National Natural Science Foundation of China (NSFC)
32171198
China
Ministry of Science and Technology (MoST, China)
2018YFA090070
China
Chinese Academy of Sciences
2020452
China
Citation
Journal: 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.
Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK I
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Electron microscopy
Microscope
FEI TITAN KRIOS
Details
Preliminary grid screening was performed manually.
Image recording
Film 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 beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Details: 8GZG, 3LA2 and 8H3Z are required for the reconstruction of the complex of NtcA-NtcB-TAC.
Final reconstruction
Applied 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 assignment
Type: PROJECTION MATCHING
Final angle assignment
Type: PROJECTION MATCHING
Final 3D classification
Software - Name: RELION
FSC plot (resolution estimation)
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Atomic model buiding 1
Refinement
Protocol: RIGID BODY FIT
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