EMDB-50294: Structure of Pol II-TC-NER-STK19 complex, focused on Pol II-ELOF1

Basic information

Entry

Database: EMDB / ID: EMD-50294

• Title: Structure of Pol II-TC-NER-STK19 complex, focused on Pol II-ELOF1
• Map data: sharpened map (deepEMhancer)

Sample

• Complex: Ternary complex of Pol II-TC-NER-STK19, focused on Pol II-ELOF1
  • Protein or peptide: x 14 types
  • DNA: x 2 types
  • RNA: x 1 types

• Keywords: Transcription-coupled DNA repair / TRANSCRIPTION

Function / homology

Function:

Formation of RNA Pol II elongation complex / Formation of the Early Elongation Complex / Transcriptional regulation by small RNAs / RNA Polymerase II Pre-transcription Events / TP53 Regulates Transcription of DNA Repair Genes / FGFR2 alternative splicing / RNA polymerase II transcribes snRNA genes / mRNA Capping / mRNA Splicing - Minor Pathway / Processing of Capped Intron-Containing Pre-mRNA / RNA Polymerase II Promoter Escape / RNA Polymerase II Transcription Pre-Initiation And Promoter Opening / RNA Polymerase II Transcription Initiation / RNA Polymerase II Transcription Elongation / RNA Polymerase II Transcription Initiation And Promoter Clearance / RNA Pol II CTD phosphorylation and interaction with CE / Estrogen-dependent gene expression / Formation of TC-NER Pre-Incision Complex / Dual incision in TC-NER / Gap-filling DNA repair synthesis and ligation in TC-NER / mRNA Splicing - Major Pathway / nuclear lumen / RNA polymerase II complex binding / maintenance of transcriptional fidelity during transcription elongation by RNA polymerase II / transcription by RNA polymerase III / transcription by RNA polymerase I / RNA polymerase I complex / transcription elongation by RNA polymerase I / RNA polymerase III complex / transcription-coupled nucleotide-excision repair / response to UV / RNA polymerase II, core complex / tRNA transcription by RNA polymerase III / : / DNA-directed RNA polymerase activity / DNA-directed RNA polymerase complex / transcription elongation factor complex / transcription initiation at RNA polymerase II promoter / DNA-templated transcription initiation / transcription elongation by RNA polymerase II / Transcription-Coupled Nucleotide Excision Repair (TC-NER) / ribonucleoside binding / Formation of TC-NER Pre-Incision Complex / : / : / : / fibrillar center / : / : / : / DNA-directed RNA polymerase / Dual incision in TC-NER / Gap-filling DNA repair synthesis and ligation in TC-NER / chromosome / transcription by RNA polymerase II / nucleic acid binding / chromosome, telomeric region / protein dimerization activity / protein ubiquitination / RNA-directed RNA polymerase activity / nucleotide binding / DNA-templated transcription / chromatin binding / nucleolus / mitochondrion / DNA binding / zinc ion binding / nucleoplasm / metal ion binding / nucleus / cytosol

Domain/homology:

