+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-15331 | |||||||||||||||
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Title | RNA polymerase- post-terminated, open clamp state | |||||||||||||||
Map data | ||||||||||||||||
Sample |
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Keywords | RNA polymerase / transcriptional pausing / transcription termination / regulatory RNA / TRANSCRIPTION | |||||||||||||||
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 / DNA-directed RNA polymerase complex / transcription elongation factor 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 / bacterial-type flagellum-dependent cell motility / nitrate assimilation / DNA-directed RNA polymerase complex / transcription elongation factor complex / regulation of DNA-templated transcription elongation / transcription antitermination / DNA-templated transcription initiation / cell motility / 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 / DNA-templated transcription / magnesium ion binding / DNA binding / zinc ion binding / membrane / cytoplasm / cytosol Similarity search - Function | |||||||||||||||
Biological species | Escherichia coli K-12 (bacteria) / Escherichia coli (E. coli) | |||||||||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.7 Å | |||||||||||||||
Authors | Dey S / Weixlbaumer A | |||||||||||||||
Funding support | European Union, France, 4 items
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Citation | Journal: Mol Cell / Year: 2022 Title: Structural insights into RNA-mediated transcription regulation in bacteria. Authors: Sanjay Dey / Claire Batisse / Jinal Shukla / Michael W Webster / Maria Takacs / Charlotte Saint-André / Albert Weixlbaumer / Abstract: RNA can regulate its own synthesis without auxiliary proteins. For example, U-rich RNA sequences signal RNA polymerase (RNAP) to pause transcription and are required for transcript release at ...RNA can regulate its own synthesis without auxiliary proteins. For example, U-rich RNA sequences signal RNA polymerase (RNAP) to pause transcription and are required for transcript release at intrinsic terminators in all kingdoms of life. In contrast, the regulatory RNA putL suppresses pausing and termination in cis. However, how nascent RNA modulates its own synthesis remains largely unknown. We present cryo-EM reconstructions of RNAP captured during transcription of putL variants or an unrelated sequence at a U-rich pause site. Our results suggest how putL suppresses pausing and promotes its synthesis. We demonstrate that transcribing a U-rich sequence, a ubiquitous trigger of intrinsic termination, promotes widening of the RNAP nucleic-acid-binding channel. Widening destabilizes RNAP interactions with DNA and RNA to facilitate transcript dissociation reminiscent of intrinsic transcription termination. Surprisingly, RNAP remains bound to DNA after transcript release. Our results provide the structural framework to understand RNA-mediated intrinsic transcription termination. | |||||||||||||||
History |
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-Structure visualization
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_15331.map.gz | 168 MB | EMDB map data format | |
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Header (meta data) | emd-15331-v30.xml emd-15331.xml | 28.9 KB 28.9 KB | Display Display | EMDB header |
Images | emd_15331.png | 37.8 KB | ||
Filedesc metadata | emd-15331.cif.gz | 9.6 KB | ||
Others | emd_15331_half_map_1.map.gz emd_15331_half_map_2.map.gz | 165.1 MB 165.1 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-15331 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-15331 | HTTPS FTP |
-Validation report
Summary document | emd_15331_validation.pdf.gz | 1.1 MB | Display | EMDB validaton report |
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Full document | emd_15331_full_validation.pdf.gz | 1.1 MB | Display | |
Data in XML | emd_15331_validation.xml.gz | 15 KB | Display | |
Data in CIF | emd_15331_validation.cif.gz | 17.7 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-15331 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-15331 | HTTPS FTP |
-Related structure data
Related structure data | 8ac2MC 8abyC 8abzC 8ac0C 8ac1C 8acpC 8ad1C M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_15331.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||
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Voxel size | X=Y=Z: 0.862 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Half map: #2
File | emd_15331_half_map_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: #1
File | emd_15331_half_map_2.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Sample components
-Entire : Escherichia coli RNA polymerase post-terminated complex - open cl...
Entire | Name: Escherichia coli RNA polymerase post-terminated complex - open clamp state |
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Components |
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-Supramolecule #1: Escherichia coli RNA polymerase post-terminated complex - open cl...
