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- EMDB-34477: Cryo-EM structure of the full transcription activation complex Nt... -

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Basic information

Entry
Database: EMDB / ID: EMD-34477
TitleCryo-EM structure of the full transcription activation complex NtcA-NtcB-TAC focusing on NtcA and NtcB binding sites
Map data
Sample
  • Complex: full transcription activation complex with two activators NtcA and NtcB
Keywordstranscription activation complex / TRANSCRIPTION-DNA complex
Biological speciesAnabaena (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.6 Å
AuthorsHan SJ / Jiang YL / You LL / Shen LQ / Kong WW / Zhang Y / Zhou CZ
Funding support China, 4 items
OrganizationGrant numberCountry
Chinese Academy of SciencesXDB37020301, XDA24020302 China
National Natural Science Foundation of China (NSFC)32171198 China
Ministry of Science and Technology (MoST, China)2018YFA090070 China
Chinese Academy of Sciences2020452 China
CitationJournal: Nat Struct Mol Biol / Year: 2024
Title: DNA looping mediates cooperative transcription activation.
Authors: Shu-Jing Han / Yong-Liang Jiang / Lin-Lin You / Li-Qiang Shen / Xiaoxian Wu / Feng Yang / Ning Cui / Wen-Wen Kong / Hui Sun / Ke Zhou / Hui-Chao Meng / Zhi-Peng Chen / Yuxing Chen / Yu Zhang / Cong-Zhao Zhou /
Abstract: Transcription factors respond to multilevel stimuli and co-occupy promoter regions of target genes to activate RNA polymerase (RNAP) in a cooperative manner. To decipher the molecular mechanism, here ...Transcription factors respond to multilevel stimuli and co-occupy promoter regions of target genes to activate RNA polymerase (RNAP) in a cooperative manner. To decipher the molecular mechanism, here we report two cryo-electron microscopy structures of Anabaena transcription activation complexes (TACs): NtcA-TAC composed of RNAP holoenzyme, promoter and a global activator NtcA, and NtcA-NtcB-TAC comprising an extra context-specific regulator, NtcB. Structural analysis showed that NtcA binding makes the promoter DNA bend by ∼50°, which facilitates RNAP to contact NtcB at the distal upstream NtcB box. The sequential binding of NtcA and NtcB induces looping back of promoter DNA towards RNAP, enabling the assembly of a fully activated TAC bound with two activators. Together with biochemical assays, we propose a 'DNA looping' mechanism of cooperative transcription activation in bacteria.
History
DepositionOct 9, 2022-
Header (metadata) releaseOct 4, 2023-
Map releaseOct 4, 2023-
UpdateFeb 28, 2024-
Current statusFeb 28, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_34477.png

Downloads & links

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Map

FileDownload / File: emd_34477.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.07 Å
Density
Contour LevelBy AUTHOR: 0.0074
Minimum - Maximum-0.005331564 - 0.035037395
Average (Standard dev.)0.00018979979 (±0.0014887368)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 321.00003 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_34477_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_34477_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : full transcription activation complex with two activators NtcA an...

EntireName: full transcription activation complex with two activators NtcA and NtcB
Components
  • Complex: full transcription activation complex with two activators NtcA and NtcB

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Supramolecule #1: full transcription activation complex with two activators NtcA an...

SupramoleculeName: full transcription activation complex with two activators NtcA and NtcB
type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Anabaena (bacteria) / Strain: sp. PCC 7120
Molecular weightTheoretical: 718 KDa

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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

Concentration17 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
100.0 mMKClpotassium chloride
5.0 mMMgCl2magnesium chloride
10.0 mMHEPES2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid
2.0 mMDTTDithiothreitol

Details: 10 mM HEPES pH 7.5, 100 mM KCl, 5 mM MgCl2 and 2 mM DTT
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 120 sec. / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK I

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Electron microscopy

MicroscopeFEI TITAN KRIOS
DetailsPreliminary grid screening was performed manually.
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 5047 / Average electron dose: 50.0 e/Å2
Details: Images were collected in movie-mode at 40 frames per second.
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.2 µm / Nominal defocus min: 1.2 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 402817
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

8gzg


Details: 8GZG, 3LA2 and 8H3Z are required for the reconstruction of the complex of NtcA-NtcB-TAC.
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 7.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION
Details: This map was reconstructed by 3D refinement and PostProcess focusing on the NtcA and NtcB binding sites.
Number images used: 65446
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: PROJECTION MATCHING
Final 3D classificationSoftware - Name: RELION
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: RIGID BODY FIT

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