Revealing the role of Med14 in Pol-II transcription regulation
Cevher, Murat Alper
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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 (the subunits are organized into discrete modules – head, middle, tail, and kinase) -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 pre-initiation, elongation and re initiation steps. Although the diversity in the function of Mediator lies in its flexible conformation and variable subunit organization (for instance, four subunit CDK8 module can both bind to and disassociate from Mediator), detailed mechanistic studies regarding how Mediator interacts with different activators/repressors to regulate transcription, how Mediator facilitates basal transcription through interactions with Pol II and general transcription factors (GTFs) to establish a proper PIC on promoter DNA and how the regulation of subunit exchanges/rearrangements on the Mediator results in architectural and functional outcome are not well characterized. By using Multibac baculovirus expression system, the previous studies have shown that the reconstitution of a functional 15- subunit human core Mediator complex (composed of head and middle modules together with Med14) is sufficient to support basal transcription as well as selective activator-dependent transcription in vitro both with purified factors and with nuclear extracts (in the presence of Med26) as the source of GTFs. This study has uncovered a mechanism by which human core Mediator facilitates transcription by directly interacting with Pol II via its Med14 subunit and recruiting Pol II to target gene promoters. This is the first time that i unraveled 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. This way, i shed light into how protein coding genes are universally regulated via the Mediator complex and in the future , I will be in a better position to specifically target selected genes by reconstituting the entire Mediator complex and characterizing activator-Mediator-Pol II crosstalk.