Supramolecule | Name: Escherichia coli RNA polymerase post-terminated complex - open clamp state type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#6 |
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Source (natural) | Organism: Escherichia coli K-12 (bacteria) |
Molecular weight | Theoretical: 435 KDa |
-Macromolecule #1: DNA-directed RNA polymerase subunit alpha
Macromolecule | Name: DNA-directed RNA polymerase subunit alpha / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: DNA-directed RNA polymerase |
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Source (natural) | Organism: Escherichia coli K-12 (bacteria) |
Molecular weight | Theoretical: 36.55868 KDa |
Recombinant expression | Organism: Escherichia coli BL21(DE3) (bacteria) |
Sequence | String: MQGSVTEFLK PRLVDIEQVS STHAKVTLEP LERGFGHTLG NALRRILLSS MPGCAVTEVE IDGVLHEYST KEGVQEDILE ILLNLKGLA VRVQGKDEVI LTLNKSGIGP VTAADITHDG DVEIVKPQHV ICHLTDENAS ISMRIKVQRG RGYVPASTRI H SEEDERPI ...String: MQGSVTEFLK PRLVDIEQVS STHAKVTLEP LERGFGHTLG NALRRILLSS MPGCAVTEVE IDGVLHEYST KEGVQEDILE ILLNLKGLA VRVQGKDEVI LTLNKSGIGP VTAADITHDG DVEIVKPQHV ICHLTDENAS ISMRIKVQRG RGYVPASTRI H SEEDERPI GRLLVDACYS PVERIAYNVE AARVEQRTDL DKLVIEMETN GTIDPEEAIR RAATILAEQL EAFVDLRDVR QP EVKEEKP EFDPILLRPV DDLELTVRSA NCLKAEAIHY IGDLVQRTEV ELLKTPNLGK KSLTEIKDVL ASRGLSLGMR LEN WPPASI ADE UniProtKB: DNA-directed RNA polymerase subunit alpha |
-Macromolecule #2: DNA-directed RNA polymerase subunit beta
Macromolecule | Name: DNA-directed RNA polymerase subunit beta / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed RNA polymerase |
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Source (natural) | Organism: Escherichia coli K-12 (bacteria) |
Molecular weight | Theoretical: 150.820875 KDa |
Recombinant expression | Organism: Escherichia coli BL21(DE3) (bacteria) |
Sequence | String: MVYSYTEKKR IRKDFGKRPQ VLDVPYLLSI QLDSFQKFIE QDPEGQYGLE AAFRSVFPIQ SYSGNSELQY VSYRLGEPVF DVQECQIRG VTYSAPLRVK LRLVIYEREA PEGTVKDIKE QEVYMGEIPL MTDNGTFVIN GTERVIVSQL HRSPGVFFDS D KGKTHSSG ...String: MVYSYTEKKR IRKDFGKRPQ VLDVPYLLSI QLDSFQKFIE QDPEGQYGLE AAFRSVFPIQ SYSGNSELQY VSYRLGEPVF DVQECQIRG VTYSAPLRVK LRLVIYEREA PEGTVKDIKE QEVYMGEIPL MTDNGTFVIN GTERVIVSQL HRSPGVFFDS D KGKTHSSG KVLYNARIIP YRGSWLDFEF DPKDNLFVRI DRRRKLPATI ILRALNYTTE QILDLFFEKV IFEIRDNKLQ ME LVPERLR GETASFDIEA NGKVYVEKGR RITARHIRQL EKDDVKLIEV PVEYIAGKVV AKDYIDESTG ELICAANMEL SLD LLAKLS QSGHKRIETL FTNDLDHGPY ISETLRVDPT NDRLSALVEI YRMMRPGEPP TREAAESLFE NLFFSEDRYD LSAV GRMKF NRSLLREEIE