UV-stimulated scaffold protein A / : / : / Uncharacterized conserved protein (DUF2043) / UVSSA N-terminal domain / Transcription elongation factor 1 / Transcription elongation factor 1 superfamily / Transcription elongation factor Elf1 like / ENTH/VHS / DNA-directed RNA polymerase II subunit Rpb4-like / 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 Rpb4/RPC9, core / DNA-directed RNA-polymerase II subunit / RNA polymerase Rpb1, domain 7 / RNA polymerase Rpb1, domain 7 superfamily / RNA polymerase Rpb1, domain 7 / Pol II subunit B9, C-terminal zinc ribbon / RNA polymerase RBP11 / Rpb4/RPC9 superfamily / RNA polymerase subunit Rpb4/RPC9 / RNA polymerase Rpb4 / Zinc finger TFIIS-type signature. / HRDC-like superfamily / RNA polymerase Rpb7-like , N-terminal / RNA polymerase Rpb7-like, N-terminal domain superfamily / RNA polymerase subunit Rpb7-like / SHS2 domain found in N terminus of Rpb7p/Rpc25p/MJ0397 / RNA polymerase Rpb2, domain 5 / RNA polymerase Rpb2, domain 5 / RNA polymerase Rpb2, domain 4 / RNA polymerase Rpb2, domain 4 / DNA-directed RNA polymerase, M/15kDa subunit / RNA polymerases M/15 Kd subunit / RNA polymerase subunit 9 / DNA-directed RNA polymerase M, 15kDa subunit, conserved site / RNA polymerases M / 15 Kd subunits signature. / DNA-directed RNA polymerase subunit/transcription factor S / : / RNA polymerase, Rpb8 / DNA-directed RNA polymerases I, II, and III subunit RPABC4 / RNA polymerase Rpb8 / RNA polymerase subunit 8 / RNA polymerase, Rpb5, N-terminal / RNA polymerase Rpb5, N-terminal domain superfamily / RNA polymerase Rpb5, N-terminal domain / DNA-directed RNA polymerase, subunit RPB6 / DNA-directed RNA polymerase subunit RPABC5/Rpb10 / RNA polymerases, subunit N, zinc binding site / RNA polymerase subunit RPB10 / RNA polymerases N / 8 kDa subunit / RNA polymerases N / 8 Kd subunits signature. / DNA directed RNA polymerase, 7 kDa subunit / RNA polymerase archaeal subunit P/eukaryotic subunit RPABC4 / RNA polymerase, subunit H/Rpb5, conserved site / RNA polymerases H / 23 Kd subunits signature. / RNA polymerase subunit CX / DNA-directed RNA polymerase, 30-40kDa subunit, conserved site / DNA-directed RNA polymerase subunit Rpo3/Rpb3/RPAC1 / RNA polymerases D / 30 to 40 Kd subunits signature. / DNA-directed RNA polymerase Rpb11, 13-16kDa subunit, conserved site / DNA-directed RNA polymerase subunit Rpo11 / RNA polymerases L / 13 to 16 Kd subunits signature. / Zinc finger, TFIIS-type / Transcription factor S-II (TFIIS) / Zinc finger TFIIS-type profile. / C2C2 Zinc finger / RNA polymerase subunit RPABC4/transcription elongation factor Spt4 / DNA-directed RNA polymerase, RBP11-like dimerisation domain / RNA polymerase Rpb3/Rpb11 dimerisation domain / RNA polymerase, subunit H/Rpb5 C-terminal / DNA-directed RNA polymerase subunit Rpo5/Rpb5 / RPB5-like RNA polymerase subunit superfamily / RNA polymerase Rpb5, C-terminal domain / Archaeal Rpo6/eukaryotic RPB6 RNA polymerase subunit / DNA-directed RNA polymerase, 14-18kDa subunit, conserved site / RNA polymerases K / 14 to 18 Kd subunits signature. / Ribosomal protein S1-like RNA-binding domain / S1 RNA binding domain / S1 domain / 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

Component:

RNA polymerase II, I and III subunit K / DNA-directed RNA polymerases I, II, and III subunit RPABC2 / DNA-directed RNA polymerase subunit / RNA polymerase II subunit D / DNA-directed RNA polymerases I, II, and III subunit RPABC5 / DNA-directed RNA polymerase subunit / DNA-directed RNA polymerase II subunit RPB11-a / DNA-directed RNA polymerases I, II, and III subunit RPABC3 / DNA-directed RNA polymerase II subunit RPB3 / DNA-directed RNA polymerase subunit beta / DNA-directed RNA polymerase II subunit E / Transcription elongation factor 1 homolog / DNA-directed RNA polymerase II subunit RPB9 / UV-stimulated scaffold protein A

• Biological species: Homo sapiens (human) / Sus scrofa domesticus (domestic pig)
• Method: single particle reconstruction / cryo EM / Resolution: 3.0 Å
• Authors: Lee S-H / Sixma TK

Funding support

Netherlands, 2 items

Organization	Grant number	Country
Netherlands Organisation for Scientific Research (NWO)	TOP 714.017.003	Netherlands
Oncode Institute		Netherlands

