Cooperative DNA binding by CI repressor is dispensable in a phage λ variant

Abstract

Complex gene regulatory circuits contain many interacting components. In principle, all of these components and interactions may be essential to the function of the circuit. Alternatively, some of them may be refinements to a simpler version of the circuit that improve its fitness. In this work, we have tested whether a particular property of a critical regulatory protein, CI, is essential to the behavior of the phage regulatory circuit. In the lysogenic state, CI represses the expression of the lytic genes, allowing a stable lysogenic state, by binding cooperatively to six operators. A mutant phage lacking cooperativity because of a change in cI could not form stable lysogens; however, this defect could be suppressed by the addition of mutations that altered two cis-acting sites but did not restore cooperativity. The resulting triple mutant was able to grow lytically, form stable single lysogens, and switch to lytic growth upon prophage induction, showing a threshold response in switching similar to that of wild-type . We conclude that cooperative DNA binding by CI is not essential for these properties of the circuitry, provided that suppressors increase the level of CI. Unlike wild-type lysogens, mutant lysogens were somewhat unstable under certain growth conditions. We surmise that cooperativity is a refinement to a more basic circuit, and that it affords increased stability to the lysogenic state in response to environmental variations.cooperativity ͉ evolution of regulatory circuitry ͉ gene regulatory circuit ͉ systems biology ͉ prophage induction C omplex gene regulatory circuits include many components and interactions. It is likely that these features are advantageous and have been selected for during the course of evolution. However, certain features may be refinements to the system rather than essential to its operation. In this view, a complex system might have arisen in a much simpler form, with refinements being added later as the system evolved toward its present form. This idea predicts that existing circuits might tolerate the removal of certain features (perhaps with the addition of suppressors) and still show qualitatively normal behavior. In this work, we have tested this prediction in a well characterized complex circuit, the bistable switch of phage , by removing one feature long believed to be essential for its proper operation.A cell infected with can follow either of two pathways, the lytic or lysogenic pathways. In the lytic pathway, the phage DNA replicates, and Ϸ100 new virions are made and released upon cell lysis. In the lysogenic pathway, the viral genome integrates into the host chromosome, and the lytic genes are repressed by the action of CI protein. This pathway leads to a stable regulatory state, the lysogenic state. In this state, CI binds tightly to two operators in the O R region, O R 1 and O R 2, repressing the expression of the lytic P R promoter and stimulating its own expression from another promoter, P RM (1).Although the lysogenic state is highly stable and can be main...

DNA looping provides stability and robustness to the bacteriophage λ switch

“…The mutant representing cI Y210N prmup-1 O R 2up also formed a stable lysogen for ⌬G NSB ϭ Ϫ2.8 kcal/mol, but cI Y210N prm252 O R 2up did not. These results are in reasonable agreement with those reported by Babić and Little (44). Furthermore, our results support the view that increased lysogenic CI levels could provide an alternative mechanism for maintaining a stable lysogenic state, although this mechanism has apparently not been selected by evolution.…”

“…Our simulations and the experiments of Babić and Little (44) show that a stable lysogen can be obtained in the absence of DNA looping and other cooperative interactions, by raising the level of CI. This observation is perhaps not so surprising, because the stability of genetic switches is predicted to depend exponentially on the expression levels of gene regulatory proteins (48,50).…”

“…In both cases, we removed all cooperative interactions between CI dimers, including looping, and increased the affinity of CI for O R 2 by a factor of 5. For the prm252 mutation, the basal and stimulated P RM transcription rates were increased by factors of 6 and 2.5, respectively, and for the prmup-1 mutation these factors were 10 and 5 (44). With nonspecific binding strength ⌬G NSB ϭ Ϫ2.1 kcal/mol, both these ''mutants'' formed stable lysogens, with CI levels Ϸ3 and Ϸ6 times that of the ''wild-type.''…”

The Lysis-Lysogeny Decision of Bacteriophage 933W: a 933W Repressor-Mediated Long-Distance Loop Has No Role in Regulating 933W P RM Activity

“…A similar tetramer is also formed at the O L 1 and O L 2 sites. The "side-by-side" cooperative binding by a repressor tetramer is prerequisite to forming a stable lysogens (2,20,30).Additional cooperative interactions between two tetramers, each bound to a pair of adjacent operators that are separated by Ͼ2.5 kb, occur in bacteriophages and P22 (13-15, 31). In , a repressor octamer-mediated O L -cI 8 -O R complex forms with the intervening DNA looping out (3,13,15).…”

“…A similar tetramer is also formed at the O L 1 and O L 2 sites. The "side-by-side" cooperative binding by a repressor tetramer is prerequisite to forming a stable lysogens (2,20,30).…”