Designed hyperstable Lac repressor.DNA loop topologies suggest alternative loop geometries.

Abstract

Lac repressor (LacI) forms DNA loops which are critical for efficient operator binding and transcriptional repression. Designed DNA loops formed on three constructs with lac operators bracketing phased A-tract bends were characterized by mobility shift, footprinting, and DNA cyclization and topology. Operator dyad axes point either in or out relative to the sequence-induced curvature. Possible conformations suggested from X-ray structures of LacI and LacI.DNA include "wrapping away" (WA), "simple loop" (SL), and "wrapping toward" (WT) models. The WA loop should be preferentially stabilized by the outward operators, the SL and WT loops by the inward operators. Competition experiments demonstrated increased loop stability for all the bent constructs, with the SL/WT construct supporting hyperstable loops (t1/2 of days). This offers a general approach to stabilizing multi-protein DNA complexes on short DNA. DNA cyclization of loops gave minicircle products with altered topologies. WA constructs afforded relaxed and positive topoisomers, and cyclization kinetics indicated slow interconversion of precursors to the two topoisomers. The SL/WT construct gave a relaxed topoisomer, with a small amount of negative supercoil. These results suggest that while it is possible to force the WA loop to form (as in a model proposed from the LacI.DNA structure), the most stable loop geometry is different, probably a U-shape around an extended LacI tetramer. The topological results show how a protein-induced positive supercoil can be reconciled with LacI's preference for binding negatively supercoiled DNA. We suggest that looping proteins can affect the assembly of subsequent proteins by controlling loop topology.