• Citation: Journal: Mol Cell / Year: 2024; Title: STK19 drives transcription-coupled repair by stimulating repair complex stability, RNA Pol II ubiquitylation, and TFIIH recruitment.; Authors: Anisha R Ramadhin / Shun-Hsiao Lee / Di Zhou / Anita Salmazo / Camila Gonzalo-Hansen / Marjolein van Sluis / Cindy M A Blom / Roel C Janssens / Anja Raams / Dick Dekkers / Karel Bezstarosti / Dea Slade / Wim Vermeulen / Alex Pines / Jeroen A A Demmers / Carrie Bernecky / Titia K Sixma / Jurgen A Marteijn / ; Abstract: Transcription-coupled nucleotide excision repair (TC-NER) efficiently eliminates DNA damage that impedes gene transcription by RNA polymerase II (RNA Pol II). TC-NER is initiated by the recognition of lesion-stalled RNA Pol II by CSB, which recruits the CRL4 ubiquitin ligase and UVSSA. RNA Pol II ubiquitylation at RPB1-K1268 by CRL4 serves as a critical TC-NER checkpoint, governing RNA Pol II stability and initiating DNA damage excision by TFIIH recruitment. However, the precise regulatory mechanisms of CRL4 activity and TFIIH recruitment remain elusive. Here, we reveal human serine/threonine-protein kinase 19 (STK19) as a TC-NER factor, which is essential for correct DNA damage removal and subsequent transcription restart. Cryogenic electron microscopy (cryo-EM) studies demonstrate that STK19 is an integral part of the RNA Pol II-TC-NER complex, bridging CSA, UVSSA, RNA Pol II, and downstream DNA. STK19 stimulates TC-NER complex stability and CRL4 activity, resulting in efficient RNA Pol II ubiquitylation and correct UVSSA and TFIIH binding. These findings underscore the crucial role of STK19 as a core TC-NER component.

History

Deposition	May 14, 2024	-
Header (metadata) release	Nov 13, 2024	-
Map release	Nov 13, 2024	-
Update	Jan 8, 2025	-
Current status	Jan 8, 2025	Processing site: PDBe / Status: Released

Map

• File: Download / File: emd_50294.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• Annotation: sharpened map (deepEMhancer)
• Voxel size: X=Y=Z: 1.06 Å

Density

Contour Level	By AUTHOR: 0.16
Minimum - Maximum	-0.029039634 - 2.0104222
Average (Standard dev.)	0.00093140115 (±0.020953694)

• Symmetry: Space group: 1

Details

EMDB XML:

Map geometry	Axis order X Y Z Origin 0 0 0 Dimensions 400 400 400 Spacing 400 400 400
Cell	A=B=C: 423.99997 Å α=β=γ: 90.0 °

Supplemental data

Mask #1

• File: emd_50294_msk_1.map

Additional map: raw map

• File: emd_50294_additional_1.map
• Annotation: raw map

Half map: half map A

• File: emd_50294_half_map_1.map
• Annotation: half map A

Half map: half map B

• File: emd_50294_half_map_2.map
• Annotation: half map B

Sample components

Entire : Ternary complex of Pol II-TC-NER-STK19, focused on Pol II-ELOF1

• Entire: Name: Ternary complex of Pol II-TC-NER-STK19, focused on Pol II-ELOF1

Components

• Complex: Ternary complex of Pol II-TC-NER-STK19, focused on Pol II-ELOF1
  • Protein or peptide: DNA-directed RNA polymerase II subunit RPB1
  • Protein or peptide: DNA-directed RNA polymerase II subunit RPB2
  • Protein or peptide: DNA-directed RNA polymerase II subunit RPB3
  • Protein or peptide: DNA-directed RNA polymerase II subunit RPB4
  • Protein or peptide: DNA-directed RNA polymerase II subunit E
  • Protein or peptide: DNA-directed RNA polymerases I, II, and III subunit RPABC2
  • Protein or peptide: DNA-directed RNA polymerase subunit RPB7
  • Protein or peptide: DNA-directed RNA polymerases I, II, and III subunit RPABC3
  • Protein or peptide: DNA-directed RNA polymerase II subunit RPB9
  • Protein or peptide: DNA-directed RNA polymerases I, II, and III subunit RPABC5
  • Protein or peptide: DNA-directed RNA polymerase II subunit RPB11-a
  • Protein or peptide: DNA-directed RNA polymerases I, II, and III subunit RPABC4
  • Protein or peptide: Transcription elongation factor 1 homolog
  • Protein or peptide: UV-stimulated scaffold protein A
  • DNA: Template strand DNA
  • DNA: Non-template strand DNA
  • RNA: RNA

Supramolecule #1: Ternary complex of Pol II-TC-NER-STK19, focused on Pol II-ELOF1

• Supramolecule: Name: Ternary complex of Pol II-TC-NER-STK19, focused on Pol II-ELOF1; type: complex / ID: 1 / Parent: 0 / Macromolecule list: all; Details: Focused map contains Pol II, ELOF1 and the C-terminal parts of UVSSA.
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 550 KDa

