The Escherichia coli bacteriophage lambda has played a crucial role in the development of molecular biology. Today it continues to serve as a paradigm for many diverse biological processes, including regulation of transcription and developmental biology. Several major early promoters are critical for phage (lambda) DNA replication and for the regulation of the """"""""lysis-versus-lysogenization"""""""" developmental checkpoint. Our studies have revealed that activity of the lambda PR promoter, required for normal phage DNA replication, is severely inhibited in the presence of the RNA polymerase allosteric effector ppGpp, which accumulates in the host cell under conditions of nutritional stress. We have also shown that the activity of PR is dramatically stimulated by DnaA, the host replication initiator protein. It is interesting that DnaA binding, which leads to stimulation of PR, occurs atypically downstream of the transcription start point in the initial transcribed sequence. Furthermore, DnaA transcriptional activation of PR requires interaction with the RNA polymerase - subunit. The efficiency of establishing lysogeny, i.e., viral latency, following infection is found to be highly dependent on the host's intracellular levels of the stress signal effector, ppGpp. Apparently, the phage surveys ppGpp levels as an indicator of the host cell's physiological state. The activity of the three promoters, PE, PI, and PaQ, collectively determine the efficiency of lysogenization. We find that the activities of each of these promoters are differentially regulated by ppGpp. We propose here a systematic investigation of the regulation of transcription from lambda promoters: PR, PE, PI, and PaQ by ppGpp and/or DnaA. Both in vivo and vitro studies are planned. These will begin with genetic screening for mutant promoters with altered responsiveness to ppGpp and/or DnaA followed by detailed characterization by in vitro transcription assays. In addition, already isolated and B RNA polymerase mutants that mimic or bypass ppGpp-dependent functions will be used to characterize the physiological regulation of the lambda major early promoters both in vivo and in vitro. These investigations are aimed at discovering aspects of regulation of phage lambda development by ppGpp and DnaA, as model environmentally responsive and general gene regulators. How these host effectors modify the life cycle of the bacteriophage lambda and why, is a long-term objective of this proposal.
