MCB9728765 Hochschild Many transcriptional activators in prokaryotes are known to bind near a promoter and contact RNA polymerase (RNAP). The study of natural activators has not, however, established whether a protein-protein contact between a DNA-bound activator and RNAP suffices to activate transcription. Recent work in the Principal Investigator's lab has shown that contact between a DNA-bound protein and a heterologous protein domain fused to RNAP can elicit transcriptional activation, suggesting that contact between a DNA-bound protein and any accessible surface of RNAP will activate transcription. A main objective of this research is to critically evaluate this hypothesis and to generate a genetic map of the surfaces of E. coli RNAP that are potentially accessible to DNA-bound activator proteins. To this end, a genetic method will be employed to screen random peptides for those that activate transcription when fused to a DNA-bound protein positioned upstream of a promoter. Genetic and complementary biochemical methods will be used to identify the sites on RNAP that are contacted by these peptides. As well as defining the accessible surfaces of RNAP, these artificial peptide activators may prove useful as biological reagents in their own right. Specifically, they will be used to generate a panel of in vivo inhibitors that may facilitate the identification of activation target sites for natural activators in E. coli. These studies should provide a view of the surface of E. coli RNAP as seen by transcriptional activators. In addition to offering new insight into the interactions of RNAP with regulators that control its function, the results of these investigations should be generally relevant to the study of protein-protein and peptide-protein interactions.
