Contacts between the lac repressor and the thymines in the lac operator.

Abstract

Important points of contact between the lac repressor and the lac operator [from Escherichia coli] were identified by crosslinking the repressor to bromouracil-substituted operator. Bromouracils were substituted for thymines in a 55-base-long restriction fragment containing the lac operator and 1 or the other 5'' end was labeled with 32P. UV irradiation of this fragment produced single-strand breaks at the bromouracils. Breakage at each bromouracil in the sequence was examined by denaturing the DNA and displaying the UV-generated fragments on a polyacrylamide gel. In the presence of lac repressor, UV radiation failed to break at specific sites. This was attributed to a competing reaction in which the DNA crosslinks to the repressor rather than breaking. These crosslinkable sites thus define positions at which the lac repressor protein lies close to the methyl group of a thymine in the major groove of DNA.