Quantitation of interactions between two DNA loops demonstrates loop domain insulation in E. coli cells.

Eukaryotic gene regulation involves complex patterns of long-range DNA-looping interactions between enhancers and promoters, but how these specific interactions are achieved is poorly understood. Models that posit other DNA loops--that aid or inhibit enhancer-promoter contact--are difficult to test or quantitate rigorously in eukaryotic cells. Here, we use the well-characterized DNA-looping proteins Lac repressor and phage λ CI to measure interactions between pairs of long DNA loops in E. coli cells in the three possible topological arrangements. We find that side-by-side loops do not affect each other. Nested loops assist each other's formation consistent with their distance-shortening effect. In contrast, alternating loops, where one looping element is placed within the other DNA loop, inhibit each other's formation, thus providing clear support for the loop domain model for insulation. Modeling shows that combining loop assistance and loop interference can provide strong specificity in long-range interactions.

Aspects of protein-DNA interactions: a review of quantitative thermodynamic theory for modelling synthetic circuits utilising LacI and CI repressors, IPTG and the reporter gene lacZ.

Protein-DNA interactions are central to the control of gene expression across all forms of life. The development of approaches to rigorously model such interactions has often been hindered both by a lack of quantitative binding data and by the difficulty in accounting for parameters relevant to the intracellular situation, such as DNA looping and thermodynamic non-ideality. Here, we review these considerations by developing a thermodynamically based mathematical model that attempts to simulate the functioning of an Escherichia coli expression system incorporating two of the best characterised prokaryotic DNA binding proteins, Lac repressor and lambda CI repressor. The key aim was to reproduce experimentally observed reporter gene activities arising from the expression of either wild-type CI repressor or one of three positive-control CI mutants. The model considers the role of several potentially important, but sometimes neglected, biochemical features, including DNA looping, macromolecular crowding and non-specific binding, and allowed us to obtain association constants for the binding of CI and its variants to a specific operator sequence.