PHYSICAL AND CHEMICAL STUDIES OF THE LACTOSE REPRESSOR AND ITS INTERACTION WITH THE OPERATOR DNA

[摘要] The lactose repressor protein binds specifically to the operator region of Escherichia coli DNA, physically blocking the transcription of the genes coding for the lactose catabolic enzymes. Initial work with tryptic digestion of repressor to produce a tetrameric core protein (inducer binding) and four NH(,2)-terminal peptides (DNA binding) suggested contributions of the protein regions to ligand interactions. Chemical studies, however, suggested that an alteration of this view was required. At low molar ratios of N-bromosuccinimide (NBS) to repressor, cysteine oxidation caused loss of operator DNA binding activity with simultaneous retention of inducer and nonspecific DNA binding activities. The region surrounding cysteine 107 was found to be influential in maintaining intact operator DNA binding function in repressor. Matthews demonstrated specific, inducible DNA binding affinity in core fragment by direct binding studies (Matthews (1979) J. Biol. Chem. 254, 3348). In light of this activity, application of methylation protection techniques using core protein fragment were of interest. The modification pattern with core protein contained components of the uninduced repressor-operator methylation pattern reported previously. These similarities in the pattern were restricted to the central, asymmetric region of the operator sequence. The effects of inducer and anti-inducer binding to both core and repressor were reflected by changes in the methylation patterns (the anti-inducer-repressor pattern has not been published previously). Exonuclease III digestion was used to probe the 3;;-boundary of DNA-protein complexes, and DNase I treatment to probe the susceptibility to digestion of the operator region in the intact complex. Exposure to exonuclease III in the presence of the tryptic core and intact repressor yielded different 3;;-boundaries for the complexes formed. The tryptic core protein protected the operator region from DNase I digestion, though to a lesser degree than repressor and in a more restricted region of the operator DNA. The effects of sugar binding on the availability of DNA to enzymatic action for both complexes were consistent with the expected alterations in affinity. A model for core and repressor binding to operator DNA is proposed.

[发布机构] Rice University