The regulation of transcription is fundamental to biology and cell physiology in health and in disease. In the proposed project, the biological mechanisms of the transcription reaction will be studied. Our demonstrated expertise in the characterization of the basal transcription factors will be a key to the success of these studies. Three major questions will be investigated. First, helicase activity, a fundamental requirement in the initiation of basal transcription, will be investigated for its role in basal and activated transcription and in elongation. The helicase domains of TFIIH subunits will be modified, transfected into cells, and recovered for biochemical analysis. Second, the newly identified mammalian RNA polymerase H holoenzyme will be studied. Based upon the paradigm revealed in yeast cells, the holoenzyme may be responsible for most, if not all, of mRNA production. Using the herpes viral gene activator, VP16, as a powerful model, we will characterize the factor requirements for activation of transcription with the holoenzyme. We will also determine the fate of the holoenzyme subunits during the transcription reaction. Third, the specific activation of transcription by NF-kappaB will be investigated using our highly purified system. NF-kappaB has been implicated in the activation of a wide range of promoters in response to diverse environmental cues and in the constitutive production of antibodies in mature B cells. NF- kappaB has homology to the rel oncogene family and plays a role in a network of interactions that affect transcription levels. Direct promoter activation by NF-kappaB will be recapitulated in vitro and the factor requirements compared to those of the powerful model activator, GAL4- VP16. Results from these experiments will help clarify the biochemical basis of the regulation of gene expression.
