National Health and Medical Research Council (NHMRC, Australia)
APP1125909
Australia
Citation
Journal: J Biol Chem / Year: 2021 Title: SAGA and SAGA-like SLIK transcriptional coactivators are structurally and biochemically equivalent. Authors: Klaudia Adamus / Cyril Reboul / Jarrod Voss / Cheng Huang / Ralf B Schittenhelm / Sarah N Le / Andrew M Ellisdon / Hans Elmlund / Marion Boudes / Dominika Elmlund / Abstract: The SAGA-like complex SLIK is a modified version of the Spt-Ada-Gcn5-Acetyltransferase (SAGA) complex. SLIK is formed through C-terminal truncation of the Spt7 SAGA subunit, causing loss of Spt8, one ...The SAGA-like complex SLIK is a modified version of the Spt-Ada-Gcn5-Acetyltransferase (SAGA) complex. SLIK is formed through C-terminal truncation of the Spt7 SAGA subunit, causing loss of Spt8, one of the subunits that interacts with the TATA-binding protein (TBP). SLIK and SAGA are both coactivators of RNA polymerase II transcription in yeast, and both SAGA and SLIK perform chromatin modifications. The two complexes have been speculated to uniquely contribute to transcriptional regulation, but their respective contributions are not clear. To investigate, we assayed the chromatin modifying functions of SAGA and SLIK, revealing identical kinetics on minimal substrates in vitro. We also examined the binding of SAGA and SLIK to TBP and concluded that interestingly, both protein complexes have similar affinity for TBP. Additionally, despite the loss of Spt8 and C-terminus of Spt7 in SLIK, TBP prebound to SLIK is not released in the presence of TATA-box DNA, just like TBP prebound to SAGA. Furthermore, we determined a low-resolution cryo-EM structure of SLIK, revealing a modular architecture identical to SAGA. Finally, we performed a comprehensive study of DNA-binding properties of both coactivators. Purified SAGA and SLIK both associate with ssDNA and dsDNA with high affinity (K = 10-17 nM), and the binding is sequence-independent. In conclusion, our study shows that the cleavage of Spt7 and the absence of the Spt8 subunit in SLIK neither drive any major conformational differences in its structure compared with SAGA, nor significantly affect HAT, DUB, or DNA-binding activities in vitro.
History
Deposition
Nov 25, 2020
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Header (metadata) release
Apr 28, 2021
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Map release
Apr 28, 2021
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Update
Jul 14, 2021
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Current status
Jul 14, 2021
Processing site: RCSB / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
Model: PELCO Ultrathin Carbon with Lacey Carbon / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: LACEY / Pretreatment - Type: GLOW DISCHARGE / Details: 10 mAmps
Vitrification
Cryogen name: ETHANE / Instrument: FEI VITROBOT MARK III
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Electron microscopy
Microscope
FEI TITAN KRIOS
Image recording
Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Average exposure time: 11.2 sec. / Average electron dose: 50.0 e/Å2
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron optics
Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm
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