Structure of a Complete Mediator-RNA Polymerase II Pre-Initiation Complex

Abstract

SUMMARY A complete, 52-protein, 2.5 million Dalton, Mediator-RNA polymerase II pre-initiation complex (Med-PIC) was assembled and analyzed by cryo-electron microscopy and by chemical cross-linking and mass spectrometry. The resulting complete Med-PIC structure reveals two components of functional significance, absent from previous structures, a protein kinase complex and the Mediator-activator interaction region. It thereby shows how the kinase and its target, the C-terminal domain of the polymerase, control Med-PIC interaction and transcription.

Role for the MED21-MED7 Hinge in Assembly of the Mediator-RNA Polymerase II Holoenzyme

“…These observations raise the possibility that these subunits form a subcomplex within the human head module. As discussed earlier, these findings are consistent with results of prior protein cross-linking studies that identified contacts between yeast Pol II and subunits in the moveable jaw and structural core of the yeast head module (17,18,21), and they provide further evidence for conservation of Mediator structure and function from yeast to human.…”

“…This finding was somewhat unexpected, because (i) the Pol II CTD is critical for binding of Pol II to Mediator (17) and (ii) an X-ray crystal structure of the yeast head module in complex with a peptide consisting of five copies of the CTD heptapeptide repeat demonstrated that CTD repeats contact four CTD-interacting regions (CIRs) in the head module consisting of portions of MED17, MED6, and MED8 (22). The first, CIR1, is composed entirely of helices from MED17.…”

“…Previous EM and cross-linking studies of yeast and human Mediator have established that the MED21-MED7 heterodimer is located in the middle module near the top of the MED14 stalk (6,17,18,21). Based on existing models of yeast Mediator (6,17,18,21), the MED21-MED7 hinge could, because of its location, affect the orientation or conformation of not only MED21-MED7, but also the MED7N-MED31-containing knob and the MED19-containing hook. As we have shown by EM, mutant Mediators containing either MED21 (D3K) or MED7 (R127A/H129A) exhibit increased disorder and/or flexibility of the knob and hook.…”

“…X-ray structures of the head module of yeast Mediator show that MED11, MED17, and MED22 constitute the structural core of the Head, and MED18 and MED20 form the moveable jaw (16,20). Analyses of yeast Mediator-Pol II holoenzyme (17,21), a yeast core Mediator-minimal preinitiation complex (18), and the complete Mediator-preinitiation complex (17) by cryo-EM and protein cross-linking show interactions between the moveable jaw (MED18-MED20) and RPB3-RPB11 and between MED17 and RPB4-RPB7 (Fig. 1C).…”

“…Although the precise mechanisms and roles of specific Mediator subunits in formation of the holoenzyme are poorly understood, evidence from a variety of biochemical and EM structural studies argues that it involves binding of Pol II to specific surfaces on the Mediator head and middle modules (Fig. 1C) (6,8,(13)(14)(15)(16)(17)(18). In this report, we describe experiments investigating assembly of the human holoenzyme.…”