Effects of macromolecular crowding on protein-mediated looping in DNA

The looping of DNA is a phenomenon that is ubiquitous throughout all kingdoms of life, and has been studied extensively under laboratory conditions. However, the interior of cells is crowded. Currently, there is no method to directly observe the looping of a single DNA molecule inside a cell, and it is exceedingly difficult to stably change the concentration of crowders in a living cell. By adding macromolecular crowders to a microchamber we are able to get a better picture of how DNA behaves inside the crowded cell. We have investigated the crowding effects of Dextran 70 (Dx70), as well as bovine serum albumin (BSA), at various concentrations, on protein-mediated DNA looping through tethered particle motion experiments, using 831 bp-long DNA and the lac repressor protein (LacI). We found that the probability of LacI-mediated DNA looping is reduced by Dx70 which is consistent with the notion that Dx70 stiffens DNA. We also found that a larger concentration of Dx70 was required to decrease looping probability at 200 mM KCl than were necessary for 100 mM KCl. Instead, BSA increased looping, unless Mg2+ was added which decreased looping probability. These observations show that looping is affected strongly by the choice of crowder and ionic conditions.