Mass spectrometry data for human pol II (A) and human Mediator (B) samples.

A mass spectrometry (MS) protocol was used to define the composition of Mediator and pol II that was used for EM analysis. These data confirm the purification protocols outlined in Figure 1B and Figure 1C isolated the complete, 26-subunit Mediator complex and the entire, 12-subunit pol II enzyme. Peptide identification and percent coverage was calculated following a 1% false-discovery rate (FDR) analysis.*No unique peptides corresponding to MED28 remained following the 1% FDR screen; however, two unique MED28 peptides were present in the MS data and these were manually validated and are shown in Figure S11. The 11.8% value listed for MED28 reflects the inclusion of these two peptides. Thus, it does not appear that MED28 dissociates from Mediator during the purification protocol outlined in Figure 1C. Note also that whereas CDK8 and Cyclin C were not detected in this sample, MED12 (2.6% coverage) and MED13 (0.8% coverage) were detectable; based upon the silver stained gel of VP16-Mediator (Figure 1C) as well as comparison of spectral counts and percent coverage, it is evident these subunits are substantially sub-stoichiometric relative to core Mediator subunits in this VP16-Mediator sample.