The Effects of Tension and Supercoiling on Protein-Mediated DNA Looping

Abstract

Protein-mediated DNA looping is widely found as a topological regulatory mechanism in DNA transcription and other genetic process in vivo. The loop formation probability is mainly regulated by the loop size, protein concentration, supercoiling level of the genomic DNA, and local tension generated from the action of motor protein and enzymes. The supercoiling level varies at different times in cell cycle and likely at different locations in the genome, and a change of the local negative supercoiling level has been known to affect gene expression and regulation. However, little is known about the dynamics of how supercoiling and enzyme-generated tension control gene regulation by affecting DNA looping probability and stability.We present single molecule measurements to study the effects of supercoiling and tension on a loop induced by the lambda repressor (CI protein) binding to two regions of specific sites in bacteriophage lambda DNA. The loop helps keeping the concentration of the repressor at the appropriate level to maintain the quiescent state, while guaranteeing efficient switching to virulence if necessary. Though even the lowest force prevents the wild type 2317 bp-long loop formation in vitro, the formation and breakdown of a 1051bp-long loop was observed under <1pN tension in unwound DNA at physiological CI concentrations. Furthermore, negative supercoiling stabilized the loop against increased tension, in agreement with our previous observation on a 400bp-long loop. The effect of DNA supercoiling on the formation and breakdown of different size loops is important for the quantitative understanding of the physiological role that DNA supercoiling and tension may have on loop-based gene regulation. Since the genome supercoiling level depends on the energy level and health status of a cell, our investigation sheds light on the dependence of some regulatory mechanisms on these two factors.