We are interested in the molecular mechanisms of transcriptional regulation in mammalian cells. Many of our experiments have focused on the transcription of the proviral genome of the retrovirus mouse mammary tumor virus, which is subject to both positive and negative control. A number of cellular proteins that are important for viral transcription have been identified, and we would like to define the precise roles of these proteins in establishing correct levels of viral gene expression. We are also exploring some specific questions related to the general mechanism of transcription initiation by RNA polymerase II and the biochemical details of transcriptional regulation. In particular, we are developing assays to directly assess effects of transcriptional regulatory proteins on discrete steps in the initiation process, including transcription complex assembly, separation of the two strands of template DNA at the initiation site, and promoter clearance by the polymerase as it begins RNA synthesis.