New Insights into the Phage Genetic Switch: Effects of Bacteriophage Lambda Operator Mutations on DNA Looping and Regulation of P_R, P_L, and P_RM.

ABSTRACT: One of the best understood systems in genetic regulatory biology is the so-called "genetic switch" that determines the choice the phage-encoded CI repressor binds cooperatively to tripartite operators, O_L and O_R, in a defined pattern, thus blocking the transcription at two lytic promoters, P_L and P_R, and auto-regulating the promoter, P_RM, which directs CI synthesis by the prophage. Fine-tuning of the maintenance of lysogeny is facilitated by interactions between CI dimers bound to O_R and O_L through the formation of a loop by the intervening DNA segment. By using a purified in vitro transcription system, we have genetically dissected the roles of individual operator sites in the formation of the DNA loop and thus have gained several new and unexpected insights into the system. First, although both O_R and O_L are tripartite, the presence of only a single active CI binding site in one of the two operators is sufficient for DNA loop formation. Second, in P_L, unlike in P_R, the promoter distal operator site, O_L3, is sufficient to directly repress P_L. Third, DNA looping mediated by the formation of CI octamers arising through the interaction of pairs of dimers bound to adjacent operator sites in O_R and O_L does not require O_R and O_L to be aligned "in register", that is, CI bound to "out-of-register" sub-operators, for example, O_L1~O_l2 and O_R2~O_R3, can also mediate loop formation. Finally, based on an examination of the mechanism of activation of P_RM when only O_R1 or O_R2 are wild type, we hypothesize that RNA polymerase bound at P_R interferes with DNA loop formation. Thus, the formation of DNA loops involves potential interactions between proteins bound at numerous cis-acting sites, which therefore very subtly contribute to the regulation of the "switch".

SUBMITTER: Lewis DEA 

PROVIDER: S-EPMC7757127 | biostudies-literature | 2016 Nov

REPOSITORIES: biostudies-literature