Disruptive effect of tension on protein-mediated DNA looping

DNA loops form when a single protein or protein complex binds to multiple sites on the same DNA molecule. These loops can affect the binding and activity of RNA polymerase (RNAP) and thus affect gene regulation. DNA loop formation is intrinsically related to DNA's structural properties and any mechanical constraints that it is subjected to. This thesis focuses on the particular relationship between DNA looping and tension in the substrate DNA. Both theoretical and experimental techniques are used to develop a quantitative understanding of how tension can disrupt DNA looping and thereby affect gene regulation.; By modeling DNA as a wormlike chain, calculations are presented for how continuous stretching of the substrate DNA affects the loop formation probability. It is found that when the loop size is greater than 100bp, a tension of 500 femtonewtons can increase the time required for loop closure by two orders of magnitude. Thus, it appears that tension can act as a molecular switch that controls the powerful processive motion of RNAP. In particular, since RNAP can exert forces over 20 pN before it stalls, a 'DNA tension switch' can affect transcriptional regulation by offering a mechanical advantage of two orders of magnitude. The theoretical results provide new perspective on existing research and offer a new paradigm of mechano-chemical gene regulation to explore.; The experimental section of the thesis provides a detailed description of a three-dimensional stroboscopic tethered particle microscopy technique that was developed in order to explicitly measure the disruptive effect of tension on DNA looping. In this technique, a sub-micron sized microsphere is attached to a coverslip by a single DNA molecule. The motion of the microsphere is indicative of the length of the DNA tether. In this way, DNA looping can be observed because looping results in a decrease in the effective length of the DNA tether. Novel data analysis is presented that distinguishes well-formed DNA tethers from defective ones for which the motion is dominated by aberrant surface effects. Preliminary measurements of the rate of lacR-mediated DNA looping are presented and an analysis of error sources is made.