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- EMDB-30119: CryoEM structure of Thermus thermophilus RNA polymerase elongatio... -

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Basic information

Entry
Database: EMDB / ID: EMD-30119
TitleCryoEM structure of Thermus thermophilus RNA polymerase elongation complex
Map data
Sample
  • Complex: Thermus thermophilus RNA polymerase elongation complex
    • Protein or peptide: DNA-directed RNA polymerase subunit alphaPolymerase
    • Protein or peptide: DNA-directed RNA polymerase subunit betaPolymerase
    • Protein or peptide: DNA-directed RNA polymerase subunit beta'Polymerase
    • Protein or peptide: DNA-directed RNA polymerase subunit omegaPolymerase
    • RNA: RNA
    • DNA: nontemplate strand DNA
    • DNA: template strand DNA
  • Ligand: ZINC ION
  • Ligand: MAGNESIUM ION
KeywordsTranscription / RNA polymerase
Function / homology
Function and homology information


DNA-directed RNA polymerase complex / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / protein dimerization activity / DNA-templated transcription / magnesium ion binding / DNA binding / zinc ion binding / cytoplasm
Similarity search - Function
: / DNA-directed RNA polymerase subunit beta', hybrid domain / 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 subunit beta', hybrid domain / 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-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 speciesThermus thermophilus (bacteria) / Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsShi J / Wen A
Funding support China, 1 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)31970040 China
CitationJournal: Nucleic Acids Res / Year: 2020
Title: Structural basis of Mfd-dependent transcription termination.
Authors: Jing Shi / Aijia Wen / Minxing Zhao / Sha Jin / Linlin You / Yue Shi / Shuling Dong / Xiaoting Hua / Yu Zhang / Yu Feng /
Abstract: Mfd-dependent transcription termination plays an important role in transcription-coupled DNA repair, transcription-replication conflict resolution, and antimicrobial resistance development. Despite ...Mfd-dependent transcription termination plays an important role in transcription-coupled DNA repair, transcription-replication conflict resolution, and antimicrobial resistance development. Despite extensive studies, the molecular mechanism of Mfd-dependent transcription termination in bacteria remains unclear, with several long-standing puzzles. How Mfd is activated by stalled RNA polymerase (RNAP) and how activated Mfd translocates along the DNA are unknown. Here, we report the single-particle cryo-electron microscopy structures of T. thermophilus Mfd-RNAP complex with and without ATPγS. The structures reveal that Mfd undergoes profound conformational changes upon activation, contacts the RNAP β1 domain and its clamp, and pries open the RNAP clamp. These structures provide a foundation for future studies aimed at dissecting the precise mechanism of Mfd-dependent transcription termination and pave the way for rational drug design targeting Mfd for the purpose of tackling the antimicrobial resistance crisis.
History
DepositionMar 14, 2020-
Header (metadata) releaseOct 14, 2020-
Map releaseOct 14, 2020-
UpdateMar 27, 2024-
Current statusMar 27, 2024Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6m6c
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_30119.png

Downloads & links

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Map

FileDownload / File: emd_30119.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.307 Å
Density
Contour LevelBy AUTHOR: 0.0278 / Movie #1: 0.03
Minimum - Maximum-0.36549258 - 0.5536737
Average (Standard dev.)0.00017458394 (±0.016011866)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 261.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.3071.3071.307
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z261.400261.400261.400
α/β/γ90.00090.00090.000
start NX/NY/NZ-19-94-60
NX/NY/NZ114128118
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS200200200
D min/max/mean-0.3650.5540.000

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Supplemental data

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Sample components

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Entire : Thermus thermophilus RNA polymerase elongation complex

EntireName: Thermus thermophilus RNA polymerase elongation complex
Components
  • Complex: Thermus thermophilus RNA polymerase elongation complex
    • Protein or peptide: DNA-directed RNA polymerase subunit alphaPolymerase
    • Protein or peptide: DNA-directed RNA polymerase subunit betaPolymerase
    • Protein or peptide: DNA-directed RNA polymerase subunit beta'Polymerase
    • Protein or peptide: DNA-directed RNA polymerase subunit omegaPolymerase
    • RNA: RNA
    • DNA: nontemplate strand DNA
    • DNA: template strand DNA
  • Ligand: ZINC ION
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Thermus thermophilus RNA polymerase elongation complex

SupramoleculeName: Thermus thermophilus RNA polymerase elongation complex
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#7
Source (natural)Organism: Thermus thermophilus (bacteria)

