DETERMINANTS OF SPECIFICITY IN THE LACTOSE REPRESSOR

乳糖抑制因子与乳糖操作因子的相互作用

The interaction of the lactose repressor with the lactose operator, and with DNA in general, will continue to be analyzed by genetic and biochemical means. Repressor mutants with specificities altered so as to bind tightly to altered (0-c) operators will be obtained by a novel dual selection scheme using symmetric wild-type and 0-c operators. Altered-specificity mutations in lacI will be sequenced to map out the regions of the primary protein structure which determine DNA binding. To test predictions of the helix-turn-helix model for protein recognition of DNA, specific amino acid substitutions in the lac repressor will be made using directed mutagenesis of lacI with synthetic DNA fragments. Portions of the headpiece region of the gene will be chemically synthesized to introduce base pair changes that create unique restriction sites. Small segments of the gene will then be removed and replaced with duplexes carrying known mutations. The stability of each mutant repressor, as well as its affinity for wild-type operator will be assayed in vivo and in vitro. Amino-terminal headpieces from selected repressors will be examined for specific binding to wild-type mutant operators in vitro.