RNA polymerase bound to the P R promoter of bacteriophage λ inhibits open complex formation at the divergently transcribed P RM promoter : Implications for an indirect mechanism of transcriptional activation by λ repressor

Abstract

We demonstrate that RNA polymerase bound at the P R promoter of bacteriophage lambda can repress transcription initiation from the divergently transcribed P RM promoter in vitro. Using abortive initiation and run-off transcription experiments we show that inactivating mutations introduced into either the −10 or −35 regions of P R result in a significant increase in the rate of formation of transcriptionally competent complexes at the P RM promoter. This is due primarily to an increase in the rate constant for the isomerization of closed to open complexes. Gel shift and DNase I footprinting experiments were employed to further define the mechanism by which P R sequences mediate P RM repression. From these assays we were able to conclude that the formation of an open complex at the P R promoter did not exclude RNA polymerase from binding at P RM. Rather, initiation at P RM was impaired because closed complexes must isomerize in the presence of an open complex already situated at the P R promoter. Extensive evidence has been obtained previously indicating that lambda repressor activates transcription directly by contacting RNA polymerase situated at the P RM promoter. Results presented here raise the possibility that an additional mechanism could be operative, whereby lambda repressor indirectly activates P RM transcription by excluding RNA polymerase from the P R promoter.