GSGILSKDDI IDVMKKLIDI RNGKGEVDDI DHLGNRRIRS VGEMAENQFR VGLVRVERAV KERLS LGDL DTLMPQDMIN AKPISAAVKE FFGSSQLSQF MDQNNPLSEI THKRRISALG PGGLTRERAG FEVRDVHPTH YGRVCP IET PEGPNIGLIN SLSVYAQTNE YGFLETPYRK VTDGVVTDEI HYLSAIEEGN YVIAQANSNL DEEGHFVEDL VTCRSKG ES SLFSRDQVDY MDVSTQQVVS VGASLIPFLE HDDANRALMG ANMQRQAVPT LRADKPLVGT GMERAVAVDS GVTAVAKR G GVVQYVDASR IVIKVNEDEM YPGEAGIDIY NLTKYTRSNQ NTCINQMPCV SLGEPVERGD VLADGPSTDL GELALGQNM RVAFMPWNGY NFEDSILVSE RVVQEDRFTT IHIQELACVS RDTKLGPEEI TADIPNVGEA ALSKLDESGI VYIGAEVTGG DILVGKVTP KGETQLTPEE KLLRAIFGEK ASDVKDSSLR VPNGVSGTVI DVQVFTRDGV EKDKRALEIE EMQLKQAKKD L SEELQILE AGLFSRIRAV LVAGGVEAEK LDKLPRDRWL ELGLTDEEKQ NQLEQLAEQY DELKHEFEKK LEAKRRKITQ GD DLAPGVL KIVKVYLAVK RRIQPGDKMA GRHGNKGVIS KINPIEDMPY DENGTPVDIV LNPLGVPSRM NIGQILETHL GMA AKGIGD KINAMLKQQQ EVAKLREFIQ RAYDLGADVR QKVDLSTFSD EEVMRLAENL RKGMPIATPV FDGAKEAEIK ELLK LGDLP TSGQIRLYDG RTGEQFERPV TVGYMYMLKL NHLVDDKMHA RSTGSYSLVT QQPLGGKAQF GGQRFGEMEV WALEA YGAA YTLQEMLTVK SDDVNGRTKM YKNIVDGNHQ MEPGMPESFN VLLKEIRSLG INIELEDE UniProtKB: DNA-directed RNA polymerase subunit beta |
-Macromolecule #3: DNA-directed RNA polymerase subunit beta'
Macromolecule | Name: DNA-directed RNA polymerase subunit beta' / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed RNA polymerase |
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Source (natural) | Organism: Escherichia coli K-12 (bacteria) |
Molecular weight | Theoretical: 155.237672 KDa |
Recombinant expression | Organism: Escherichia coli BL21(DE3) (bacteria) |
Sequence | String: MKDLLKFLKA QTKTEEFDAI KIALASPDMI RSWSFGEVKK PETINYRTFK PERDGLFCAR IFGPVKDYEC LCGKYKRLKH RGVICEKCG VEVTQTKVRR ERMGHIELAS PTAHIWFLKS LPSRIGLLLD MPLRDIERVL YFESYVVIEG GMTNLERQQI L TEEQYLDA ...String: MKDLLKFLKA QTKTEEFDAI KIALASPDMI RSWSFGEVKK PETINYRTFK PERDGLFCAR IFGPVKDYEC LCGKYKRLKH RGVICEKCG VEVTQTKVRR ERMGHIELAS PTAHIWFLKS LPSRIGLLLD MPLRDIERVL YFESYVVIEG GMTNLERQQI L TEEQYLDA LEEFGDEFDA KMGAEAIQAL LKSMDLEQEC EQLREELNET NSETKRKKLT KRIKLLEAFV QSGNKPEWMI LT VLPVLPP DLRPLVPLDG GRFATSDLND LYRRVINRNN RLKRLLDLAA PDIIVRNEKR MLQEAVDALL DNGRRGRAIT GSN KRPLKS LADMIKGKQG RFRQNLLGKR VDYSGRSVIT VGPYLRLHQC GLPKKMALEL FKPFIYGKLE LRGLATTIKA AKKM VEREE AVVWDILDEV IREHPVLLNR APTLHRLGIQ AFEPVLIEGK AIQLHPLVCA AYNADFDGDQ MAVHVPLTLE AQLEA RALM MSTNNILSPA NGEPIIVPSQ DVVLGLYYMT RDCVNAKGEG MVLTGPKEAE RLYRSGLASL HARVKVRITE YEKDAN GEL VAKTSLKDTT VGRAILWMIV PKGLPYSIVN QALGKKAISK MLNTCYRILG LKPTVIFADQ IMYTGFAYAA RSGASVG ID DMVIPEKKHE IISEAEAEVA EIQEQFQSGL VTAGERYNKV IDIWAAANDR