The activation of oncogenic cellular genes involved in transformation and of many other genes seems to be, at least in some cases, at the level of transcription. This proposal centers on the analysis of initiation of transcription by RNA polymerases II and III in vitro systems which faithfully reproduce the site and requirements for in vivo initiation of transcription. We will use a combination of straight biochemical fractionation approaches with a genetic approach which takes advantage of template and transcriptional machinery mutants as well as nucleotide analogs. The RNA polymerase II work will focus on the proteins involved in initiation by analyzing in detail the biochemical (enzyme with cofactors) as well as genetic (i.e., in the template sequence) features required for the formation of the transcription initiation complex. We will use transcriptional extracts from HeLa cells and templates consisting of oncogenic adenovirus DNA inserted in bacterial plasmids or phages. Novel features of the initiation process, like for example, unwinding of the DNA double helix or the mechanism of action of the inhibitor of transcription initiation, DRB, will be some of the partial processes that will be analyzed in detail in transcriptional initiation complexes. The unwinding assay will also be used for the VAI RNA genes transcribed by RNA polymerase III. The formation of a transcriptional complex, which distinguishes between the initiation and elongation phases of transcription, revealed a large molar excess of initiation events. This excess could be related to a mechanism of gene activation by an increase in the amounts of elongation factors or might be an artifact of the in vitro system. Point mutants in the DNA template will be generated by site-specific mutagenesis on M-13 cloning vectors which allow rapid sequence analysis of the mutants and in vitro transcription with the replicative form of the recombinant DNA phage. Site-specific point mutants allow to better establish the role of each element of the promoter sequence, since the environment around the mutation remains constant. The interactions between the mutant or wild-type promoter sequences and the elements of the transcriptional machinery, cellular RNA polymerase II Alpha-amanitin resistant mutants and transcriptionally resistant DRB resistant mutants will be analyzed. Mutations in the RNA polymerase or factors should produce some abnormal interactions. We will continue our efforts in establishing the mechanism of action of the transcription initiation inhibitor DRB and the biochemical basis for these cell mutants.
