Characterization of the Enhancer and Promoter Elements of the Human TAF(II)55 Gene
To understand the mechanisms by which activators and cofactors function on TATA-less promoters, we also purified a transcriptional cofactor complex, Mediator, by first establishing a HeLa-derived cell line (hMED7-7) that expresses the epitope-tagged Med7 component of human Mediator. From the derived nuclear extracts, or the 500 mM and the 850 mM KCl P11 phosphocellulose column fractions of the derived nuclear extracts, three forms of Mediator complexes containing previously characterized Mediator subunits were purified. The 500 mM fraction-derived Mediator contains Cdk8/Cyclin C, while the 850 mM fraction-derived Mediator lacks the protein pair and likely corresponds to the previously characterized positive cofactor 2 (PC2). The associated RNA polymerase II was also depleted from the Mediator complexes with an immobilized antibody against the largest subunit of RNA polymerase II. The availability of these human Mediator complexes allows us to address the functional redundancy and/or transcriptional synergy among three general cofactors (TAFIIs, Mediator and PC4) commonly implicated in activator-dependent transcription from both TATA-containing and TATA-less promoters.
University of Illinois - Urbana-Champaign
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University of Illinois at Urbana-Champaign