8–19 Detailed analysis of the regulatory region of the Lac operon has revealed surprising complexity. Instead of a single binding site for the Lac repressor, as might be expected, there are three sites termed operators: O1, O2, and O3, arrayed along the DNA as shown in Figure Q8–7. To probe the functions of these three sites, you make a series of constructs in which various combinations of operator sites are present. You examine their ability to repress expression of β-galactosidase, using either tetrameric (wild type) or dimeric (mutant) forms of the Lac repressor. The dimeric form of the repressor can bind to a single operator (with the same affinity as the tetramer) with each monomer binding to half the site. The tetramer, the form normally expressed in cells, can bind to two sites simultaneously. When you measure repression of β-galactosidase expression, you find the results shown in Figure Q8–7, with higher numbers indicating more effective repression.
concentration of X
Figure Q8–6 Perturbations of a bistable system (Problem 8–18). As shown by the green lines, after perturbation 1 the system returns to its original stable state (green dot at left), and after perturbation 2, the system moves to the other stable state (green dot at right). Perturbation 3 moves the system to the precise boundary between the two stable states (orange dot).
A. Which single operator site is the most important for repression? How can you tell?
b.
Do combinations of operator sites (Figure Q8–7, constructs 1, 2, 3, and 5) substantially increase repression by the dimeric repressor? Do combinations of operator sites substantially increase repression by the tetrameric repressor? If the two repressors behave differently, offer an explanation for the difference.
C.
The wild-type repressor binds O3 very weakly when it is by itself on a segment of DNA. However, if O1 is included on the same segment of DNA, the repressor binds O3 quite well. How can that be?
Figure Q8–7 Repression of β-galactosidase by promoter regions that contain different combinations of Lac repressor binding sites (Problem 8–19). The base-pair (bp) separation of the three operator sites is shown. Numbers at right refer to the level of repression, with higher numbers indicating more effective repression by dimeric (2-mer) or tetrameric (4-mer) repressors. (From S. Oehler et al., EMBO J. 9:973–979, 1990. With permission from John Wiley and Sons.)
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