The large T antigen of SV40 has been associated with a number of regulatory functions such as initiation of viral replication, repression of viral transcription, stimulation of cellular DNA and RNA synthesis, and initiation and maintenance of transformation. Although the effects of T antigen on these diverse metabolic processes has been extensively documented, the biochemical basis and molecular mechanism governing these multifunctional activities of T antigen are not well understood.
The aim of this research is to understand how T antigen carries out these diverse biochemical functions. We previously described a series of experimens which indicated that the interaction of T antigen at specific binding sites plays a direct role in repressing transcription of early viral mRNA in an in vitro reconstituted system. Here we propose to investigate the mechanism of SV40 autoregulation. First, we will map by in vitro mutagenesis the region of SV40 DNA required to promote transcription of early and late genes both in vitro and in vivo. Next, we will attempt to purify RNA polymerase II and its selectivity factors in order to perform specific holoenzyme DNA binding studies at the promoter sequences. The mechanisms of transcriptional selectivity provided by the specificity determinants will be tested in vitro both by binding studies and by transcription. Finally, we will study the interaction of T antigen with RNA polymerase and its specificity factors at the early at late promoter-binding sites.
