In Silico, In Vitro, and In Vivo Estimation of J-Factors for LAC Repressor-Mediated DNA Loop Formation

Abstract

Transcription is often regulated by regulatory proteins with specific binding sites that can be many kilo-bases away from promoters. This is made possible by long range, DNA looping interactions. According to the “loop domain model”, the separation of promoters and binding sites into separate topological domains can block promoter-regulator communication and this has been shown in bulk, in vitro experiments. However, the efficiency and regulation of long-range DNA looping are not well understood. To investigate looping, simulations, tethered particle motion (TPM) experiments, and measurements of reporter gene repression were used to estimate J-factors (Jloop) for lac repressor-mediated DNA loops. The Jloop values are consistent with previously published experimental results using DNA circularization assays and theoretical Gaussian polymer and Shimada and Yamakawa models. Jloop and repression decrease for the larger loops as expected. Comparison of simulations and TPM experiments enables fine tuning of the simulation. Instead in vitro titrations of DNA looping as a function of protein concentration suggest that loops in vivo operate in a narrow range of effective concentrations of just a few picomolar LacI.