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Macromolecule #1: DNA-directed RNA polymerase subunit alpha

MacromoleculeName: DNA-directed RNA polymerase subunit alpha / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: DNA-directed RNA polymerase
Source (natural)Organism: Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579) (bacteria)
Strain: HB8 / ATCC 27634 / DSM 579
Molecular weightTheoretical: 35.056164 KDa
SequenceString: MLDSKLKAPV FTVRTQGREY GEFVLEPLER GFGVTLGNPL RRILLSSIPG TAVTSVYIED VLHEFSTIPG VKEDVVEIIL NLKELVVRF LNPSLQTVTL LLKAEGPKEV KARDFLPVAD VEIMNPDLHI ATLEEGGRLN MEVRVDRGVG YVPAEKHGIK D RINAIPVD ...String:
MLDSKLKAPV FTVRTQGREY GEFVLEPLER GFGVTLGNPL RRILLSSIPG TAVTSVYIED VLHEFSTIPG VKEDVVEIIL NLKELVVRF LNPSLQTVTL LLKAEGPKEV KARDFLPVAD VEIMNPDLHI ATLEEGGRLN MEVRVDRGVG YVPAEKHGIK D RINAIPVD AVFSPVRRVA FQVEDTRLGQ RTDLDKLTLR IWTDGSVTPL EALNQAVEIL REHLTYFSNP QAAAVAAPEE AK EPEAPPE QEEELDLPLE ELGLSTRVLH SLKEEGIESV RALLALNLKD LKNIPGIGER SLEEIKEALE KKGFTLKE

UniProtKB: DNA-directed RNA polymerase subunit alpha

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Macromolecule #2: DNA-directed RNA polymerase subunit beta

MacromoleculeName: DNA-directed RNA polymerase subunit beta / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed RNA polymerase
Source (natural)Organism: Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579) (bacteria)
Strain: HB8 / ATCC 27634 / DSM 579
Molecular weightTheoretical: 125.436539 KDa
SequenceString: MEIKRFGRIR EVIPLPPLTE IQVESYRRAL QADVPPEKRE NVGIQAAFRE TFPIEEEDKG KGGLVLDFLE YRLGEPPFPQ DECREKDLT YQAPLYARLQ LIHKDTGLIK EDEVFLGHIP LMTEDGSFII NGADRVIVSQ IHRSPGVYFT PDPARPGRYI A SIIPLPKR ...String:
MEIKRFGRIR EVIPLPPLTE IQVESYRRAL QADVPPEKRE NVGIQAAFRE TFPIEEEDKG KGGLVLDFLE YRLGEPPFPQ DECREKDLT YQAPLYARLQ LIHKDTGLIK EDEVFLGHIP LMTEDGSFII NGADRVIVSQ IHRSPGVYFT PDPARPGRYI A SIIPLPKR GPWIDLEVEP NGVVSMKVNK RKFPLVLLLR VLGYDQETLA RELGAYGELV QGLMDESVFA MRPEEALIRL FT LLRPGDP PKRDKAVAYV YGLIADPRRY DLGEAGRYKA EEKLGIRLSG RTLARFEDGE FKDEVFLPTL RYLFALTAGV PGH EVDDID HLGNRRIRTV GELMTDQFRV GLARLARGVR ERMLMGSEDS LTPAKLVNSR PLEAAIREFF SRSQLSQFKD ETNP LSSLR HKRRISALGP GGLTRERAGF DVRDVHRTHY GRICPVETPE GANIGLITSL AAYARVDELG FIRTPYRRVV GGVVT DEVV YMTATEEDRY TIAQANTPLE GNRIAAERVV ARRKGEPVIV SPEEVEFMDV SPKQVFSVNT NLIPFLEHDD ANRALM GSN MQTQAVPLIR AQAPVVMTGL EERVVRDSLA ALYAEEDGEV AKVDGNRIVV RYEDGRLVEY PLRRFYRSNQ GTALDQR PR VVVGQRVRKG DLLADGPASE NGFLALGQNV LVAIMPFDGY NFEDAIVISE ELLKRDFYTS IHIERYEIEA RDTKLGPE R ITRDIPHLSE AALRDLDEEG VVRIGAEVKP GDILVGRTSF KGESEPTPEE RLLRSIFGEK ARDVKDTSLR VPPGEGGIV VRTVRLRRGD PGVELKPGVR EVVRVYVAQK RKLQVGDKLA NRHGNKGVVA KILPVEDMPH LPDGTPVDVI LNPLGVPSRM NLGQILETH LGLAGYFLGQ RYISPIFDGA KEPEIKELLA QAFEVYFGKR KGEGFGVDKR EVEVLRRAEK LGLVTPGKTP E EQLKELFL QGKVVLYDGR TGEPIEGPIV VGQMFIMKLY HMVEDKMHAR STGPYSLITQ QPLGGKAQFG GQRFGEMEVW AL EAYGAAH TLQEMLTLKS DDIEGRNAAY EAIIKGEDVP EPSVPESFRV LVKELQALAL DVQTLDEKDN PVDIFEGLAS KR

UniProtKB: DNA-directed RNA polymerase subunit beta

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Macromolecule #3: DNA-directed RNA polymerase subunit beta'

MacromoleculeName: DNA-directed RNA polymerase subunit beta' / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed RNA polymerase
Source (natural)Organism: Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579) (bacteria)
Strain: HB8 / ATCC 27634 / DSM 579
Molecular weightTheoretical: 170.997391 KDa
SequenceString: MKKEVRKVRI ALASPEKIRS WSYGEVEKPE TINYRTLKPE RDGLFDERIF GPIKDYECAC GKYKRQRFEG KVCERCGVEV TKSIVRRYR MGHIELATPA AHIWFVKDVP SKIGTLLDLS ATELEQVLYF SKYIVLDPKG AILNGVPVEK RQLLTDEEYR E LRYGKQET ...String:
MKKEVRKVRI ALASPEKIRS WSYGEVEKPE TINYRTLKPE RDGLFDERIF GPIKDYECAC GKYKRQRFEG KVCERCGVEV TKSIVRRYR MGHIELATPA AHIWFVKDVP SKIGTLLDLS ATELEQVLYF SKYIVLDPKG AILNGVPVEK RQLLTDEEYR E LRYGKQET YPLPPGVDAL VKDGEEVVKG QELAPGVVSR LDGVALYRFP RRVRVEYVKK ERAGLRLPLA AWVEKEAYKP GE ILAELPE PYLFRAEEEG VVELKELEEG AFLVLRREDE PVATYFLPVG MTPLVVHGEI VEKGQPLAEA KGLLRMPRQV RAA QVEAEE EGETVYLTLF LEWTEPKDYR VQPHMNVVVP EGARVEAGDK IVAAIDPEEE VIAEAEGVVH LHEPASILVV KARV YPFED DVEVSTGDRV APGDVLADGG KVKSDVYGRV EVDLVRNVVR VVESYDIDAR MGAEAIQQLL KELDLEALEK ELLEE MKHP SRARRAKARK RLEVVRAFLD SGNRPEWMIL EAVPVLPPDL RPMVQVDGGR FATSDLNDLY RRLINRNNRL KKLLAQ GAP EIIIRNEKRM LQEAVDALLD NGRRGAPVTN PGSDRPLRSL TDILSGKQGR FRQNLLGKRV DYSGRSVIVV GPQLKLH QC GLPKRMALEL FKPFLLKKME EKGIAPNVKA ARRMLERQRD IKDEVWDALE EVIHGKVVLL NRAPTLHRLG IQAFQPVL V EGQSIQLHPL VCEAFNADFD GDQMAVHVPL SSFAQAEARI QMLSAHNLLS PASGEPLAKP SRDIILGLYY ITQVRKEKK GAGLEFATPE EALAAHERGE VALNAPIKVA GRETSVGRLK YVFANPDEAL LAVAHGIVDL QDVVTVRYMG KRLETSPGRI LFARIVAEA VEDEKVAWEL IQLDVPQEKN SLKDLVYQAF LRLGMEKTAR LLDALKYYGF TFSTTSGITI GIDDAVIPEE K KQYLEEAD RKLLQIEQAY EMGFLTDRER YDQILQLWTE TTEKVTQAVF KNFEENYPFN PLYVMAQSGA RGNPQQIRQL CG LRGLMQK PSGETFEVPV RSSFREGLTV LEYFISSHGA RKGGADTALR TADSGYLTRK LVDVTHEIVV READCGTTNY ISV PLFQPD EVTRSLRLRK RADIEAGLYG RVLAREVEVL GVRLEEGRYL SMDDVHLLIK AAEAGEIQEV PVRSPLTCQT RYGV CQKCY GYDLSMARPV SIGEAVGIVA AQSIGEPGTQ LTMRTFHTGG VAGAADITQG LPRVIELFEA RRPKAKAVIS EIDGV VRIE ETEEKLSVFV ESEGFSKEYK LPKEARLLVK DGDYVEAGQP LTRGAIDPHQ LLEAKGPEAV ERYLVEEIQK VYRAQG VKL HDKHIEIVVR QMMKYVEVTD PGDSRLLEGQ VLEKWDVEAL NERLIAEGKT PVAWKPLLMG VTKSALSTKS WLSAASF QN TTHVLTEAAI AGKKDELIGL KENVILGRLI PAGTGSDFVR FTQVVDQKTL KAIEEARKEA VEAKERPAAR RGVKREQP G KQA