Macromolecule #1: DNA-directed RNA polymerase II subunit RPB1

• Macromolecule: Name: DNA-directed RNA polymerase II subunit RPB1 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
• Source (natural): Organism: Sus scrofa domesticus (domestic pig) / Organ: thymus

Sequence

String:

MHGGGPPSGD SACPLRTIKR VQFGVLSPDE LKRMSVTEGG IKYPETTEGG RPKLGGLMDP RQGVIERTG RCQTCAGNMT ECPGHFGHIE LAKPVFHVGF LVKTMKVLRC VCFFCSKLLV D SNNPKIKD ILAKSKGQPK KRLTHVYDLC KGKNICEGGE EMDNKFGVEQ PEGDEDLTKE KG HGGCGRY QPRIRRSGLE LYAEWKHVNE DSQEKKILLS PERVHEIFKR ISDEECFVLG MEP RYARPE WMIVTVLPVP PLSVRPAVVM QGSARNQDDL THKLADIVKI NNQLRRNEQN GAAA HVIAE DVKLLQFHVA TMVDNELPGL PRAMQKSGRP LKSLKQRLKG KEGRVRGNLM GKRVD FSAR TVITPDPNLS IDQVGVPRSI AANMTFAEIV TPFNIDRLQE LVRRGNSQYP GAKYII RDN GDRIDLRFHP KPSDLHLQTG YKVERHMCDG DIVIFNRQPT LHKMSMMGHR VRILPWS TF RLNLSVTTPY NADFDGDEMN LHLPQSLETR AEIQELAMVP RMIVTPQSNR PVMGIVQD T LTAVRKFTKR DVFLERGEVM NLLMFLSTWD GKVPQPAILK PRPLWTGKQI FSLIIPGHI NCIRTHSTHP DDEDSGPYKH ISPGDTKVVV ENGELIMGIL CKKSLGTSAG SLVHISYLEM GHDITRLFY SNIQTVINNW LLIEGHTIGI GDSIADSKTY QDIQNTIKKA KQDVIEVIEK A HNNELEPT PGNTLRQTFE NQVNRILNDA RDKTGSSAQK SLSEYNNFKS MVVSGAKGSK IN ISQVIAV VGQQNVEGKR IPFGFKHRTL PHFIKDDYGP ESRGFVENSY LAGLTPTEFF FHA MGGREG LIDTAVKTAE TGYIQRRLIK SMESVMVKYD ATVRNSINQV VQLRYGEDGL AGES VEFQN LATLKPSNKA FEKKFRFDYT NERALRRTLQ EDLVKDVLSN AHIQNELERE FERMR EDRE VLRVIFPTGD SKVVLPCNLL RMIWNAQKIF HINPRLPSDL HPIKVVEGVK ELSKKL VIV NGDDPLSRQA QENATLLFNI HLRSTLCSRR MAEEFRLSGE AFDWLLGEIE SKFNQAI AH PGEMVGALAA QSLGEPATQM TLNTFHYAGV SAKNVTLGVP RLKELINISK KPKTPSLT V FLLGQSARDA ERAKDILCRL EHTTLRKVTA NTAIYYDPNP QSTVVAEDQE WVNVYYEMP DFDVARISPW LLRVELDRKH MTDRKLTMEQ IAEKINAGFG DDLNCIFNDD NAEKLVLRIR IMNSDENKM QEEEEVVDKM DDDVFLRCIE SNMLTDMTLQ GIEQISKVYM HLPQTDNKKK I IITEDGEF KALQEWILET DGVSLMRVLS EKDVDPVRTT SNDIVEIFTV LGIEAVRKAL ER ELYHVIS FDGSYVNYRH LALLCDTMTC RGHLMAITRH GVNRQDTGPL MKCSFEETVD VLM EAAAHG ESDPMKGVSE NIMLGQLAPA GTGCFDLLLD AEKCKYGMEI PTNIPGLGAA GPTG MFFGS APSPMGGISP AMTPWNQGAT PAYGAWSPSV GSGMTPGAAG FSPSAASDAS GFSPG YSPA WSPTPGSPGS PGPSSPYIPS PGGAMSPSYS PTSPAYEPRS PGGYTPQSPS YSPTSP SYS PTSPSYSPTS PNYSPTSPSY SPTSPSYSPT SPSYSPTSPS YSPTSPSYSP TSPSYSP TS PSYSPTSPSY SPTSPSYSPT SPSYSPTSPS YSPTSPSYSP TSPSYSPTSP SYSPTSPS Y SPTSPNYSPT SPNYTPTSPS YSPTSPSYSP TSPNYTPTSP NYSPTSPSYS PTSPSYSPT SPSYSPSSPR YTPQSPTYTP SSPSYSPSSP SYSPTSPKYT PTSPSYSPSS PEYTPTSPKY SPTSPKYSP TSPKYSPTSP TYSPTTPKYS PTSPTYSPTS PVYTPTSPKY SPTSPTYSPT S PKYSPTSP TYSPTSPKGS TYSPTSPGYS PTSPTYSLTS PAISPDDSDE EN

