lac repressor forms loops with linear DNA carrying two suitably spaced lac operators.

Abstract

Tetrameric lac repressor may bind to two lac operators on one DNA fragment and induce the intervening DNA to form a loop. Electron microscopy, non-denaturing polyacrylamide gel electrophoresis, and DNase I protection experiments were used to demonstrate such DNA loops, where the distance between the centres of symmetry of the two lac operators varies between 63 and 535 bp. Formation of a DNA loop is favoured by correct phasing of the two lac operators and a low concentration of both components of the reaction. When a large excess of lac repressor over DNA is used, a 'tandem' structure is observed, in which both lac operators are occupied independently by two repressor tetramers. When the concentrations of both lac repressor and lac operator are high, a 'sandwich' structure is observed, in which two DNA molecules are connected by two lac repressor tetramers in trans.

Biological consequences of tightly bent DNA: The other life of a macromolecular celebrity

“…132 This result agrees with the observations by Hsieh et al using the filter binding assay, whose quantitative results are shown in Figure 8. 133 This disagreement in the behavior of the looping free energy as the distance between operators decreases between the in vivo and in vitro experiments is a stark reminder of the challenge of reconciling the in vitro and in vivo pictures of DNA mechanics in general, and proteinmediated looping in specifically.…”

Modeling the Lac repressor-operator assembly: The influence of DNA looping on Lac repressor conformation

“…As we explain below, knowledge of the location of cleavage enhancement or suppression sites can be used to obtain information about the structure of looped DNA in solution. Our analysis of the DNase I footprinting profiles of short, LacRmediated loops (20) indicates that, under the conditions of these experiments, the tetramer adopts the E state.…”

“…Thus, the cutting activity of DNase I should not be affected by loop formation, and, indeed, no enhancement of DNase I cutting is observed for the wild-type O1-O3 loop (29) and the pHK59 loop (20).…”

“…4 Inset shows details of the dependence of ⌬G on N for loops of lengths 150 -170 bp. Experimental measurements of the relative abundance of such loops in gel-mobility assays (20) indicate that the lengths of the most stable loops are 158 and 168 bp and that the lengths of the least stable loops are 153 and 163 bp. The close match of these lengths with the calculated locations of minima and maxima of ⌬G for the P1 E loop strongly suggests that loops of type P1 E are observed in the cited experiment and hence that LacR adopts a f lexible, open conformation in the protein-DNA assembly.…”

“…For four of these, A1, A2, P1, and P2, the LacR is in its V-shaped conformation; for P1 E the LacR has its extended form. Employing a base-pair-level theory of sequence-dependent DNA elasticity, we have calculated the deformational free energy of the DNA in the complex for each type of loop and used the results to relate protein and DNA fine structure to a wide range of heretofore-unconnected experimental observations, including (i) loop stabilities (20,30), (ii) permanganate sensitivity (22), and (iii) DNase I cutting patterns (20). This work provides an understanding of the effects of chain length, base-pair sequence, protein binding, and supercoiling on DNA looping preferences and, in principle, allows one to design new ways to test specific looped structures in the laboratory.…”