Looping probability as a function of loop length and sequence.

<p>(<b>A</b>) Schematic of the tethered particle motion (TPM) assay for measuring looping. In TPM, a bead is tethered to the surface of a microscope coverslip by a linear DNA. The motion of the bead serves as a readout for the state of the tether: if the DNA tether contains two binding sites for a looping protein such as the Lac repressor, and the looping protein is present and binds both sites simultaneously, forming a loop, the motion of the bead is reduced in a detectable fashion <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075799#pone.0075799-Schafer1" target="_blank">[47]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075799#pone.0075799-Nelson2" target="_blank">[50]</a>. The motion of the bead is observed over time, and the looping probability for a particular DNA is defined as the time spent in the looped (reduced motion) state, divided by the total observation time. (<b>B</b>) Schematic of the “no promoter” (left) and “with-promoter” (right) constructs used in this work. “Loop length” is defined as the inner edge-to-edge distance between operators (excluding the operators themselves, but including the promoter, if present). (<b>C</b>) Looping probabilities for the five sequences described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075799#pone-0075799-t001" target="_blank">Table 1</a>, without the bacterial <i>lac</i>UV5 promoter sequence as part of the loop. (<b>D</b>) Looping probabilities for the same five sequences but with the promoter sequence in the loop. Righthand panels in (C) and (D) show the same data as lefthand panels, except magnified around loop lengths 100–110 bp. The five sequences do not all share the same maxima of looping (colored arrows), not even the TA and 5S sequences that share similar sequence features (see Figures S1 and S2 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075799#pone.0075799.s001" target="_blank">File S1</a>), though each sequence has the same maximum with and without the promoter (as far as can be determined with the current data; note that the with-promoter maximum for the TA sequence could be at 105 or 106, as those points are within error). All E8 and TA data (in particular, those outside of the 101–108 bp range) were previously described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075799#pone.0075799-Johnson1" target="_blank">[35]</a>.