Bacteriophage 933W repressor protein binding and autoregulation within the immunity region

Lambdoid bacteriophages encode a phage specific repressor protein that establishes and maintains the phage's lysogenic state by repressing transcription of genes needed for lytic growth while also activating transcription of its own gene. This repressor protein regulates gene expression at appropriate promoters by differentially binding multiple repressor-binding sites located in two operator regions of the phage, OR and OL. In this thesis, operator site binding and DNA recognition by the repressor of lambdoid phage 933W, a prophage within the Shiga toxin-encoding E. coli strain EDL933, was examined. Homology modeling of the 933W repressor-DNA complex, together with gel mobility shift and DNA footprinting analysis of altered DNA operator sites, was used to define the DNA specificity determinants for 933W repressor binding. These determinants are located at positions 2-5 in each operator half site. Additionally, 933W repressor's intrinsic affinities for individual natural operator sites are similar to those previously determined affinities for these sites in the context of the intact operators. This result indicates that 933W repressor does not cooperatively bind adjacent operator sites.;In the well studied lambda bacteriophage, cooperative interactions between repressor dimers are necessary for regulating binding at operator sites in OR and OL as well as for forming an octamer between the two operator regions through a DNA loop structure. Ultimately, cooperative binding is necessary for autoregulation of lambda repressor expression. Unlike lambda and other lambdoid phages, 933W lacks an O L3 site and therefore is hypothesized not use a long range loop for repressor autoregulation. The results of in vivo footprinting and quantitative RT-PCR showed that OR3 occupancy and PRM repression in 933W is independent of the presence of the OL region, suggesting this phage evolved in a manner different from lambda phage. Analysis of repressor-mediated in vitro transcription of the 933W OR promoters suggests how this phage is able to properly autoregulate repressor gene expression in absence of cooperative binding. It appears 933W phage takes advantage of close intrinsic repressor-operator site affinities appropriately regulate transcription from PRM and therefore maintain stable, yet inducible, lysogenic growth.