UniProtKB: DNA-directed RNA polymerase subunit

Macromolecule #2: DNA-directed RNA polymerase II subunit RPB2

• Macromolecule: Name: DNA-directed RNA polymerase II subunit RPB2 / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
• Source (natural): Organism: Sus scrofa domesticus (domestic pig)

Sequence

String:

MKPLRPENPP KAELKGLLFV QQHCKTRLHS VAVTRPRGAA TEESTPKLRM CSTNLSQALA YFRAGANLTA ASLWAGFRGS RRIFWERRKR KSLCLALRPG GACWRWLLAV SCVSLRGLGA PGSCANMYDA DEDMQYDEDD DEITPDLWQE ACWIVISSYF DEKGLVRQQL DSFDEFIQMS VQRIVEDAPP IDLQAEAQHA SGEVEEPPRY LLKFEQIYLS KPTHWERDGA PSPMMPNEAR LRNLTYSAPL YVDITKTVIK EGEEQLQTQH QKTFIGKIPI MLRSTYCLLN GLTDRDLCEL NECPLDPGGY FIINGSEKVL IAQEKMATNT VYVFAKKDSK YAYTGECRSC LENSSRPTST IWVSMLARGG QGAKKSAIGQ RIVATLPYIK QEVPIIIVFR ALGFVSDRDI LEHIIYDFED PEMMEMVKPS LDEAFVIQEQ NVALNFIGSR GAKPGVTKEK RIKYAKEVLQ KEMLPHVGVS DFCETKKAYF LGYMVHRLLL AALGRRELDD RDHYGNKRLD LAGPLLAFLF RGMFKNLLKE VRIYAQKFID RGKDFNLELA IKTRIISDGL KYSLATGNWG DQKKAHQARA GVSQVLNRLT FASTLSHLRR LNSPIGRDGK LAKPRQLHNT LWGMVCPAET PEGHAVGLVK NLALMAYISV GSQPSPILEF LEEWSMENLE EISPAAIADA TKIFVNGCWV GIHKDPEQLM NTLRKLRRQM DIIVSEVSMI RDIREREIRI YTDAGRICRP LLIVEKQKLL LKKRHIDQLK EREYNNYSWQ DLVASGVVEY IDTLEEETVM LAMTPDDLQE KEVAYCSTYT HCEIHPSMIL GVCASIIPFP DHNQSPRNTY QSAMGKQAMG VYITNFHVRM DTLAHVLYYP QKPLVTTRSM EYLRFRELPA GINSIVAIAS YTGYNQEDSV IMNRSAVDRG FFRSVFYRSY KEQESKKGFD QEEVFEKPTR ETCQGMRHAI YDKLDDDGLI APGVRVSGDD VIIGKTVTLP ENEDELEGTN RRYTKRDCST FLRTSETGIV DQVMVTLNQE GYKFCKIRVR SVRIPQIGDK FASRHGQKGT CGIQYRQEDM PFTCEGITPD IIINPHAIPS RMTIGHLIEC LQGKVSANKG EIGDATPFND AVNVQKISNL LSDYGYHLRG NEVLYNGFTG RKITSQIFIG PTYYQRLKHM VDDKIHSRAR GPIQILNRQP MEGRSRDGGL RFGEMERDCQ IAHGAAQFLR ERLFEASDPY QVHVCNLCGI MAIANTRTHT YECRGCRNKT QISLVRMPYA CKLLFQELMS MSIAPRMMSV

UniProtKB: DNA-directed RNA polymerase subunit beta

Macromolecule #3: DNA-directed RNA polymerase II subunit RPB3

• Macromolecule: Name: DNA-directed RNA polymerase II subunit RPB3 / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
• Source (natural): Organism: Sus scrofa domesticus (domestic pig) / Organ: thymus

