Extended Data Figure 8. Functional validation of Mediator-RNAPII contacts.

a, Left, coomassie blue-stained SDS-PAGE analysis (4–20% gradient gel) of purified wild-type, and ΔMed31 S cerevisiae Mediator. Right, effect of Med31 deletion on interaction of Sc Mediator with RNAPII. Wild-type and ΔMed31 ScMED were purified by TAP-tagging. Protein elutes from a calmodulin resin were analyzed. b, The x-ray structure of Sc Med7N/31 (PDB 3FBI) was fitted into Sp holoenzyme cryo-EM map. (Sc Med31 in green and Sc Med7N In orange). ScMED Med31 residues expected to be in close contact with the CTD were mutated as indicated. Head, Middle, and Med14 in the Sp holoenzyme cryo-EM map shown in transparent pink, cyan and green, respectively. c, Med31 sequence conservation in Sc, Sp, and human Mediators. d, Effect of Med31 point mutations on Mediator interaction with RNAPII. Y41E and T44P Med31 mutations resulted in a considerable decrease in RNAPII interaction. Mutation Y38A had a smaller but still considerable effect. Q45–46AA double mutation had no effect on RNAPII binding. A K83A mutation of a lysine located far from the CTD had no effect on RNAPII interaction either. e, 2D class averages showing that Med31 point mutations that affect RNAPII interactions do not result in destabilization of the knob (knob highlighted by yellow arrowhead in magnified insets). WT and ΔMed31 class averages shown for comparison.