Reconstitution of RNA Polymerase II subunits and analysis of their interaction with PIC components

Mediator Complex is a global coactivator of RNA Pol II transcription machinery. It transmits the regulatory signals from enhancer to promoter region. In the absence of Mediator Complex, Pol II transcription is defected. It has been shown that Mediator Complex binds to the CTD domain of Pol II as well as other components of PIC (pre-initiation complex), and thus it forms the transcription loop that enables communication between enhancer region and the promoter. Our lab has found the Mediator Complex subunit (which will be mentioned as MEDX since the data is not published yet) which interacts with the purified endogenous Pol II. After these findings, we aimed to find the subunit of Pol II that interacts with MEDX. For that purpose, we cloned 8 out of 12 subunits of Pol II and so far only reconstituted 5 subunits of it (RPB4, RPB5, RPB6, RPB9 and RPB12) and did biochemical analysis with these proteins. Immunoprecipitation of MEDX with 5 subunits of Pol II showed that 4 of these 5 subunits of Pol II seemed to have pulled down MEDX eve after stringent wash conditions. However, we cannot conclude that MEDX interacts with these 4 subunits, since we are lacking other controls. Also, we will continue characterizing the other subunits of Pol II to find the subunit that interacts with the Mediator subunit MEDX. As the transcription loop is mediated by activators/Mediator/Pol II-PIC we also started cloning two model activators to observe activator mediated polymerase interactions. For this purpose we started cloning and expressing two nuclear hormone receptors: Androgen Receptor and Mineralocorticoid Receptors. Nuclear Receptors are a huge family of activators which transmits signals from the environment of the cell by the help of their ligands, hormones to the transcription machinery. Upon ligand binding, these activators enter the nucleus and bind to the enhancer sequences upstream of the promoter region. Then they recruit Mediator Complex which will start the formation of PIC at the promoter region. We therefore obtained AR from Dr. Nathan Lack and cloned MR. We expressed these proteins in insect cells and characterize PIC formation.