RNA polymerase II (Pol II) catalyzes the expression of all protein- encoding genes. Data suggest that gene transcription is regulated by a form of the Pol II enzyme known as the Pol II holoenzyme, which is a massive complex of the core Pol II bound to a subset of basal transcription factors and co-activators. A co-activator is a protein that mediates the signal from enhance binding regulatory proteins to the basal machinery. Advances in the biochemistry of transcription regulators has revealed much about how the basal transcription factors function, but little is known about how the enhancer bound regulators communicate with the basal machinery through the co-activators. The focus of this project is to identify mechanisms by which co-activators and regulated modifications influence pol II holoenzyme function. We have found that DNA topoisomerase IIalpha acts as a co-activator in the Pol II holoenzyme. While this enzyme has been known to be critical in the replication of the genome, its transcription function has not been demonstrated We found that DNA topoisomerase II functions in transcription by a chromatin-dependent co-activator activity.
The first aim of this project will identify the mechanism by which DNA topoisomerase IIALPHA regulates transcription on chromatin templates and identify genes whose expression is regulated by topoisomerase IIalpha.
The second aim of this project will develop a reliable purification of the Pol II holoenzyme in order to characterize its polypeptide composition, modifications to specific subunits which may affect function, and delineate whether subpopulations exist of the Pol II holoenzyme. Together, these aims will define this critical mRNA synthesizing machine of the cell, identify how modifications and subpopulations perhaps create Pol II holoenzyme complexes with specialized function and elucidate how topoisomerase II regulates transcription.
