Autorepressor properties of the pi-initiation protein encoded by plasmid R6K

Abstract

A DNA fusion containing the promoter of the pir gene of plasmid R6K that encodes for the pi-initiation protein and the beta-galactosidase gene of Escherichia coli (lacZ) is described. The synthesis of beta-galactosidase promoted by this pir-lac fusion was almost completely inhibited when an R6K sequence containing the pir gene was provided in trans in E. coli. Transcription in vitro from the pir promoter but not the trp promoter of E. coli, was inhibited by purified pi protein indicating that the pi protein alone is responsible for repression of its own gene and that the effect is promoter specific. The DNA-protein interaction sites in the pir regulatory region have been determined for the pi protein and E. coli RNA polymerase using the DNase I protection method. The binding sites for these two proteins overlap for three helical turns. Competition DNA binding experiments show that the pi protein will displace bound RNA polymerase. From these studies we conclude that repression of the pir gene is accomplished by binding of the pi protein and this association blocks access of RNA polymerase to the pir promoter region.