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- PDB-7p5x: Mycobacterial RNAP with transcriptional activator PafBC -

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Basic information

Entry
Database: PDB / ID: 7p5x
TitleMycobacterial RNAP with transcriptional activator PafBC
Components
  • (DNA-directed RNA polymerase subunit ...Polymerase) x 4
  • PafC
  • RNA polymerase sigma factor SigA
  • RNA polymerase-binding protein RbpA
  • Transcriptional regulator-like protein
  • recA-op non-template strand
  • recA-op template strand
KeywordsTRANSCRIPTION / activator / sigma adaption / RNA polymerase / RNAP / PafB / PafC / PafBC / Mycobacterium / smegmatis
Function / homology
Function and homology information


bacterial-type RNA polymerase core enzyme binding / sigma factor activity / DNA-directed RNA polymerase complex / DNA-templated transcription initiation / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / protein dimerization activity / response to antibiotic / DNA-templated transcription ...bacterial-type RNA polymerase core enzyme binding / sigma factor activity / DNA-directed RNA polymerase complex / DNA-templated transcription initiation / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / protein dimerization activity / response to antibiotic / DNA-templated transcription / positive regulation of DNA-templated transcription / magnesium ion binding / DNA binding / zinc ion binding / cytoplasm
Similarity search - Function
PafC, HTH domain / PafC helix-turn-helix domain / Protein PafC / WYL domain / WYL domain / RNA polymerase-binding protein RbpA / RbpA superfamily / RNA polymerase-binding protein / Sigma-70 factors family signature 1. / RNA polymerase sigma factor RpoD, C-terminal ...PafC, HTH domain / PafC helix-turn-helix domain / Protein PafC / WYL domain / WYL domain / RNA polymerase-binding protein RbpA / RbpA superfamily / RNA polymerase-binding protein / Sigma-70 factors family signature 1. / RNA polymerase sigma factor RpoD, C-terminal / RNA polymerase sigma factor RpoD / RNA polymerase sigma-70 region 1.2 / Sigma-70 factor, region 1.2 / RNA polymerase sigma-70 region 3 / Sigma-70 region 3 / Sigma-70 factors family signature 2. / RNA polymerase sigma-70 / RNA polymerase sigma-70 region 4 / Sigma-70, region 4 / RNA polymerase sigma-70 region 2 / RNA polymerase sigma-70 like domain / Sigma-70 region 2 / RNA polymerase sigma factor, region 2 / RNA polymerase sigma factor, region 3/4-like / 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 / Winged helix-like DNA-binding domain superfamily
Similarity search - Domain/homology
DNA / DNA (> 10) / DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit alpha / RNA polymerase sigma factor SigA / DNA-directed RNA polymerase subunit omega / RNA polymerase-binding protein RbpA / Protein pafC / Transcriptional regulator-like protein / DNA-directed RNA polymerase subunit beta
Similarity search - Component
Biological speciesMycolicibacterium smegmatis MC2 155 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsMueller, A.U. / Kummer, E. / Schilling, C.M. / Ban, N. / Weber-Ban, E.
Funding support Switzerland, 1items
OrganizationGrant numberCountry
Swiss National Science Foundation310030_185250 Switzerland
CitationJournal: Sci Adv / Year: 2021
Title: Transcriptional control of mycobacterial DNA damage response by sigma adaptation.
Authors: Andreas U Müller / Eva Kummer / Charlotte M Schilling / Nenad Ban / Eilika Weber-Ban /
Abstract: Transcriptional activator PafBC is the key regulator of the mycobacterial DNA damage response and controls around 150 genes, including genes involved in the canonical SOS response, through an unknown ...Transcriptional activator PafBC is the key regulator of the mycobacterial DNA damage response and controls around 150 genes, including genes involved in the canonical SOS response, through an unknown molecular mechanism. Using a combination of biochemistry and cryo–electron microscopy, we demonstrate that PafBC in the presence of single-stranded DNA activates transcription by reprogramming the canonical −10 and −35 promoter specificity of RNA polymerase associated with the housekeeping sigma subunit. We determine the structure of this transcription initiation complex, revealing a unique mode of promoter recognition, which we term “sigma adaptation.” PafBC inserts between DNA and sigma factor to mediate recognition of hybrid promoters lacking the −35 but featuring the canonical −10 and a PafBC-specific −26 element. Sigma adaptation may constitute a more general mechanism of transcriptional control in mycobacteria.
History
DepositionJul 15, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 22, 2021Provider: repository / Type: Initial release

