Reconstitution of the partial tail module of the human mediator complex

Abstract

RNA Polymerase II (Pol II) regulates and maintains every aspect of the cell through the act of transcription of protein coding genes. Transcription of protein coding genes depends on extrinsic and intrinsic signals to the Pol II through which it generates a calibrated response. Disturbance of the calibrated response through environmental and genetic factors are the source of many disease phenotypes including cancer. These intrinsic and extrinsic signals are relayed to the Pol II through the Mediator Complex. Mediator Complex is a 30 subunit 2-MDa protein complex that transduces signals from activators and repressors to mediate Pol II generated transcriptional response through which all processes of the cell are regulated. The Mediator Complex consist of 4 modules head middle kinase and tail. Tail module of the mediator complex is a 7 protein module recruits the mediator to the upstream activating sequences through its direct interactions with activators. Therefore reconstitution of the Mediator’s tail subunit is essential to mechanistically understand the Mediator’s regulation of Pol II and how activators cause transcriptional responses in the cell. Through MultiBac expression system we have generated each tail subunit and a partial tail complex of the mediator. Our data indicates that Med16-Med23-Med24-Med25 forms a 4 protein subcomplex through direct interactions of each protein with the tail subunit Med16 in the absence of Med14, the architectural scaffold of the Mediator. Presence of Med14 causes Med25 to be absent from this subcomplex and a subcomplex of Med14-Med16-Med23-Med24 is generated again through the direct interaction of each subunit with Med16. The remaining 3 subunits of the tail module Med15 Med27 and Med29 found to be separate from the remaining 4 subunits as they have failed to be elucidated in any of the immunoprecipitations performed. The results overall suggest the elucidation of a partial tail module. Purification of a full tail module may require additional Mediator Complex proteins.