Relevance - The development and health of cellular organisms is dependent on the proper functioning of large, complex gene regulatory networks that orchestrate the appropriate combinations of genes to be expressed in each cell. Our ability to understand, predict and manipulate the behaviour of these networks - an ability that will underpin the next century of medicine - is currently very limited. The approach of this project is to examine well characterized and experimentally accessible simple model organisms - viruses that infect bacteria - to uncover fundamental principles of gene network function. Project Summary - This project extends our studies of the ability of the gene regulatory networks of two unrelated but similarly functioning temperate bacteriophages, lambda and 186, to maintain and make efficient transitions between alternative, stable expression states: lysis and lysogeny. Here we focus on the contribution of transcriptional interference (Tl) to decisiveness in these networks. Tl is the interference of one transcriptional process on another in cis, for example, inactivation through collisions of converging RNA polymerases. Tl is a poorly understood gene regulatory mechanism present in all organisms, and is associated with human disease. In 186, Tl between closely spaced convergent promoters and the relief of that Tl by repression of one of the promoters provides positive feedback and contributes to switching between expression modes. We wish to test the generality of this mechanism. In both phages, the choice to enter lysogeny may also involve Tl between a pair of converging promoters 300 bp apart. We will test whether Tl provides ultrasensitivity, an all-or-nothing character, to this interaction and contributes to decisiveness. This mechanism will also be examined by construction and analysis of synthetic switch circuits. The behavior of these transcriptional circuits will be studied using in vivo reporter constructs in which circuit components are manipulated genetically or biochemically. Two basic mechanisms of Tl, removal of initiation intermediates at the promoter and collisions of elongating polymerases, will be examined in further detail by biochemical and reporter analyses of a range of two-promoter constructs. The data will be used to test and extend our current mathematical model of Tl in prokaryotes, with the hope of providing a foundation for the investigation of this phenomenon in more complex organisms.