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Structure visualization

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Assembly

Deposited unit
AA: DNA-directed RNA polymerase subunit alpha
AB: DNA-directed RNA polymerase subunit alpha
AC: DNA-directed RNA polymerase subunit beta
AD: DNA-directed RNA polymerase subunit beta'
AE: DNA-directed RNA polymerase subunit omega
AF: RNA polymerase sigma factor SigA
AJ: RNA polymerase-binding protein RbpA
AO: recA-op non-template strand
AP: recA-op template strand
AX: PafC
AY: Transcriptional regulator-like protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)545,24513
Polymers545,11411
Non-polymers1312
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: assay for oligomerization
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area64200 Å2
ΔGint-306 kcal/mol
Surface area161470 Å2

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Components

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

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


Mass: 37959.441 Da / Num. of mol.: 2 / Source method: isolated from a natural source
Source: (natural) Mycolicibacterium smegmatis MC2 155 (bacteria)
References: UniProt: A0QSL8, DNA-directed RNA polymerase
#2: Protein DNA-directed RNA polymerase subunit beta / Polymerase / RNAP subunit beta / RNA polymerase subunit beta / Transcriptase subunit beta


Mass: 128680.141 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Mycolicibacterium smegmatis MC2 155 (bacteria)
References: UniProt: P60281, DNA-directed RNA polymerase
#3: Protein DNA-directed RNA polymerase subunit beta' / Polymerase / RNAP subunit beta' / RNA polymerase subunit beta' / Transcriptase subunit beta'


Mass: 146712.891 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Mycolicibacterium smegmatis MC2 155 (bacteria)
References: UniProt: A0QS66
#4: Protein DNA-directed RNA polymerase subunit omega / Polymerase / RNAP omega subunit / RNA polymerase omega subunit / Transcriptase subunit omega


Mass: 11544.763 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Mycolicibacterium smegmatis MC2 155 (bacteria)
Strain: ATCC 700084 / mc(2)155 / References: UniProt: A0QWT1, DNA-directed RNA polymerase

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Protein , 4 types, 4 molecules AFAJAXAY

#5: Protein RNA polymerase sigma factor SigA


Mass: 51573.551 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycolicibacterium smegmatis MC2 155 (bacteria)
Strain: ATCC 700084 / mc(2)155 / Gene: sigA, MSMEG_2758 / Production host: Escherichia coli (E. coli) / References: UniProt: A0QW02
#6: Protein RNA polymerase-binding protein RbpA


Mass: 13078.731 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycolicibacterium smegmatis MC2 155 (bacteria)
Strain: ATCC 700084 / mc(2)155 / Gene: rbpA, MSMEG_3858, MSMEI_3768 / Production host: Escherichia coli (E. coli) / References: UniProt: A0QZ11
#9: Protein PafC


Mass: 34044.402 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: residues 82-123 have been modelled as poly-alanine
Source: (gene. exp.) Mycolicibacterium smegmatis MC2 155 (bacteria)
Strain: ATCC 700084 / mc(2)155 / Gene: MSMEG_3888 / Production host: Escherichia coli (E. coli) / References: UniProt: A0QZ41
#10: Protein Transcriptional regulator-like protein


Mass: 36081.465 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: residues 84-128 have been modelled as poly-alanine
Source: (gene. exp.) Mycolicibacterium smegmatis MC2 155 (bacteria)
Strain: ATCC 700084 / mc(2)155 / Gene: pafB, MSMEI_3799 / Production host: Escherichia coli (E. coli) / References: UniProt: I7G3U5

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DNA chain , 2 types, 2 molecules AOAP

#7: DNA chain recA-op non-template strand


Mass: 23733.148 Da / Num. of mol.: 1 / Source method: obtained synthetically
Source: (synth.) Mycolicibacterium smegmatis MC2 155 (bacteria)
#8: DNA chain recA-op template strand


Mass: 23746.215 Da / Num. of mol.: 1 / Source method: obtained synthetically
Source: (synth.) Mycolicibacterium smegmatis MC2 155 (bacteria)

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Non-polymers , 1 types, 2 molecules

#11: 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: DNA-directed RNA polymerase with transcriptional activator PafBC
Type: COMPLEX / Entity ID: #1-#10 / Source: MULTIPLE SOURCES
Molecular weightExperimental value: NO
Buffer solutionpH: 7.8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE-PROPANE

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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
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 128190 / Symmetry type: POINT

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