The long term objective of this proposal is to understand the molecular mechanism by which the adenovirus oncogene early region 1a (E1a) induces transcriptional activation. E1a does not bind to DNA directly and may exert its effect by interaction and or modification of a cellular transcriptional factor. Thus, we have chosen to examine and purify the protein factors that are responsible for early region 3 transcription, an adenovirus gene absolutely dependent on E1a for expression. In addition to RNA polymerase II, other general transcription factors like TFII, B, E, D and A, and gene specific factors are required. Some of the latter are sequence specific DNA binding proteins. The experiments described here focus on the isolation and characterization of a protein which binds to a DNA region necessary both for transcription and E1a response, located 85 to 105 nucleotides upstream from the transcription start site. The unique properties that we will exploit for the purification are the partial assay and selectivity provided by binding to specific DNA sequences, by interaction of this protein with E1a protein and the modern methods of protein HPLC. Specific DNA binding columns derived from synthetic oligonucleotides, as well as E1a affinity columns, will be used in combination with FPLC techniques to purify the specific proteins. Footprinting, gel retardation, and in vitro transcription reconstitution assays will be used to follow the activity of this protein. We will investigate the mode of interaction of the purified factor from unifected or infected cells with E3 DNA and with E1a protein in vitro to determine which properties of the E3 transcription factor are modified by E1a during adenovirus infection. Thus, using a partially purified transcription system we will try to reproduce the regulation effected by E1a on the viral gene E3. The elucidation of this regulatory mechanism will clarify the nature of the E1a interaction with viral and cellular genes and the resulting viral induced transformation event.
