NIH Research Festival
Background: One of the best understood systems in genetic regulatory biology is the life-cycles of Bacteriophage Lambda in E. coli. The phage grows lytic or lysogenic during bacterial infection. The prophage state is maintained by differential binding of phage CI repressor to two operators, OL and OR, blocking transcription from two lytic promoters, PL and PR, and auto-regulating the promoter, PRM, which directs CI synthesis. Fine-tuning of prophage state to achieve extreme stability of the lysogenic state is facilitated by interactions between CI bound to OR and OL, and DNA loop formation. We analyzed critical elements of CI-OR/OL interactions that were unknown and contribute to prophage repression. Methods: Using in vitro transcription system, we genetically dissected the roles of individual operator in loop formation and their effects on PL and PR repression. Results: Although OR and OL are tripartite, a single CI binding site in each operator is sufficient for looping. In PL, not in PR, the promoter distal operator site, OL3, is sufficient to directly repress PL. Looping mediated by formation of CI octamers bound to adjacent operator sites in OR and OL does not require OR and OL to be “in register”. Looping enhances PRM activation. Conclusion: The formation of loop does not require full set of interactions between OR and OL as believed previously; Looping and associated repression of the lytic promoters needed for prophage maintenance occur in various sub-optimal states preventing complete de-repression of lytic promoters required for the switching to lytic growth.
Scientific Focus Area: Molecular Biology and Biochemistry
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