VSKAMMDNLQ TETVINRDGQ EEKQVSFN S IYMMADSGAR GSAAQIRQLA GMRGLMAKPD GSIIETPITA NFREGLNVLQ YFISTHGARK GLADTALKTA NSGYLTRRL VDVAQDLVVT EDDCGTHEGI MMTPVIEGGD VKEPLRDRVL GRVTAEDVLK PGTADILVPR NTLLHEQWCD LLEENSVDAV KVRSVVSCD TDFGVCAHCY GRDLARGHII NKGEAIGVIA AQSIGEPGTQ LTMRTFHIGG AASRAAAESS IQVKNKGSIK L SNVKSVVN SSGKLVITSR NTELKLIDEF GRTKESYKVP YGAVLAKGDG EQVAGGETVA NWDPHTMPVI TEVSGFVRFT DM IDGQTIT RQTDELTGLS SLVVLDSAER TAGGKDLRPA LKIVDAQGND VLIPGTDMPA QYFLPGKAIV QLEDGVQISS GDT LARIPQ ESGGTKDITG GLPRVADLFE ARRPKEPAIL AEISGIVSFG KETKGKRRLV ITPVDGSDPY EEMIPKWRQL NVFE GERVE RGDVISDGPE APHDILRLRG VHAVTRYIVN EVQDVYRLQG VKINDKHIEV IVRQMLRKAT IVNAGSSDFL EGEQV EYSR VKIANRELEA NGKVGATYSR DLLGITKASL ATESFISAAS FQETTRVLTE AAVAGKRDEL RGLKENVIVG RLIPAG TGY AYHQDRMRRR AAGEAPAAPQ VTAEDASASL AELLNAGLGG SDN UniProtKB: DNA-directed RNA polymerase subunit beta' |
-Macromolecule #4: DNA-directed RNA polymerase subunit omega
Macromolecule | Name: DNA-directed RNA polymerase subunit omega / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed RNA polymerase |
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Source (natural) | Organism: Escherichia coli K-12 (bacteria) |
Molecular weight | Theoretical: 10.249547 KDa |
Recombinant expression | Organism: Escherichia coli BL21(DE3) (bacteria) |
Sequence | String: MARVTVQDAV EKIGNRFDLV LVAARRARQM QVGGKDPLVP EENDKTTVIA LREIEEGLIN NQILDVRERQ EQQEQEAAEL QAVTAIAEG RR UniProtKB: DNA-directed RNA polymerase subunit omega |
-Macromolecule #5: DNA Non-template strand
Macromolecule | Name: DNA Non-template strand / type: dna / ID: 5 / Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: Escherichia coli (E. coli) |
Molecular weight | Theoretical: 91.015141 KDa |
Sequence | String: (DC)(DA)(DG)(DT)(DC)(DA)(DC)(DG)(DA)(DC) (DG)(DT)(DT)(DG)(DT)(DA)(DA)(DA)(DA)(DC) (DG)(DA)(DC)(DG)(DG)(DC)(DC)(DA)(DG) (DT)(DG)(DA)(DA)(DT)(DT)(DC)(DG)(DA)(DG) (DC) (DT)(DC)(DG)(DG)(DT)(DA) ...String: (DC)(DA)(DG)(DT)(DC)(DA)(DC)(DG)(DA)(DC) (DG)(DT)(DT)(DG)(DT)(DA)(DA)(DA)(DA)(DC) (DG)(DA)(DC)(DG)(DG)(DC)(DC)(DA)(DG) (DT)(DG)(DA)(DA)(DT)(DT)(DC)(DG)(DA)(DG) (DC) (DT)(DC)(DG)(DG)(DT)(DA)(DC)(DC) (DA)(DA)(DA)(DA)(DA)(DT)(DA)(DA)(DA)(DT) (DT)(DT) (DC)(DC)(DT)(DT)(DA)(DA)(DA) (DG)(DT)(DT)(DC)(DA)(DC)(DT)(DA)(DA)(DC) (DT)(DT)(DA) (DT)(DG)(DA)(DT)(DG)(DT) (DA)(DG)(DT)(DG)(DA)(DG)(DC)(DT)(DT)(DT) (DT)(DT)(DA)(DT) (DA)(DC)(DC)(DC)(DA) (DT)(DA)(DA)(DA)(DA)(DT)(DG)(DT)(DA)(DC) (DT)(DA)(DT)(DT)(DG) (DG)(DT)(DA)(DC) (DT)(DT)(DT)(DA)(DC)(DA)(DT)(DT)(DA)(DA) (DT)(DG)(DA)(DA)(DC)(DT) (DT)(DT)(DA) (DA)(DG)(DT)(DA)(DC)(DA)(DT)(DC)(DA)(DT) (DA)(DA)(DG)(DC)(DC)(DC)(DA) (DT)(DC) (DG)(DA)(DG)(DA)(DG)(DG)(DG)(DA)(DC)(DA) (DC)(DG)(DG)(DG)(DG)(DA)(DA)(DA) (DC) (DA)(DC)(DC)(DA)(DC)(DC)(DA)(DT)(DG)(DC) (DT)(DT)(DA)(DT)(DA)(DA)(DT)(DA)(DA) (DT)(DT)(DC)(DT)(DG)(DC)(DC)(DG)(DG)(DA) (DG)(DC)(DG)(DA)(DC)(DC)(DG)(DC)(DA)(DC) (DT)(DG)(DT)(DG)(DG)(DT)(DT)(DT)(DA) (DC)(DC)(DA)(DG)(DA)(DT)(DG)(DG)(DC)(DG) (DT) (DG)(DT)(DG)(DT)(DC)(DC)(DC)(DA) (DA)(DT)(DC)(DT)(DT)(DT)(DC)(DA)(DC)(DA) (DA)(DC) (DA)(DT)(DT)(DA)(DG)(DC)(DG) (DA)(DG)(DA)(DA)(DG)(DG)(DC)(DT)(DT)(DT) (DT)(DT)(DT) (DG)(DT)(DT)(DT)(DT)(DT) (DA)(DG)(DT)(DC)(DA)(DC)(DG)(DG)(DC) |
-Macromolecule #6: DNA Template strand
Macromolecule | Name: DNA Template strand / type: dna / ID: 6 / Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: Escherichia coli (E. coli) |
Molecular weight | Theoretical: 91.180203 KDa |
Sequence | String: (DG)(DC)(DC)(DG)(DT)(DG)(DA)(DC)(DT)(DA) (DA)(DA)(IGU)(IGU)(DC)(DA)(DA)(DA)(DA) (DA)(DA)(DG)(DC)(DC)(DT)(DT)(DC)(DT)(DC) (DG)(DC)(DT)(DA)(DA)(DT)(DG)(DT)(DT)(DG) (DT)(DG)(DA)(DA)(DA)(DG)(DA) ...String: (DG)(DC)(DC)(DG)(DT)(DG)(DA)(DC)(DT)(DA) (DA)(DA)(IGU)(IGU)(DC)(DA)(DA)(DA)(DA) (DA)(DA)(DG)(DC)(DC)(DT)(DT)(DC)(DT)(DC) (DG)(DC)(DT)(DA)(DA)(DT)(DG)(DT)(DT)(DG) (DT)(DG)(DA)(DA)(DA)(DG)(DA)(DT)(DT) (DG)(DG)(DG)(DA)(DC)(DA)(DC)(DA)(DC)(DG) (DC) (DC)(DA)(DT)(DC)(DT)(DG)(DG)(DT) (DA)(DA)(DA)(DC)(DC)(DA)(DC)(DA)(DG)(DT) (DG)(DC) (DG)(DG)(DT)(DC)(DG)(DC)(DT) (DC)(DC)(DG)(DG)(DC)(DA)(DG)(DA)(DA)(DT) (DT)(DA)(DT) (DT)(DA)(DT)(DA)(DA)(DG) (DC)(DA)(DT)(DG)(DG)(DT)(DG)(DG)(DT)(DG) (DT)(DT)(DT)(DC) (DC)(DC)(DC)(DG)(DT) (DG)(DT)(DC)(DC)(DC)(DT)(DC)(DT)(DC)(DG) (DA)(DT)(DG)(DG)(DG) (DC)(DT)(DT)(DA) (DT)(DG)(DA)(DT)(DG)(DT)(DA)(DC)(DT)(DT) (DA)(DA)(DA)(DG)(DT)(DT) (DC)(DA)(DT) (DT)(DA)(DA)(DT)(DG)(DT)(DA)(DA)(DA)(DG) (DT)(DA)(DC)(DC)(DA)(DA)(DT) (DA)(DG) (DT)(DA)(DC)(DA)(DT)(DT)(DT)(DT)(DA)(DT) (DG)(DG)(DG)(DT)(DA)(DT)(DA)(DA) (DA) (DA)(DA)(DG)(DC)(DT)(DC)(DA)(DC)(DT)(DA) (DC)(DA)(DT)(DC)(DA)(DT)(DA)(DA)(DG) (DT)(DT)(DA)(DG)(DT)(DG)(DA)(DA)(DC)(DT) (DT)(DT)(DA)(DA)(DG)(DG)(DA)(DA)(DA)(DT) (DT)(DT)(DA)(DT)(DT)(DT)(DT)(DT)(DG) (DG)(DT)(DA)(DC)(DC)(DG)(DA)(DG)(DC)(DT) (DC) (DG)(DA)(DA)(DT)(DT)(DC)(DA)(DC) (DT)(DG)(DG)(DC)(DC)(DG)(DT)(DC)(DG)(DT) (DT)(DT) (DT)(DA)(DC)(DA)(DA)(DC)(DG) (DT)(DC)(DG)(DT)(DG)(DA)(DC)(DT)(DG) |
-Macromolecule #7: MAGNESIUM ION
Macromolecule | Name: MAGNESIUM ION / type: ligand / ID: 7 / Number of copies: 1 / Formula: MG |
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Molecular weight | Theoretical: 24.305 Da |
