A long-term objective of the proposed research is to understand the mechanism of transcription initiation and promoter site selection in eucaryotes. In order to achieve these objectives, the study of transcription of Adenovirus 2 (Ad 2) DNA in an in vitro system will be continued. The major components of the system under investigation are wheat germ, human placenta and calf thymus polymerases, class II, and purified DNA of Adenovirus 2. Our previous research has indicated that these polymerases: (1) can form strong binary complexes with Ad 2 DNA, (2) the complexes formed are localized at specific sites on the DNA molecule which are identical for all eucaryotic polymerases studied but different from those for E. coli polymerase, (3) binary complexes exist in two different states, open (RS), able to initiate in the presence of poly I and rifamycin AF/013, and closed (I) which are inactivated by these inhibitors, (4) only a small fraction of polymerase can form binary complexes and transcribe double-stranded Ad 2 DNA. This suggests that the mechanism for initiation of RNA synthesis and promoter site selection is similar for these eucaryotic polymerases to that proposed for E. coli polymerase (Chamberlin, 1976). The proposed research will concentrate on answering the following questions: (1) What is the precise position of in vitro binding sites on DNA, and how are these sites related to in vivo promoters? (2) What is the precise mechanism of promoter selection by polymerase II? and (3) What are the catalytic properties and the polypeptide composition of the polymerase which is capable of recognizing promoters and synthesizing RNA? First, we will isolate fragments with specific promoters. Second, the kinetic parameters of interactions of the polymerases with individual promoters will be measured. Third, the enzyme capable of forming binary complexes will be separated from the remaining enzyme using exclusion chromatography, and its properties studied. Fourth, the DNA base sequence protected by polymerases will be determined. Fifth, an attempt will be made to define conditions (e.g., temperature, template modification, presence of additional proteins) in which in vitro transcription mimics the in vivo situation.