Abstract
The dissociation of the repressor-operator complex from a series of negatively supercoiled plasmid DNAs was examined as a function of the sequence context, orientation, and spacing. The plasmids were grouped into four classes, each with common sequence context. The highest dissociation rate constants were observed for the plasmids containing only a single operator (or pseudooperator) sequence, while approximately 10-fold lower rate constants were measured for plasmids with the I gene pseudooperator in conjunction with either the Z gene pseudooperator or the primary operator. Comparison of the behavior of these two classes of plasmids demonstrated the importance of two operator sequences and supported a model of DNA loop formation to stabilize the repressor-operator complex (Whitson, P. A., and Matthews, K. S. (1986) Biochemistry 25, 3845-3852; Whitson, P. A., Olson, J. S., and Matthews, K. S. (1986) Biochemistry 25, 3852-3858; Whitson, P. A., Hsieh, W. T., Wells, R. D., and Matthews, K. S. (1987) J. Biol. Chem. 262, 4943-4946; Krämer, H., Niemöller, M., Amouyal, M., Revet, B., von Wilcken-Bergmann, B., and Müller-Hill, B. (1987) EMBO J. 6, 1481-1491). The third class, with intermediate dissociation rate constants, was comprised of plasmids which contained the primary operator and the higher affinity pseudooperator normally located in the Z gene. Neither the additional presence of the I gene pseudooperator nor the orientation of the primary operator relative to the Z gene pseudooperator significantly affected the dissociation rate constants. The binding characteristics of this group of plasmids demonstrated the essential role of the Z gene pseudooperator in the formation of intramolecular ternary complex and suggested an in vivo function for this pseudooperator. Plasmids containing two primary operator sequences were the class with lowest dissociation rate constants from lac repressor, and minimal effects of salt or spacing on dissociation of this class were observed. These data are consistent with formation of an intramolecular complex with a looped DNA segment stabilized by the combination of increased local concentration of binding sites and torsional stresses on the DNA which favor binding in supercoiled DNA.
Highlights
- for the plasmids containingonly a single operator sequence, while 10-fold lower rate constants were measured forplasmids with theI gene pseudooperator in conjunction with either the Z gene pseudooperator or the primary operator
Sociation rate constants, was comprised of plasmids We have examined the effects of DNA topology on the which contained the primary operator and the higher interaction of the lactose repressor protein and operaDtoNrA
Neither the additional presenocfethe I gene pseudoop- action have indicated significanteffects of salt concentration, erator nor theorientation of the primary operator relative to theZ gene pseudooperator significantly affectedthe dissociation rate constants.Thebinding characteristics of this groupof plasmids demonstrated the essential role of the Z gene pseudooperator in the formation of intramolecular ternarycomplex and sugtemperature, and size and sequence context of the operator region
Summary
Escherichia coli CSH 46 by the method of Rosenberg et al (1977) as pLA322-8(di) 0-4100-0 8200bp, a plasmid dimer of pLA322-8. Time courses were to 42 h, depending on the dissociation rate Analyses of these data were based on the equilibrium binding where possible (Whitson and Matthews, 1986); for the longer time courses, the inducer-sensitive bindingwas subtracted from each time included plasmids containing the Igene pseudooperator in conjunction with thZegene pseudooperator or native operator sequences(pRW1502 [Oi-O,] and pRW1506[Oi-01). These plasmids exhibited 10-fold lower dissociation rate constants point andthe rate determinedfrom a log plotof the curve as described than those withonly a single operator.
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