II translocation state. In its nucleotide-free state, RECQL5 mechanically twists the downstream DNA in the EC, and upon nucleotide binding, it undergoes a conformational change that allosterically induces Pol II towards a post-translocation state. We propose this mechanism may help restart Pol II elongation and therefore contribute to reduction of transcription stress.
History
DepositionNov 25, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 5, 2025Provider: repository / Type: Initial release
Revision 1.0Mar 5, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Mar 5, 2025Data content type: Additional map / Part number: 1 / Data content type: Additional map / Provider: repository / Type: Initial release
Revision 1.0Mar 5, 2025Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Mar 5, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Mar 5, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Mar 5, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Mar 5, 2025Data content type: Mask / Part number: 1 / Data content type: Mask / Provider: repository / Type: Initial release
Revision 1.0Mar 5, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Jul 16, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin / Item: _em_admin.last_update
Revision 1.2Sep 24, 2025Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: DNA-directed RNA polymerase II subunit RPB1
U: ATP-dependent DNA helicase Q5


Theoretical massNumber of molelcules
Total (without water)326,4452
Polymers326,4452
Non-polymers00
Water00
1


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

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Components

#1: Protein DNA-directed RNA polymerase II subunit RPB1 / RNA polymerase II subunit B1 / DNA-directed RNA polymerase II subunit A / DNA-directed RNA ...RNA polymerase II subunit B1 / DNA-directed RNA polymerase II subunit A / DNA-directed RNA polymerase III largest subunit / RNA-directed RNA polymerase II subunit RPB1


Mass: 217420.047 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: P24928, DNA-directed RNA polymerase
#2: Protein ATP-dependent DNA helicase Q5 / DNA 3'-5' helicase RecQ5 / DNA helicase / RecQ-like type 5 / RecQ5 / RecQ protein-like 5


Mass: 109024.859 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RECQL5, RECQ5 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: O94762, DNA 3'-5' helicase
Has protein modificationN

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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: Stalled Human RNA polymerase II Elongation Complex bound to an apo RECQL5 helicase (RECQL5 IRI Module-focused classified)
Type: COMPLEX / Entity ID: all / Source: MULTIPLE SOURCES
Molecular weightValue: 0.49 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
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 FIELD / Nominal defocus max: 1800 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM softwareName: PHENIX / Version: 1.20.1_4487 / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 103214 / Symmetry type: POINT
RefinementHighest resolution: 2.8 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0042216
ELECTRON MICROSCOPYf_angle_d0.5652986
ELECTRON MICROSCOPYf_dihedral_angle_d4.246292
ELECTRON MICROSCOPYf_chiral_restr0.041328
ELECTRON MICROSCOPYf_plane_restr0.004395

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