Studies on gene control regions XII. The functional significance of a Lac operator constitutive mutation

Abstract

The functional significance of a lac operator constitutive mutation has been determined. The transition adenine-thymine to guanine-cytosine was shown to be a constitutive mutation simply because thymine contains the functionally important 5-methyl group whereas cytosine does not. The remainder of the base pair is of no consequence. The experimental approach was to synthesize various modified operators containing cytosine, 5-methyl-cytosine, and 5-bromocytosine. The synthetic operator containing a guanine-cytosine base pair displays an eightfold reduction in stability with lac repressor whereas the operator containing 5-methylcytosine binds repressor at least as tightly as does the wild type sequence. Results published previously have shown that a similar decrease in stability of the repressor-operator complex can be obtained simply by substituting uracil for thymine or by inverting the base pair to thymine-adenine. All these results taken together implicate the thymine 5-methyl as the only important functional group recognized by the lac repressor at this base pair. Further confirmation of this conclusion was obtained by substitution of 5-bromocytosine and 5-bromouracil at this base pair. Both altered the stability of the repressor-operator complex by about the same percent suggesting that the bromine atom was the important determinant of complex stability for 5-bromopyrimidine analogs.