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- PDB-8i23: Clostridium thermocellum RNA polymerase transcription open comple... -

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Basic information

Entry
Database: PDB / ID: 8i23
TitleClostridium thermocellum RNA polymerase transcription open complex with SigI1 and its promoter
Components
  • (DNA (80-mer)) x 2
  • (DNA-directed RNA polymerase subunit ...) x 4
  • RNA polymerase sigma factor SigI1
KeywordsTRANSCRIPTION/DNA / TRANSCRIPTION OPEN COMPLEX / SIGI / TRANSCRIPTION / TRANSCRIPTION-DNA complex
Function / homologyDNA / DNA (> 10)
Function and homology information
Biological speciesAcetivibrio thermocellus DSM1313 (bacteria)
Acetivibrio thermocellus DSM 1313 (bacteria)
Acetivibrio thermocellus (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / negative staining / cryo EM / Resolution: 3.03 Å
AuthorsLi, J. / Zhang, H. / Li, D. / Feng, Y. / Zhu, P.
Funding support China, 1items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)32070125 China
CitationJournal: Nat Commun / Year: 2023
Title: Structure of the transcription open complex of distinct σ factors.
Authors: Jie Li / Haonan Zhang / Dongyu Li / Ya-Jun Liu / Edward A Bayer / Qiu Cui / Yingang Feng / Ping Zhu /
Abstract: Bacterial σ factors of the σ-family are widespread in Bacilli and Clostridia and are involved in the heat shock response, iron metabolism, virulence, and carbohydrate sensing. A multiplicity of σ ...Bacterial σ factors of the σ-family are widespread in Bacilli and Clostridia and are involved in the heat shock response, iron metabolism, virulence, and carbohydrate sensing. A multiplicity of σ paralogues in some cellulolytic bacteria have been shown to be responsible for the regulation of the cellulosome, a multienzyme complex that mediates efficient cellulose degradation. Here, we report two structures at 3.0 Å and 3.3 Å of two transcription open complexes formed by two σ factors, SigI1 and SigI6, respectively, from the thermophilic, cellulolytic bacterium, Clostridium thermocellum. These structures reveal a unique, hitherto-unknown recognition mode of bacterial transcriptional promoters, both with respect to domain organization and binding to promoter DNA. The key characteristics that determine the specificities of the σ paralogues were further revealed by comparison of the two structures. Consequently, the σ factors represent a distinct set of the σ-family σ factors, thus highlighting the diversity of bacterial transcription.
History
DepositionJan 13, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Oct 11, 2023Provider: repository / Type: Initial release
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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: RNA polymerase sigma factor SigI1
O: DNA (80-mer)
P: DNA (80-mer)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)431,90311
Polymers431,7488
Non-polymers1553
Water00
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-directed RNA polymerase subunit ... , 4 types, 5 molecules ABCDE

#1: Protein DNA-directed RNA polymerase subunit alpha


Mass: 35071.125 Da / Num. of mol.: 2 / Mutation: 0 / Source method: isolated from a natural source
Source: (natural) Acetivibrio thermocellus DSM 1313 (bacteria)
Strain: DSM1313 / References: DNA-directed RNA polymerase
#2: Protein DNA-directed RNA polymerase subunit beta


Mass: 140110.359 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Acetivibrio thermocellus DSM1313 (bacteria)
Strain: DSM1313 / References: DNA-directed RNA polymerase
#3: Protein DNA-directed RNA polymerase subunit beta'


Mass: 132698.844 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Acetivibrio thermocellus DSM 1313 (bacteria)
Strain: DSM1313 / References: DNA-directed RNA polymerase
#4: Protein DNA-directed RNA polymerase subunit omega


Mass: 8783.143 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Acetivibrio thermocellus DSM1313 (bacteria)
Strain: DSM1313 / References: DNA-directed RNA polymerase

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Protein , 1 types, 1 molecules F

#5: Protein RNA polymerase sigma factor SigI1


Mass: 30714.949 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Acetivibrio thermocellus DSM1313 (bacteria)
Strain: DSM1313 / Production host: Escherichia coli BL21(DE3) (bacteria)

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DNA chain , 2 types, 2 molecules OP

#6: DNA chain DNA (80-mer) / Promoter DNA non-template strand


Mass: 24897.059 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Acetivibrio thermocellus (bacteria)
#7: DNA chain DNA (80-mer) / Promoter DNA template strand


Mass: 24401.629 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Acetivibrio thermocellus (bacteria)

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Non-polymers , 2 types, 3 molecules

#8: Chemical ChemComp-ZN / ZINC ION


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


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg

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Details

Has ligand of interestN
Has protein modificationN

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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: Clostridium thermocellum RNA polymerase transcription open complex with SigI1 its promoter
Type: COMPLEX / Entity ID: #1-#7 / Source: MULTIPLE SOURCES
Molecular weight
IDEntity assembly-IDValue (°)UnitsExperimental value
110.43 MDaNO
210.35 MDaNO
310.03 MDaNO
41MEGADALTONSNO
Source (natural)Organism: Acetivibrio thermocellus DSM 1313 (bacteria)
Buffer solutionpH: 8
Details: 20 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM DTT, 0.2 mM EDTA, 5% (v/v) glycerol, 10 mM MgCl2
SpecimenConc.: 23.54 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: YES / Vitrification applied: YES / Details: This sample was monodisperse.
EM stainingType: NEGATIVE / Material: Uranyl Acetate
VitrificationCryogen name: ETHANE

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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 FIELD / Nominal magnification: 22500 X / Calibrated magnification: 22500 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1500 nm / Calibrated defocus min: 1500 nm / Calibrated defocus max: 2500 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 77 K / Temperature (min): 77 K
Image recordingAverage exposure time: 0.25 sec. / Electron dose: 1.875 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
Image scansWidth: 3838 / Height: 3710 / Movie frames/image: 32 / Used frames/image: 1-32

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM softwareName: PHENIX / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.03 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 150741 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00326940
ELECTRON MICROSCOPYf_angle_d0.53436836
ELECTRON MICROSCOPYf_dihedral_angle_d17.1544415
ELECTRON MICROSCOPYf_chiral_restr0.0434196
ELECTRON MICROSCOPYf_plane_restr0.0044391

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