Sequence

String:

MPYANQPTVR ITELTDENVK FIIENTDLAV ANSIRRVFIA EVPIIAIDWV QIDANSSVLH DEFIAHRLGL IPLTSDDIVD KLQYSRDCTC EEFCPECSVE FTLDVRCNED QTRHVTSRDL ISNSPRVIPV TSRNRDNDPS DYVEQDDILI VKLRKGQELR LRAYAKKGFG KEHAKWNPTA GVAFEYDPDN ALRHTVYPKP EEWPKSEYSE LDEDESQAPY DPNGKPERFY YNVESCGSLR PETIVLSALS GLKKKLSDLQ TQLSHEIQSD VLTIN

UniProtKB: DNA-directed RNA polymerase II subunit RPB3

Macromolecule #4: DNA-directed RNA polymerase II subunit RPB4

• Macromolecule: Name: DNA-directed RNA polymerase II subunit RPB4 / type: protein_or_peptide / ID: 4 / Enantiomer: LEVO
• Source (natural): Organism: Sus scrofa domesticus (domestic pig) / Organ: thymus

Sequence

String:

MAAGGSDPRA GDVEEDASQL IFPKEFETAE TLLNSEVHML LEHRKQQNES AEDEQELSEV FMKTLNYTAR FSRFKNRETI ASVRSLLLQK KLHKFELACL ANLCPETAEE SKALIPSLEG RFEDEELQQI LDDIQTKRSF QY

UniProtKB: RNA polymerase II subunit D

Macromolecule #5: DNA-directed RNA polymerase II subunit E

• Macromolecule: Name: DNA-directed RNA polymerase II subunit E / type: protein_or_peptide / ID: 5 / Enantiomer: LEVO
• Source (natural): Organism: Sus scrofa domesticus (domestic pig) / Organ: thymus

Sequence

String:

MDDEEETYRL WKIRKTIMQL CHDRGYLVTQ DELDQTLEEF KAQFGDKPSE GRPRRTDLTV LVAHNDDPTD QMFVFFPEEP KVGIKTIKVY CQRMQEENIT RALIVVQQGM TPSAKQSLVD MAPKYILEQF LQQELLINIT EHELVPEHVV MTKEEVTELL ARYKLRENQL PRIQAGDPVA RYFGIKRGQV VKIIRPSETA GRYITYRLVQ

UniProtKB: DNA-directed RNA polymerase II subunit E

Macromolecule #6: DNA-directed RNA polymerases I, II, and III subunit RPABC2

• Macromolecule: Name: DNA-directed RNA polymerases I, II, and III subunit RPABC2; type: protein_or_peptide / ID: 6 / Enantiomer: LEVO
• Source (natural): Organism: Sus scrofa domesticus (domestic pig) / Organ: thymus

Sequence

String:

MSDNEDNFDG DDFDDVEEDE GLDDLENAEE EGQENVEILP SGERPQANQK RITTPYMTKY ERARVLGTRA LQIAMCAPVM VELEGETDPL LIAMKELKAR KIPIIIRRYL PDGSYEDWGV DELIISD

UniProtKB: DNA-directed RNA polymerases I, II, and III subunit RPABC2

Macromolecule #7: DNA-directed RNA polymerase subunit RPB7

• Macromolecule: Name: DNA-directed RNA polymerase subunit RPB7 / type: protein_or_peptide / ID: 7 / Enantiomer: LEVO
• Source (natural): Organism: Sus scrofa domesticus (domestic pig) / Organ: thymus

Sequence

String:

MFYHISLEHE ILLHPRYFGP NLLNTVKQKL FTEVEGTCTG KYGFVIAVTT IDNIGAGVIQ PGRGFVLYPV KYKAIVFRPF KGEVVDAVVT QVNKVGLFTE IGPMSCFISR HSIPSEMEFD PNSNPPCYKT MDEDIVIQQD DEIRLKIVGT RVDKNDIFAI GSLMDDYLGL VS

UniProtKB: DNA-directed RNA polymerase subunit

Macromolecule #8: DNA-directed RNA polymerases I, II, and III subunit RPABC3

• Macromolecule: Name: DNA-directed RNA polymerases I, II, and III subunit RPABC3; type: protein_or_peptide / ID: 8 / Enantiomer: LEVO
• Source (natural): Organism: Sus scrofa domesticus (domestic pig)