-Macromolecule #8: ZINC ION
Macromolecule | Name: ZINC ION / type: ligand / ID: 8 / Number of copies: 2 / Formula: ZN |
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Molecular weight | Theoretical: 65.409 Da |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 12 mg/mL |
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Buffer | pH: 8 Details: 20 mM Tris-glutamate pH 8.0, 50 mM K-glutamate, 10 mM Mg-glutamate, 0.001 mM ZnCl2, 2mM DTT |
Grid | Model: Quantifoil R1.2/1.3 / Material: GOLD / Support film - Material: GOLD / Support film - topology: HOLEY |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 283 K / Instrument: FEI VITROBOT MARK IV Details: Quantifoil UltrAuFoil R1.2/1.3 300 mesh holey gold grids were plasma cleaned on a Model 1070 (Fischione Instruments) for 30 sec at 70% power and with an 80% Argon and 20% Oxygen mixture ...Details: Quantifoil UltrAuFoil R1.2/1.3 300 mesh holey gold grids were plasma cleaned on a Model 1070 (Fischione Instruments) for 30 sec at 70% power and with an 80% Argon and 20% Oxygen mixture prior to the application of 0.004 ml of sample. Grids were plunge frozen into liquid ethane using a Vitrobot mark IV (FEI) with 95% chamber humidity at 283K.. |
Details | The co-transcriptionally halted complexes for cryo-EM analysis were prepared in vitro by mixing E. coli RNAP holoenzyme with templates containing the DNA base analogue isoG. The RNAP ECs halted at the U-rich pause (G122). The complexes were prepared in 0.03 ml reactions and contained: 0.02 mM of E. coli RNAP, 0.080 mM of E. coli Sigma70, 0.02 mM of template DNA, 20 mM Tris-glutamate pH 8.0, 50 mM K-glutamate, 10 mM Mg-glutamate, 0.001 mM ZnCl2, 2mM DTT and 6 mM of each ATP, GTP, CTP and UTP. Initially, all the components except NTPs were added and incubated for 10 min at 310K. To allow transcription elongation, NTPs were added and the sample was incubated for 20 to 40 min at 310K. The final products were purified on a Superose 6 Increase 3.2/300 gel filtration column equilibrated in Glutamate buffer. Samples were concentrated to 10-12 mg/mL using an Amicon Ultra 0.5mL centrifugal filter unit (30 KDa MWCO). Before grid freezing, 8 mM of CHAPSO was added to freshly prepared sample to overcome preferred particle orientation. |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Average exposure time: 2.2 sec. / Average electron dose: 52.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.8 µm |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |