Cell - 8 September 2016

Figure S5. Mediator-Pol II Interaction Studies, Related toFigure 5
(A) Surface plasmon resonance (SPR) curves for the interaction of full-length Mediator complex (analyte) with RNA polymerase II variants immobilized on the
sensor chip surface (ligands).
(B and C) Equivalent binding curves forDTail Mediator and Med Head analytes, respectively. The pol II ligand type, analyte concentrations and measured kinetic
constants are indicated for each panel. All Mediator complexes bind pol II variants harboring a CTD whereas pol II variants lacking a CTD show no detectible
binding.
(D) The interaction of Mediator (panel 1) and TFIIF (panels 2–4) analytes with WT pol II, mutant CTD pol II andDCTD pol II ligands. First panel: single-cycle kinetic
analysis of the interaction of Mediator with immobilized pol II variants with Mediator concentrations at each injection step indicated. Whereas Mediator binds WT
pol II but neither mutant CTD norDCTD pol II variants, the TFIIF positive control binds to all three pol II ligand surfaces with equivalent affinity demonstrating
equivalent binding activity (panels 2–4).
(E) The kinetic analysis of pol II variant analytes binding to an immobilized Mediator ligand surface provides further support for the CTD dependenceof the
Mediator-pol II interaction. All kinetic data were processed using double reference subtraction (blank analyte injection andDligand reference lane). The pol II
(ligand)-TFIIF (analyte) and Mediator (ligand)-pol II (analyte) interaction curves were fit using the ‘Langmuir (1:1)’ model with all other curves modeled using a
‘Heterogeneous Ligand’ model, consistent with the availability of multiple equivalent CTD heptad repeats.