A central question of molecular biology is how regulatory proteins recognize specific nucleic acid sequences, and the lactose repressor has long been a prime example of such a regulatory protein. The lactose repressor interaction, with the lactose operator and with DNA in general, will be analyzed by genetic and biochemical means. Repressor mutants with specificities altered so as to bind tightly to altered (oc) operators will be obtained by a novel dual selection scheme. Altered-specificity mutantions in lacI, as well as existing weak- and tight-binding mutations will be sequenced to map out the regions of the primary protein structure which determine DNA binding. Purified repressor headpiece (residues I-59) and core (residues 60-360) fragments will be purified from partial tryptic digests and tested for specific operator binding. The headpice test will use pure operator DNA fragments and the standard filter-binding system. The core test will utilize chromatography through operator-DNA cellulose columns. We will attempt to clone and express fragments of the lac1 gene encoding the headpiece and core domains of the repressor. If lac operon repression results, we will select for mutations which enhance repression by headpiece or by core. The levels of expression of lacI headpiece genes and the in vivo stability of the products will be determined. Mutant repressors will be tested for their binding affinities against a panel of oc operators, and the rate studies will be used to look for changes in the diffusional properties of the altered repressors. Large amounts of headpiece fragment from wild-type and selected mutant repressors will be prepared for crystallization trials.