J. Mol. Biol. (1991) 222, 479-494 RNA Polymerase Bound to the PR Promoter of Bacteriophage h Inhibits Open Complex Formation at the Divergently Transcribed PRM Promoter Implications for an Indirect Mechanism of Transcriptional Activation by 3, Repressor Pamela A. Hershberger and Pieter L. deHasetht Department of Biochemistry School of Medicine, Case Western Reserve University Cleveland, OH 44106, U.S.A. (Received 4 March 1991; accepted 26 July 1991) We demonstrate that RNA polymerase bound at the PRpromoter of bacteriophage lambda can repress transcription initiation from the divergently transcribed PRM 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 Pa result in a significant increase in the rate of formation of transcriptionally competent complexes at the PRM 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 Pa sequencesmediate PRMrepression. From these assayswe were able to conclude that the formation of an open complex at the Pa promoter did not exclude RNA polymerase from binding at PRM. Rather, initiation at PRM was impaired because closed complexes must isomerize in the presence of an open complex already situated at the Pa promoter. Extensive evidence has been obtained previously indicating that lambda repressor activates transcription directly by contacting RNA polymerase situated at the PRMpromoter. Results presented here raise the possibility that an additional mechanism could be operative, whereby lambda repressor indirectly activates PRM transcription by excluding RNA polymerase from the Pz promoter. Keywords: bacteriophage A; indirect activation; genetic switch; Pa and PRM promoters; polymerase dimers 1. Introduction which the repressor protein is not synthesized, RNA Bacteriophage ;1 may establish either a lytic or lysogenic infection in Escherichia coli. Which of the two pathways is selected depends upon a complex interplay of phage-encoded and host proteins. Ultimately, the developmental decision is imple- mented at the rightward control region of the phage genome (for a review, see Ptashne, 1986). Located within the control region are the divergently oriented Pz and Pa, promoters and the three opera- tor regions to which the regulatory proteins ,? repressor and Cro bind (see Fig. 1). Phage develop- ment is governed by which of these promoters is active upon bacterial infection. Under conditions in polymerase preferentially utilizes the Pz promoter. This results in the synthesis of Cro and other pro- teins required for the establishment of a lytic infec- tion. If, however, repressor is present it binds with highest affinity to OR1 (Johnson et al., 1979). Since the sequences of the Pa promoter and the repressor binding site overlap in this region (see Fig. l), the binding of repressor excludes RNA polymerase from Pa (Hawley et al., 1985) thereby blocking lytic development. Due to co-operative protein-protein interactions between repressor dimers, Oa2 fills concomitantly with Oal (Johnson et al., 1979). Protein dimers bound at 0,2 are believed to directly contact RNA polymerase situated at the PRM t Author to whom all correspondence should be addressed. 0022%283(i/91/230479-16 003.00/O promoter (Hochschild et al., 1983). It is thought that PRM transcription is activated by this inter- action (Meyer & Ptashne, 1980). 479 c 1991 Academic Press Limited