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Characterization of mediator complexes from HeLa cell nuclear extract
G Wang1, G T Cantin, J L Stevens
1Molecular Biology Institute, University of California, Los Angeles, California 90095-1570, USA.
Molecular and Cellular Biology
|June 21, 2001
Summary
Mammalian Mediator complexes exist primarily as a single, large (~2-MDa) form in vivo, not multiple smaller forms. This large Mediator complex, containing multiple subunits, plays a key role in gene transcription regulation.
Area of Science:
- Molecular Biology
- Biochemistry
- Gene Regulation
Background:
- Multiple mammalian multiprotein complexes, including TRAP/SMCC, NAT, DRIP, ARC, and human Mediator, contain homologs of yeast Mediator subunits.
- These complexes have been described in varying sizes, from high-molecular-mass (1-2 MDa) to smaller forms (~500-700 kDa).
Purpose of the Study:
- To determine if these described Mediator complexes exist in vivo as a single form or multiple forms.
- To characterize the size and composition of mammalian Mediator complexes in HeLa cell nuclear extracts.
Main Methods:
- Gel filtration chromatography (Superose 6) of HeLa cell nuclear extract.
- Immunoblotting using antisera specific for Mediator subunits.
- Quantitative immunoblotting and immunoprecipitation.
Main Results:
- A single major size class of high-molecular-mass (~2-MDa) complexes containing multiple mammalian Mediator subunits was detected.
- No significant peak was observed at ~500-700 kDa, suggesting smaller complexes are less abundant or are dissociation products.
- Two classes of Mediator complexes were identified: one containing CDK8/cyclin C and another lacking it.
- The ~2-MDa complexes stimulated activated transcription in vitro.
Conclusions:
- Mammalian Mediator complexes predominantly exist as a large (~2-MDa) entity in vivo.
- The abundance of Mediator subunits is comparable to RNA polymerase II and general transcription factors.
- The Mediator complex, particularly its larger form, is crucial for activating transcription.