Figure 1.

LacI-mediated loops trap supercoiling and resist torsion. The formation of plectonemes in an O1-400-O2 construct under 0.25 pN of tension without LacI produced a typical extension-vs-turns curve (black). When LacI was added to the buffer, the maxima of subsequent extension-vs-turns curves were often reduced by ΔZ due to loop formation by LacI and shifted according to the number of trapped supercoils. (A) In this example, after unwinding by -20 turns, an extended plectoneme reduced the extension of the tether (state I) with or without LacI. The relative shift of the maxima of the extension-vs-turns curves acquired with (red) or without (black) LacI while winding from –20 to –2 turns indicates that two negative supercoils were trapped within the loop (state II). After further winding to +2 turns to reach state III, the loop ruptured, and the extension became identical to that without LacI (state IV). Further winding to +20 produced extensions equal to those observed without LacI. (B) Schematic representations of supercoiled configurations compatible with the red extension-vs-turns curve in A. At –20 turns a large plectoneme is formed in which LacI can secure a loop between juxtaposed operators (state I). Winding the tether from –20 to –2 turns, relaxed flanking plectonemic gyres but not those within the LacI-mediated loop, which shifted the maximum of the extension-vs-turns curve from 0 to –2 turns (state II). Further winding to +2 turns produced (+) plectonemes in the flanking DNA, while the plectoneme within the loop remained negatively (–) supercoiled (state III). In this state, the LacI junction sustains maximal torsional stress between oppositely supercoiled DNA segments. When the loop spontaneously ruptured, negative supercoiling within immediately relaxed an equal amount of positive supercoiling in the flanking DNA giving a more extended tether with a smaller plectoneme (state IV). Thereafter, further winding extended the plectoneme and the extension-vs-turns curve superimposed with that of an unlooped tether as a large plectoneme with positive supercoils formed (state V). LacI might secure loops between juxtaposed operators in this configuration that could be detected by a shift in the extension-vs-turns maximum during unwinding. (C) Repeatedly winding and unwinding DNA tethers in the presence of LacI and noting the shifts in the extension-vs-turns curves produced distributions of the number of supercoils trapped by the LacI-mediated loops in tethers under 0.25 (red) or 0.45 (blue) pN of tension. While the distribution of trapped supercoils is symmetric about zero for tethers under 0.25 pN of tension for equal numbers of winding and unwinding cycles, negative supercoils are more often trapped in loops formed at 0.45 pN. Note also that few loops formed without trapping any supercoiling.