Sequence

String:

MAGILFEDIF DVKDIDPEGK KFDRVSRLHC ESESFKMDLI LDVNIQIYPV DLGDKFRLVI ASTLYEDGTL DDGEYNPTDD RPSRADQFEY VMYGKVYRIE GDETSTEAAT RLSAYVSYGG LLMRLQGDAN NLHGFEVDSR VYLLMKKLAF

UniProtKB: DNA-directed RNA polymerases I, II, and III subunit RPABC3

Macromolecule #9: DNA-directed RNA polymerase II subunit RPB9

• Macromolecule: Name: DNA-directed RNA polymerase II subunit RPB9 / type: protein_or_peptide / ID: 9 / Enantiomer: LEVO
• Source (natural): Organism: Sus scrofa domesticus (domestic pig) / Organ: thymus

Sequence

String:

MEPDGTYEPG FVGIRFCQEC NNMLYPKEDK ENRILLYACR NCDYQQEADN SCIYVNKITH EVDELTQIIA DVSQDPTLPR TEDHPCQKCG HKEAVFFQSH SARAEDAMRL YYVCTAPHCG HRWTE

UniProtKB: DNA-directed RNA polymerase II subunit RPB9

Macromolecule #10: DNA-directed RNA polymerases I, II, and III subunit RPABC5

• Macromolecule: Name: DNA-directed RNA polymerases I, II, and III subunit RPABC5; type: protein_or_peptide / ID: 10 / Enantiomer: LEVO
• Source (natural): Organism: Sus scrofa domesticus (domestic pig) / Organ: thymus

Sequence

String:

MIIPVRCFTC GKIVGNKWEA YLGLLQAEYT EGDALDALGL KRYCCRRMLL AHVDLIEKLL NYAPLEK

UniProtKB: DNA-directed RNA polymerases I, II, and III subunit RPABC5

Macromolecule #11: DNA-directed RNA polymerase II subunit RPB11-a

• Macromolecule: Name: DNA-directed RNA polymerase II subunit RPB11-a / type: protein_or_peptide / ID: 11 / Enantiomer: LEVO
• Source (natural): Organism: Sus scrofa domesticus (domestic pig) / Organ: thymus

Sequence

String:

MNAPPAFESF LLFEGEKKIT INKDTKVPNA CLFTINKEDH TLGNIIKSQL LKDPQVLFAG YKVPHPLEHK IIIRVQTTPD YSPQEAFTNA ITDLISELSL LEERFRVAIK DKQEGIE

UniProtKB: DNA-directed RNA polymerase II subunit RPB11-a

Macromolecule #12: DNA-directed RNA polymerases I, II, and III subunit RPABC4

• Macromolecule: Name: DNA-directed RNA polymerases I, II, and III subunit RPABC4; type: protein_or_peptide / ID: 12 / Enantiomer: LEVO
• Source (natural): Organism: Sus scrofa domesticus (domestic pig) / Organ: thymus

Sequence

String:

MDTQKDVQPP KQQPMIYICG ECHTENEIKS RDPIRCRECG YRIMYKKRTK RLVVFDAR

UniProtKB: RNA polymerase II, I and III subunit K

Macromolecule #13: Transcription elongation factor 1 homolog

• Macromolecule: Name: Transcription elongation factor 1 homolog / type: protein_or_peptide / ID: 13 ; Details: The first two residues (Gly, Ala) are residual residues after TEV protease treatment. ELOF1 sequence starts from residue 3 (Met).; Enantiomer: LEVO
• Source (natural): Organism: Homo sapiens (human)
• Recombinant expression: Organism: Escherichia coli (E. coli)

Sequence

String:

GAMGRRKSKR KPPPKKKMTG TLETQFTCPF CNHEKSCDVK MDRARNTGVI SCTVCLEEFQ TPITYLSEPV DVYSDWIDAC EAANQ

UniProtKB: Transcription elongation factor 1 homolog

Macromolecule #14: UV-stimulated scaffold protein A

• Macromolecule: Name: UV-stimulated scaffold protein A / type: protein_or_peptide / ID: 14 / Details: The construct contains a His tag at the N-terminus / Enantiomer: LEVO
• Source (natural): Organism: Homo sapiens (human)
• Recombinant expression: Organism: Spodoptera frugiperda (fall armyworm)

