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- PDB-6jcx: Mycobacterium tuberculosis transcription initiation complex with ... -

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Basic information

Entry
Database: PDB / ID: 6jcx
TitleMycobacterium tuberculosis transcription initiation complex with ECF sigma factor sigma H and 6nt RNA
Components
  • (DNA-directed RNA polymerase subunit ...) x 4
  • DNA (5'-D(*TP*GP*CP*AP*TP*CP*CP*GP*TP*GP*AP*GP*TP*CP*GP*AP*GP*GP*GP*T)-3')
  • DNA (5'-D(*TP*TP*GP*TP*GP*GP*GP*AP*GP*CP*TP*GP*TP*CP*AP*CP*GP*GP*AP*TP*GP*CP*A)-3')
  • ECF RNA polymerase sigma factor SigH
  • RNA (5'-R(*CP*CP*UP*CP*GP*A)-3')
KeywordsTRANSCRIPTION / Mycobacterium tuberculosis / RNA polymerase / sigma H / transcription initiation
Function / homology
Function and homology information


response to nitrosative stress / Antimicrobial action and antimicrobial resistance in Mtb / sigma factor activity / bacterial-type RNA polymerase core enzyme binding / cytosolic DNA-directed RNA polymerase complex / DNA-directed RNA polymerase complex / peptidoglycan-based cell wall / DNA-templated transcription initiation / ribonucleoside binding / DNA-directed RNA polymerase ...response to nitrosative stress / Antimicrobial action and antimicrobial resistance in Mtb / sigma factor activity / bacterial-type RNA polymerase core enzyme binding / cytosolic DNA-directed RNA polymerase complex / DNA-directed RNA polymerase complex / peptidoglycan-based cell wall / DNA-templated transcription initiation / ribonucleoside binding / DNA-directed RNA polymerase / DNA-directed RNA polymerase activity / cellular response to heat / response to heat / response to oxidative stress / protein dimerization activity / response to antibiotic / DNA-templated transcription / regulation of DNA-templated transcription / magnesium ion binding / DNA binding / zinc ion binding / plasma membrane / cytosol / cytoplasm
Similarity search - Function
RNA polymerase sigma-70, actinobacteria / RNA polymerase sigma factor 70, region 4 type 2 / Sigma-70, region 4 / RNA polymerase sigma factor 70, ECF, conserved site / Sigma-70 factors ECF subfamily signature. / RNA polymerase sigma-70 like / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1 funnel domain / RNA polymerase, RBP11-like subunit / Enzyme I; Chain A, domain 2 ...RNA polymerase sigma-70, actinobacteria / RNA polymerase sigma factor 70, region 4 type 2 / Sigma-70, region 4 / RNA polymerase sigma factor 70, ECF, conserved site / Sigma-70 factors ECF subfamily signature. / RNA polymerase sigma-70 like / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1 funnel domain / RNA polymerase, RBP11-like subunit / Enzyme I; Chain A, domain 2 / Topoisomerase I; Chain A, domain 4 / DNA-directed RNA polymerase, insert domain / RNA Polymerase Alpha Subunit; Chain A, domain 2 / 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 / Gyrase A; domain 2 / 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 beta subunit, bacterial-type / DNA-directed RNA polymerase, alpha subunit / Beta Complex / 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, beta subunit, protrusion / RNA polymerase beta subunit / RNA polymerase Rpb1, domain 1 / RNA polymerase, alpha subunit / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 4 / RNA polymerase Rpb1, domain 2 / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 4 / DNA-directed RNA polymerase, insert domain superfamily / RNA polymerase Rpb2, domain 2 / RNA polymerase Rpb2, domain 2 / RNA polymerase, N-terminal / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase I subunit A N-terminus / RNA polymerase, RBP11-like subunit / 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 / 2-Layer Sandwich / Orthogonal Bundle / Mainly Beta / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
DNA / DNA (> 10) / RNA / ECF RNA polymerase sigma factor SigH / DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit beta / DNA-directed RNA polymerase subunit alpha
Similarity search - Component
Biological speciesMycobacterium tuberculosis H37Rv (bacteria)
synthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.903 Å
AuthorsLi, L. / Zhang, Y.
Funding support China, 1items
OrganizationGrant numberCountry
National Natural Science Foundation of China31670067 China
CitationJournal: Nucleic Acids Res / Year: 2019
Title: Structures and mechanism of transcription initiation by bacterial ECF factors.
Authors: Chengli Fang / Lingting Li / Liqiang Shen / Jing Shi / Sheng Wang / Yu Feng / Yu Zhang /
Abstract: Bacterial RNA polymerase (RNAP) forms distinct holoenzymes with extra-cytoplasmic function (ECF) σ factors to initiate specific gene expression programs. In this study, we report a cryo-EM structure ...Bacterial RNA polymerase (RNAP) forms distinct holoenzymes with extra-cytoplasmic function (ECF) σ factors to initiate specific gene expression programs. In this study, we report a cryo-EM structure at 4.0 Å of Escherichia coli transcription initiation complex comprising σE-the most-studied bacterial ECF σ factor (Ec σE-RPo), and a crystal structure at 3.1 Å of Mycobacterium tuberculosis transcription initiation complex with a chimeric σH/E (Mtb σH/E-RPo). The structure of Ec σE-RPo reveals key interactions essential for assembly of E. coli σE-RNAP holoenzyme and for promoter recognition and unwinding by E. coli σE. Moreover, both structures show that the non-conserved linkers (σ2/σ4 linker) of the two ECF σ factors are inserted into the active-center cleft and exit through the RNA-exit channel. We performed secondary-structure prediction of 27,670 ECF σ factors and find that their non-conserved linkers probably reach into and exit from RNAP active-center cleft in a similar manner. Further biochemical results suggest that such σ2/σ4 linker plays an important role in RPo formation, abortive production and promoter escape during ECF σ factors-mediated transcription initiation.
History
DepositionJan 30, 2019Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0May 29, 2019Provider: repository / Type: Initial release
Revision 1.1Jun 5, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed ..._citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Jul 31, 2019Group: Data collection / Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Nov 22, 2023Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / struct_conn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id / _struct_conn.ptnr2_label_seq_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: DNA-directed RNA polymerase subunit alpha
B: DNA-directed RNA polymerase subunit alpha
C: DNA-directed RNA polymerase subunit beta
D: DNA-directed RNA polymerase subunit beta'
E: DNA-directed RNA polymerase subunit omega
F: ECF RNA polymerase sigma factor SigH
H: DNA (5'-D(*TP*GP*CP*AP*TP*CP*CP*GP*TP*GP*AP*GP*TP*CP*GP*AP*GP*GP*GP*T)-3')
G: DNA (5'-D(*TP*TP*GP*TP*GP*GP*GP*AP*GP*CP*TP*GP*TP*CP*AP*CP*GP*GP*AP*TP*GP*CP*A)-3')
I: RNA (5'-R(*CP*CP*UP*CP*GP*A)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)408,82712
Polymers408,6729
Non-polymers1553
Water3,333185
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area52100 Å2
ΔGint-263 kcal/mol
Surface area126440 Å2
MethodPISA
Unit cell
Length a, b, c (Å)125.139, 161.001, 128.846
Angle α, β, γ (deg.)90.000, 117.060, 90.000
Int Tables number4
Space group name H-MP1211

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Components

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

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


Mass: 40279.039 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis H37Rv (bacteria)
Strain: H37Rv / Gene: rpoA / Plasmid: pETduet / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: P9WGZ1, DNA-directed RNA polymerase
#2: Protein DNA-directed RNA polymerase subunit beta / RNAP subunit beta / RNA polymerase subunit beta / Transcriptase subunit beta


Mass: 129602.344 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis H37Rv (bacteria)
Strain: H37Rv / Gene: rpoB / Plasmid: pACYCduet / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: P9WGY9, DNA-directed RNA polymerase
#3: Protein DNA-directed RNA polymerase subunit beta' / RNAP subunit beta' / RNA polymerase subunit beta' / Transcriptase subunit beta'


Mass: 147068.094 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis H37Rv (bacteria)
Strain: H37Rv / Gene: rpoC / Details (production host): pACYCduet / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: P9WGY7, DNA-directed RNA polymerase
#4: Protein DNA-directed RNA polymerase subunit omega / RNAP omega subunit / RNA polymerase omega subunit / Transcriptase subunit omega


Mass: 11851.140 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis H37Rv (bacteria)
Strain: H37Rv / Gene: rpoZ / Plasmid: pETduet / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: P9WGY5, DNA-directed RNA polymerase

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DNA chain , 2 types, 2 molecules HG

#6: DNA chain DNA (5'-D(*TP*GP*CP*AP*TP*CP*CP*GP*TP*GP*AP*GP*TP*CP*GP*AP*GP*GP*GP*T)-3')


Mass: 6206.001 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#7: DNA chain DNA (5'-D(*TP*TP*GP*TP*GP*GP*GP*AP*GP*CP*TP*GP*TP*CP*AP*CP*GP*GP*AP*TP*GP*CP*A)-3')


Mass: 7152.606 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Protein / RNA chain , 2 types, 2 molecules FI

#5: Protein ECF RNA polymerase sigma factor SigH / ECF sigma factor SigH / Alternative RNA polymerase sigma factor SigH / RNA polymerase sigma-H ...ECF sigma factor SigH / Alternative RNA polymerase sigma factor SigH / RNA polymerase sigma-H factor / Sigma-H factor


Mass: 24382.180 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis H37Rv (bacteria)
Strain: H37Rv / Gene: sigH / Plasmid: pTOLO-EX5 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: P9WGH9
#8: RNA chain RNA (5'-R(*CP*CP*UP*CP*GP*A)-3')


Mass: 1851.165 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 3 types, 188 molecules

#9: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn
#10: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#11: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 185 / Source method: isolated from a natural source / Formula: H2O

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.83 Å3/Da / Density % sol: 56.51 %
Crystal growTemperature: 295 K / Method: vapor diffusion, hanging drop
Details: 0.08M Magnesium acetate, 0.05M sodium cacodylate pH 6.5, 15% PEG 400

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SSRF / Beamline: BL19U1 / Wavelength: 0.9789 Å
DetectorType: DECTRIS EIGER X 9M / Detector: PIXEL / Date: Jun 18, 2018
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9789 Å / Relative weight: 1
ReflectionResolution: 2.9→50 Å / Num. obs: 98120 / % possible obs: 98 % / Redundancy: 5.9 % / Biso Wilson estimate: 67.42 Å2 / CC1/2: 0.988 / Rmerge(I) obs: 0.125 / Rpim(I) all: 0.056 / Rrim(I) all: 0.138 / Χ2: 0.862 / Net I/av σ(I): 11.695 / Net I/σ(I): 5.2
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsMean I/σ(I) obsNum. unique obsCC1/2Rpim(I) allRrim(I) allΧ2% possible all
2.9-2.9550.8771.21740380.6540.420.9780.73281.3
2.95-34.90.81844260.6760.3950.9140.72788.5
3-3.0650.78246980.7050.3790.8750.77193.8
3.06-3.125.30.74348330.7490.3510.8260.7897.2
3.12-3.195.90.71449810.7890.3240.7880.78399.7
3.19-3.276.20.58749690.8680.2590.6440.80199.9
3.27-3.356.30.47649670.9170.2070.5210.762100
3.35-3.446.30.39149890.9380.1690.4280.77499.9
3.44-3.546.20.31349960.9560.1370.3430.781100
3.54-3.6560.24250310.9720.1080.2660.81100
3.65-3.785.80.18450040.980.0840.2040.83100
3.78-3.946.10.15349890.9880.0680.1680.849100
3.94-4.116.40.12449990.9910.0530.1350.896100
4.11-4.336.40.10149910.9930.0440.110.93399.7
4.33-4.66.30.08550200.9950.0370.0930.975100
4.6-4.965.90.07749990.9940.0340.0850.973100
4.96-5.466.20.07749990.9950.0330.0840.976100
5.46-6.246.40.07550490.9950.0320.0820.99999.9
6.24-7.865.80.05950370.9970.0260.0641.009100
7.86-505.80.04451050.9970.0190.0480.94999.5

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Processing

Software
NameVersionClassification
PHENIX1.14_3260refinement
HKL-2000data scaling
PDB_EXTRACT3.24data extraction
HKL-2000data reduction
PHENIXphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 5zx3

5zx3


Resolution: 2.903→36.087 Å / SU ML: 0.41 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 30.66
RfactorNum. reflection% reflection
Rfree0.2561 2441 2.5 %
Rwork0.2102 --
obs0.2114 97692 97.46 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso max: 193.14 Å2 / Biso mean: 77.6787 Å2 / Biso min: 20.97 Å2
Refinement stepCycle: final / Resolution: 2.903→36.087 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms23946 1009 3 185 25143
Biso mean--70.06 61.51 -
Num. residues----3153
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 17

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
2.9029-2.96210.34971080.32164550465880
2.9621-3.02650.35831570.32065141529890
3.0265-3.09680.34571610.30935397555895
3.0968-3.17420.3681240.30315675579999
3.1742-3.260.32421580.291157035861100
3.26-3.35590.33041510.25757165867100
3.3559-3.46410.30971270.237957885915100
3.4641-3.58780.27661630.226857345897100
3.5878-3.73130.27541490.219556895838100
3.7313-3.9010.27691260.211157905916100
3.901-4.10630.22851490.192457055854100
4.1063-4.36320.22871330.177457475880100
4.3632-4.69940.21351450.17157385883100
4.6994-5.17110.21941480.181657685916100
5.1711-5.91660.27281550.206157355890100
5.9166-7.44350.27381360.211958165952100
7.4435-36.08940.18381510.16635559571095

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