In vitro RNA polymerase interaction with a restriction fragment containing the Escherichia coli origin of replication

The interaction of RNA polymerase with a restriction fragment containing the origin of Escherichia coli replication (oriC) was examined by methods used to investigate transcription promoter activities. Interactions of RNA polymerase with oriC were determined and characterized by agarose gel exclusion under conditions of polymerase binding and RNA synthesis initiation. These interactions were further demonstrated and defined by nitrocellulose retention experiments under various reaction conditions. The binding of RNA polymerase to the oriC fragment was compared to binding to the tetracycline promoter (tet), a known strong promoter of transcription. Specific localization of the RNA polymerase-oriC interaction was determined by restriction protection experiments. The binding of RNA polymerase was determined to be located near the HindIII site of oriC. These methods allowed the observation and characterization of a specific association of RNA polymerase with the origin of E. coli DNA replication.