Pol II transkripsiyon regulasyonunda MED14 fonksiyonunun belirlenmesi

Transcription of protein coding genes by RNA polymerase II (Pol II) is a multi-step process each of which requires a series of factors including coactivators. 30 subunit, 2mDa human Mediator complex is the key coactivator in transcription not only for facilitating the establishment of pre initiation complex (PIC) by acting as a bridge between gene-specific activators and Pol II but also and more importantly for regulating the Pol II activity at all transcription steps. This study identifies human Mediator complex subunit of MED14 is the critical factor in transcription by directly interacting with Pol II. By using Multibac Baculovirus expression system and reconstitution based approach of study human Mediator, Pol II was shown to be directly interacted with N terminus of MED14 and MED14 was responsible for the recruitment of Pol II to the promoter DNA. Immobilized template recruitment assay as well as viability assays showed that, MED14 is indispensable for transcription start through direct interaction with Pol II. It is the first time that the mechanism of how Pol II binds to Mediator and how it is being recruited to target gene promoters for proper PIC assembly and transcription have been unraveled. This way, we shed light into how protein coding genes are universally regulated via the Mediator complex and in the future, we will be in a better position to specifically target selected genes by reconstituting the entire Mediator complex and characterizing activator- Mediator-Pol II crosstalk.