Characterization of Mediator Complexes from HeLa Cell Nuclear Extract

The Saccharomyces cerevisiae Mediator complex was originally identified because of its ability to stimulate activated transcription in vitro. Many of the subunits of the purified Mediator complex (24) are encoded by SRB genes, first characterized as suppressors of a deletion in the C-terminal heptapeptide repeat (CTD) of the large subunit of RNA polymerase II (Pol II) (23). Additional Mediator subunits are encoded by genes initially identified in other genetic screens for mutations affecting gene control and are named accordingly (e.g., RGR-1). Mediator subunits not characterized previously were called Med1, Med2, etc. (24). Mediator subunits in yeast have also been purified as part of a still larger holoenzyme complex including Pol II and several general transcription factors (GTFs) (23). The Pol II holoenzyme analyzed by Young and colleagues (see reference 23 for a review) includes Srb8, Srb9, Srb10, and Srb11, whereas the Mediator complex studied by Myers et al. (24) lacks these subunits. The Srb8 to Srb11 subunits form a functional subcomplex of the holoenzyme required for repression by several yeast repressors (3, 11, 23). These subunits are regulated differently from other yeast Mediator and holoenzyme subunits. The intracellular Srb10 concentration falls dramatically as yeast cells deplete nutrients from their media, whereas the concentrations of other Mediator subunits do not (11). Recently, Liu et al. analyzed yeast Mediator complexes in a nuclear extract, avoiding ion-exchange chromatography and high salt concentration to avoid dissociation of subunits (18). Under these conditions they found that the majority of each Mediator subunit, including Srb8 to Srb11, was associated with Pol II in a complex of ∼1.9 MDa that lacks GTFs. A less abundant complex of ∼0.55 MDa included a subset of Mediator subunits. Several mammalian multiprotein complexes have been identified that have several subunits homologous to components of the yeast Mediator and several subunits that are not clearly related to yeast proteins (21). Broadly speaking, two size classes of complexes have been identified. Complexes of ∼2 MDa, such as the TRAP/SMCC (12), NAT (32), DRIP (30), ARC (25), and human Mediator (2) complexes, share an overlapping set of components. Smaller complexes (∼500 to 700 kDa) containing Srb/Med homologs have also been identified, including the murine Mediator (13), CRSP (31), and PC2 (20) complexes. These mammalian Mediator-like complexes were identified and purified by different biochemical procedures. TRAP (7), DRIP (30), ARC (25), and human Mediator (2) were purified on the basis of their ability to bind to activation domains during affinity chromatography. SMCC (8) and NAT (32) were purified based on their content of CDK8, which is a homolog of yeast Srb10. Functions of these Mediator complexes were assayed in different in vitro transcription systems that varied in the purity of the GTFs and the use of naked DNA versus chromatin templates. Most of these complexes, including ARC, DRIP, PC2, and CRSP, greatly stimulated activated transcription (21). NAT and SMCC repressed activated transcription in assays with highly purified factors (8, 32), but SMCC activated transcription when TFIIH was omitted (8). This repression has been attributed to the phosphorylation of the cyclin H subunit of TFIIH by the CDK8 kinase within the Mediator complexes (1). The human Mediator complex inhibited activated transcription in a highly purified system but stimulated high levels of activated transcription in reactions with partially purified GTFs (2). While the different mammalian Mediator-like complexes so far described share many subunits, they also differ with regard to their reported subunit composition (21). This raises the question whether there are multiple distinct Mediator-like complexes in mammalian cells that may differ in their functional properties or whether there is in fact one or a small number of mammalian Mediator complexes. In the latter case, the apparent differences in subunit composition reported by different laboratories might result from relatively minor differences in the methods used to characterize the subunits or from different methods of purification that partially dissociate a single large complex. To estimate the number of different complexes containing Mediator subunits in HeLa cells, we subjected unfractionated HeLa cell nuclear extract to gel filtration chromatography at low salt concentration to avoid the dissociation of subunits. Protein complexes in eluted fractions were characterized by immunoblotting with several antibodies specific for Mediator subunits, including components of both size classes of Mediator complexes described. A single peak of ∼2 MDa containing each of the several Mediator subunits was observed. No significant peak was observed at ∼500 to 700 kDa, indicating that either this size class of Mediator complex is much less abundant than the ∼2-MDa size class or that the ∼500 to 700-kDa Mediator complexes described above were derived from the larger size class by dissociation during the multiple steps of column chromatography used in their purification. Mediator subunits CDK8, cyclin C, and hSur2 were also observed in lower-molecular-mass complexes, but only the ∼2-MDa size class significantly stimulated activated transcription. High-resolution gel filtration and immunoprecipitation analyses indicated that there are at least two subclasses of ∼2-MDa Mediator complexes, one containing CDK8 and cyclin C and one lacking these subunits. A total of ∼300,000 hSur2 subunits per cell were present in the ∼2-MDa Mediator complexes; this number is approximately equal to the number of Pol II molecules per HeLa cell (15).