Hybrid tetramers of native and core lactose repressor protein. Assessment of operator and nonspecific DNA binding parameters and their relationship.
Hybrid tetramers of lac repressor and its trypsin-resistant core protein were produced by mild proteolytic digestion and isolated by chromatography on phosphocellulose. These tetramers were used in binding studies to probe the relationship of the NH2 terminus and core domains in operator DNA binding and to explore subunit participation in both operator and nonspecific DNA binding. The purity of each tetramer was demonstrated by several lines of evidence, including physical characterization and measurement of the binding activities of the hybrid tetramer preparations. Each tetramer displayed measurable operator binding activity, and the dissociation constants of the tetramers for lambdaplac DNA and a 29 base operator fragment increase with the loss of each NH2 terminus. These studies strongly suggest that each NH2 terminus of the repressor interacts with the operator DNA, although the four NH2 termini do not appear to make equal contacts. The contributions of the NH2 terminus to operator binding do no appear to be identical with those for nonspecific DNA binding. In addition, the binding of the NH2 termini to nonspecific DNA can be described by postulating four identical, noninteracting sites. Although the rate of dissociation of the repressor-operator is increased by a factor of 2 upon removal of each NH2 terminus, calculations using the measured dissociation rate and equilibrium dissociation constant indicate that the rate of association of repressor to operator is decreased approximately 5-fold. These data suggest that the contacts made by the NH2 termini may greatly facilitate association with DNA. Competition studies of nonspecific DNA with operator DNA binding confirm the existence of two operator DNA binding sites on each tetramer and suggest that the contacts of a single NH2 terminus are not identical for the two operator binding sites.
D001667:Binding, Competitive; D004247:DNA / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D046911:Macromolecular Substances; D009876:Operon; D011485:Protein Binding; D055503:Protein Multimerization; D012097:Repressor Proteins / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*
D046911:Macromolecular Substances; D012097:Repressor Proteins; D014157:Transcription Factors; D004247:DNA