Sequence

String:

MAHHHHHHSA ALEVLFQGPG MDQKLSKLVE ELTTSGEPRL NPEKMKELKK ICKSSEEQLS RAYRLLIAQL TQEHAEIRLS AFQIVEELFV RSHQFRMLVV SNFQEFLELT LGTDPAQPLP PPREAAQRLR QATTRAVEGW NEKFGEAYKK LALGYHFLRH NKKVDFQDTN ARSLAERKRE EEKQKHLDKI YQERASQAER EMQEMSGEIE SCLTEVESCF RLLVPFDFDP NPETESLGMA SGMSDALRSS CAGQVGPCRS GTPDPRDGEQ PCCSRDLPAS AGHPRAGGGA QPSQTATGDP SDEDEDSDLE EFVRSHGLGS HKYTLDVELC SEGLKVQENE DNLALIHAAR DTLKLIRNKF LPAVCSWIQR FTRVGTHGGC LKRAIDLKAE LELVLRKYKE LDIEPEGGER RRTEALGDAE EDEDDEDFVE VPEKEGYEPH IPDHLRPEYG LEAAPEKDTV VRCLRTRTRM DEEVSDPTSA AAQLRQLRDH LPPPSSASPS RALPEPQEAQ KLAAERARAP VVPYGVDLHY WGQELPTAGK IVKSDSQHRF WKPSEVEEEV VNADISEMLR SRHITFAGKF EPVQHWCRAP RPDGRLCERQ DRLKCPFHGK IVPRDDEGRP LDPEDRAREQ RRQLQKQERP EWQDPELMRD VEAATGQDLG SSRYSGKGRG KKRRYPSLTN LKAQADTARA RIGRKVFAKA AVRRVVAAMN RMDQKKHEKF SNQFNYALN

UniProtKB: UV-stimulated scaffold protein A

Macromolecule #15: Template strand DNA

• Macromolecule: Name: Template strand DNA / type: dna / ID: 15 / Classification: DNA
• Source (natural): Organism: Homo sapiens (human)

Sequence

String:

GATCAAGCTC AAGCGCTCTG CTCCTTCTCC CATCCTCTCG ATGGCTATGA GATCAACTAG

Macromolecule #16: Non-template strand DNA

• Macromolecule: Name: Non-template strand DNA / type: dna / ID: 16 / Classification: DNA
• Source (natural): Organism: Homo sapiens (human)

Sequence

String:

CTAGTTGATC TCATATTTCA TTCCTACTCA GGAGAAGGAG CAGAGCGCTT GAGCTTGATC

Macromolecule #17: RNA

• Macromolecule: Name: RNA / type: rna / ID: 17
• Source (natural): Organism: Homo sapiens (human)

Sequence

String:

GAAUAUAUAU ACAAAAUCGA GAGGA

Experimental details

Structure determination

• Method: cryo EM
• Processing: single particle reconstruction
• Aggregation state: particle

Sample preparation

• Concentration: 0.15 mg/mL

Buffer

pH: 7.5
Component:

Concentration	Formula	Name
20.0 mM	HEPES	HEPES
100.0 mM	NaCl	sodium chloride
2.0 mM	MgCl2	magnesium chloride
10.0 uM	ZnSO4	zinc sulfate
1.0 mM	TCEP	tris(2-carboxyethyl)phosphine

• Grid: Model: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY
• Vitrification: Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
• Details: The final concentration of Pol II is around 0.15 mg/ml. The other components were added in different molar ratio. This sample was glutaraldehyde crosslinked.

Electron microscopy

• Microscope: FEI TITAN KRIOS
• Specialist optics: Energy filter - Slit width: 20 eV
• Details: Collected on Krios 1 at Netherlands Center for Electron Nanoscopy (NeCEN)
• Image recording: Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 2 / Number real images: 13029 / Average exposure time: 3.43 sec. / Average electron dose: 50.0 e/Ų; Details: Two datasets were collected from the same sample using the same parameters.
• Electron beam: Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
• Electron optics: C2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.2 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 81000
• Sample stage: Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
• Experimental equipment: Model: Titan Krios / Image courtesy: FEI Company

Image processing

• Particle selection: Number selected: 2866913 / Details: Particles were combined from two datasets.
• Startup model: Type of model: OTHER
• Final reconstruction: Resolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 159581
• Initial angle assignment: Type: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
• Final angle assignment: Type: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC