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- PDB-7uwe: CryoEM Structure of E. coli Transcription-Coupled Ribonucleotide ... -

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Basic information

Entry
Database: PDB / ID: 7uwe
TitleCryoEM Structure of E. coli Transcription-Coupled Ribonucleotide Excision Repair (TC-RER) complex
Components
  • (DNA (29-MER)) x 2
  • (DNA-directed RNA polymerase subunit ...Polymerase) x 4
  • RNA (18-MER)
  • Ribonuclease HII
KeywordsTRANSFERASE/HYDROLASE/DNA/RNA / Transcription-coupled RER / TRANSCRIPTION / TRANSFERASE-HYDROLASE-DNA-RNA complex
Function / homology
Function and homology information


RNA catabolic process / ribonuclease H / 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 ...RNA catabolic process / ribonuclease H / 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 / DNA-directed RNA polymerase 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 / RNA-DNA hybrid ribonuclease activity / manganese ion binding / response to heat / intracellular iron ion homeostasis / protein dimerization activity / response to antibiotic / DNA-templated transcription / magnesium ion binding / DNA binding / RNA binding / zinc ion binding / membrane / cytosol / cytoplasm
Similarity search - Function
Ribonuclease HII / Ribonuclease (RNase) H type-2 domain profile. / Ribonuclease HII/HIII / Ribonuclease HII/HIII domain / Ribonuclease HII / 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 ...Ribonuclease HII / Ribonuclease (RNase) H type-2 domain profile. / Ribonuclease HII/HIII / Ribonuclease HII/HIII domain / Ribonuclease HII / 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 / Ribonuclease H superfamily / Ribonuclease H-like superfamily
Similarity search - Domain/homology
DNA / DNA (> 10) / RNA / RNA (> 10) / DNA-directed RNA polymerase subunit alpha / DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit beta / Ribonuclease HII
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsHao, Z.T. / Grower, M. / Bharati, B. / Proshkin, S. / Epshtein, V. / Svetlov, V. / Nudler, E. / Shamovsky, I.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM126891 United States
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: Cell / Year: 2023
Title: RNA polymerase drives ribonucleotide excision DNA repair in E. coli.
Authors: Zhitai Hao / Manjunath Gowder / Sergey Proshkin / Binod K Bharati / Vitaly Epshtein / Vladimir Svetlov / Ilya Shamovsky / Evgeny Nudler /
Abstract: Ribonuclease HII (RNaseHII) is the principal enzyme that removes misincorporated ribonucleoside monophosphates (rNMPs) from genomic DNA. Here, we present structural, biochemical, and genetic evidence ...Ribonuclease HII (RNaseHII) is the principal enzyme that removes misincorporated ribonucleoside monophosphates (rNMPs) from genomic DNA. Here, we present structural, biochemical, and genetic evidence demonstrating that ribonucleotide excision repair (RER) is directly coupled to transcription. Affinity pull-downs and mass-spectrometry-assisted mapping of in cellulo inter-protein cross-linking reveal the majority of RNaseHII molecules interacting with RNA polymerase (RNAP) in E. coli. Cryoelectron microscopy structures of RNaseHII bound to RNAP during elongation, with and without the target rNMP substrate, show specific protein-protein interactions that define the transcription-coupled RER (TC-RER) complex in engaged and unengaged states. The weakening of RNAP-RNaseHII interactions compromises RER in vivo. The structure-functional data support a model where RNaseHII scans DNA in one dimension in search for rNMPs while "riding" the RNAP. We further demonstrate that TC-RER accounts for a significant fraction of repair events, thereby establishing RNAP as a surveillance "vehicle" for detecting the most frequently occurring replication errors.
History
DepositionMay 3, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 31, 2023Provider: repository / Type: Initial release
Revision 1.1Jun 7, 2023Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.page_first

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: DNA (29-MER)
B: DNA (29-MER)
R: RNA (18-MER)
G: DNA-directed RNA polymerase subunit alpha
H: DNA-directed RNA polymerase subunit alpha
I: DNA-directed RNA polymerase subunit beta
J: DNA-directed RNA polymerase subunit beta'
K: DNA-directed RNA polymerase subunit omega
C: Ribonuclease HII
hetero molecules


Theoretical massNumber of molelcules
Total (without water)434,78212
Polymers434,6269
Non-polymers1553
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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DNA chain , 2 types, 2 molecules AB

#1: DNA chain DNA (29-MER)


Mass: 8840.689 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)
#2: DNA chain DNA (29-MER)


Mass: 8813.646 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)

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DNA-directed RNA polymerase subunit ... , 4 types, 5 molecules GHIJK

#4: Protein DNA-directed RNA polymerase subunit alpha / Polymerase / RNAP subunit alpha / RNA polymerase subunit alpha / Transcriptase subunit alpha


Mass: 36558.680 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: rpoA, pez, phs, sez, b3295, JW3257
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: P0A7Z4, DNA-directed RNA polymerase
#5: Protein DNA-directed RNA polymerase subunit beta / Polymerase / RNAP subunit beta / RNA polymerase subunit beta / Transcriptase subunit beta


Mass: 150820.875 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: rpoB, Z5560, ECs4910
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: P0A8V4, DNA-directed RNA polymerase
#6: Protein DNA-directed RNA polymerase subunit beta' / Polymerase / RNAP subunit beta' / RNA polymerase subunit beta' / Transcriptase subunit beta'


Mass: 155366.781 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: rpoC, tabB, b3988, JW3951
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: P0A8T7, DNA-directed RNA polymerase
#7: Protein DNA-directed RNA polymerase subunit omega / Polymerase / RNAP omega subunit / RNA polymerase omega subunit / Transcriptase subunit omega


Mass: 10249.547 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: rpoZ, Z5075, ECs4524
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: P0A802, DNA-directed RNA polymerase

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RNA chain / Protein , 2 types, 2 molecules RC

#3: RNA chain RNA (18-MER)


Mass: 5859.580 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)
#8: Protein Ribonuclease HII / RNase HII


Mass: 21558.021 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli)
Gene: rnhB, ABE90_006955, ACN68_08240, ACN81_28465, ACU57_18505, AT845_002192, AWP93_20140, BANRA_00657, BANRA_01142, BANRA_03354, BG944_001858, BHS81_00720, BJI68_15710, BJJ90_21290, BK292_13740, ...Gene: rnhB, ABE90_006955, ACN68_08240, ACN81_28465, ACU57_18505, AT845_002192, AWP93_20140, BANRA_00657, BANRA_01142, BANRA_03354, BG944_001858, BHS81_00720, BJI68_15710, BJJ90_21290, BK292_13740, BMT50_03940, BO068_002873, BOH76_17645, BON63_15375, BON64_03455, BON67_09920, BON69_16465, BON70_27225, BON71_14810, BON73_05390, BON74_02650, BON77_22000, BON80_16390, BON89_11100, BON93_03865, BON95_12960, BTQ06_06560, BvCmsF30A_01255, BvCmsKSNP073_05569, BvCmsNSP072_03655, BVL39_02115, C5N07_08650, C5Y87_12645, C9114_06955, CA593_02795, CG831_000806, CIG67_16650, CO706_23125, CV83915_01391, CWS33_09690, D0X26_09345, D3Y67_16475, D9H94_14105, DAH17_03135, DAH18_22235, DAH20_13890, DAH22_03575, DAH27_18705, DAH28_17945, DAH29_14760, DAH30_10540, DAH31_15680, DAH32_15180, DAH34_04115, DAH35_21445, DAH36_21020, DAH37_16260, DAH41_15695, DEN87_21140, DEN89_20355, DEN90_18695, DEN91_15555, DEN92_14745, DEN93_18240, DEN94_16175, DEN95_12235, DEN96_06260, DEN97_04355, DEN98_04360, DEN99_03185, DEO00_05260, DEO01_06085, DEO02_09465, DEO03_16395, DEO04_22160, DEO05_22030, DEO06_22675, DEO07_22500, DEO08_20735, DEO09_21885, DEO10_22145, DEO11_22040, DEO12_12305, DEO13_08570, DEO14_11170, DEO15_08350, DEO18_03480, DEO19_04140, DEO20_19790, DIV22_06340, DN627_10175, DRW19_04855, DXT69_01400, DXT70_17000, E0I42_19050, E2113_05780, E2117_05455, E2119_15650, E2122_04525, E2131_12130, E2135_11705, E4K51_05720, E5M02_17430, E5P23_04780, E5P24_16135, E5P25_12930, E5P26_03445, E5P27_14095, E5P28_14045, E5P29_07870, E5P30_07200, E5P35_01175, E5P36_00585, E5P51_07020, E5S34_18450, E5S35_00100, E5S36_11195, E5S37_18845, E5S39_17415, E5S42_18175, E5S43_01730, E5S44_09555, E5S45_04795, E5S47_12390, E5S48_12785, E5S51_08350, E5S54_18395, E5S56_11640, EAI46_03575, EAX79_05490, EC1094V2_3668, EC3234A_2c01650, EC95NR1_04369, EHD79_10665, EHH55_16915, EI021_19915, EIZ93_07855, EKI52_10365, EL79_3691, EL80_3638, ELT41_02045, ELV08_10210, ELX85_10375, EYV17_16515, EYV18_21815, F0L67_09075, F2N31_00095, F9V24_09570, FDM60_08750, FOI11_012770, FOI11_22910, FQ007_15880, FTV90_09235, FV293_02825, FWK02_16565, G9448_10045, GIB53_10190, GKF86_01260, GKF89_14710, GP650_06585, GP662_15385, GP954_09955, GP979_14795, GQA06_14505, GQE64_01325, GQF59_21085, GRW05_07765, GRW57_08780, GRW81_09880, GUC01_17950, HHH44_003649, HKA49_001638, HV209_01610, HVW19_15045, HX136_20530, I6H02_18720, IH772_14505, J0541_003901, J4S20_003213, J5U05_002998, JE86ST02C_01770, JE86ST05C_01800, JFD_01269, JNP96_05105, NCTC10418_06054, NCTC11181_01347, NCTC11341_03667, NCTC13216_02729, NCTC8008_03658, NCTC8179_01396, NCTC8500_04527, NCTC8960_01547, NCTC9036_04009, NCTC9037_04174, NCTC9045_04695, NCTC9073_03377, NCTC9111_04250, NCTC9117_05034, NCTC9706_01322, ND22_002535, PGD_03824, RG28_03840, SAMEA3472067_01428, SAMEA3751407_04799, WP2S18E08_37600
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: W8T723, ribonuclease H

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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: Elongation Complex(EC)-RNaseH2 complex / Type: COMPLEX / Entity ID: #1-#8 / Source: MULTIPLE SOURCES
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: OTHER

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 62 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

CTF correctionType: NONE
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 142145 / Symmetry type: POINT

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