UniProtKB: DNA-directed RNA polymerase subunit beta'

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Macromolecule #4: DNA-directed RNA polymerase subunit omega

MacromoleculeName: DNA-directed RNA polymerase subunit omega / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed RNA polymerase
Source (natural)Organism: Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579) (bacteria)
Strain: HB8 / ATCC 27634 / DSM 579
Molecular weightTheoretical: 11.533316 KDa
SequenceString:
MAEPGIDKLF GMVDSKYRLT VVVAKRAQQL LRHGFKNTVL EPEERPKMQT LEGLFDDPNA VTWAMKELLT GRLVFGENLV PEDRLQKEM ERLYPVEREE

UniProtKB: DNA-directed RNA polymerase subunit omega

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Macromolecule #5: RNA

MacromoleculeName: RNA / type: rna / ID: 5 / Number of copies: 1
Source (natural)Organism: Thermus thermophilus (bacteria)
Molecular weightTheoretical: 6.509968 KDa
SequenceString:
GCAUUCAAAG CGGAGAGGUA

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Macromolecule #6: nontemplate strand DNA

MacromoleculeName: nontemplate strand DNA / type: dna / ID: 6 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Thermus thermophilus (bacteria)
Molecular weightTheoretical: 19.366436 KDa
SequenceString: (DC)(DG)(DA)(DA)(DA)(DA)(DG)(DA)(DA)(DG) (DC)(DT)(DT)(DT)(DG)(DC)(DT)(DT)(DA)(DA) (DT)(DA)(DA)(DT)(DC)(DC)(DA)(DT)(DA) (DT)(DG)(DG)(DT)(DT)(DG)(DG)(DG)(DC)(DT) (DA) (DC)(DC)(DT)(DC)(DT)(DC) ...String:
(DC)(DG)(DA)(DA)(DA)(DA)(DG)(DA)(DA)(DG) (DC)(DT)(DT)(DT)(DG)(DC)(DT)(DT)(DA)(DA) (DT)(DA)(DA)(DT)(DC)(DC)(DA)(DT)(DA) (DT)(DG)(DG)(DT)(DT)(DG)(DG)(DG)(DC)(DT) (DA) (DC)(DC)(DT)(DC)(DT)(DC)(DC)(DA) (DT)(DG)(DA)(DC)(DG)(DG)(DC)(DG)(DA)(DA) (DT)(DA) (DC)(DC)(DC)

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Macromolecule #7: template strand DNA

MacromoleculeName: template strand DNA / type: dna / ID: 7 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Thermus thermophilus (bacteria)
Molecular weightTheoretical: 19.290348 KDa
SequenceString: (DG)(DG)(DG)(DT)(DA)(DT)(DT)(DC)(DG)(DC) (DC)(DG)(DT)(DG)(DT)(DA)(DC)(DC)(DT)(DC) (DT)(DC)(DC)(DT)(DA)(DG)(DC)(DC)(DC) (DA)(DA)(DC)(DC)(DA)(DT)(DA)(DT)(DG)(DG) (DA) (DT)(DT)(DA)(DT)(DT)(DA) ...String:
(DG)(DG)(DG)(DT)(DA)(DT)(DT)(DC)(DG)(DC) (DC)(DG)(DT)(DG)(DT)(DA)(DC)(DC)(DT)(DC) (DT)(DC)(DC)(DT)(DA)(DG)(DC)(DC)(DC) (DA)(DA)(DC)(DC)(DA)(DT)(DA)(DT)(DG)(DG) (DA) (DT)(DT)(DA)(DT)(DT)(DA)(DA)(DG) (DC)(DA)(DA)(DA)(DG)(DC)(DT)(DT)(DC)(DT) (DT)(DT) (DT)(DC)(DG)

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Macromolecule #8: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 8 / Number of copies: 2 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #9: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 9 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.9
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 59.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 558003

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