BIOCHEMICAL AND BIOPHYSICAL STUDIES OF THE LACTOSE REPRESSOR AND OPERATOR DNA
Doctor of Philosophy
Lactose repressor from Escherichia coli regulates the expression of the lac operon. The protein binds specifically to the operator region and nonspecifically to the rest of genome. The repression is released upon addition of inducer. Modification of repressor with tetranitromethane results in loss of both operator and nonspecific DNA binding activity with maintenance of most of the inducer binding capacity. The tyrosine residues modified are primarily tyrosines 7 and 17. These two residues may be involved in or close to the operator binding site. Modification of core protein also results in loss of operator DNA binding, though to a lesser extent. The charge on the nitro-tyrosine at neutral pH apparently prohibits operator DNA binding. The presence of nonspecific DNA protects tyrosine residues from modification and prevents loss of operator DNA binding activity. Reduction of nitro-tyrosine to amino-tyrosine by sodium dithionite restores the neutral charge; and the amino group does not influence nonspecific DNA binding, but interference with operator DNA binding is observed. Steric restraints at tyrosines 7 and 17 are apparently more stringent for specific vs nonspecific DNA binding. Modification of the protein with dansyl chloride results in loss of operator DNA binding at low molar ratio of reagent/monomer. Loss of nonspecific DNA binding can be observed at higher molar excess, while IPTG binding is not affected. Lysine residues are the only modified amino acids detectable. Four DNS-lysine peptides are observed by high pressure liquid chromatography: one residue modified is in the amino terminus; the other three are in the core region. Energy transfer occurs between a DNS incorporated in the core region and tryptophan 201 in the repressor. Cross-linking studies have been used to explore the contact sites between the repressor and the operator DNA fragment. Cross-linking is observed between p-azidophenacyl bromide-modified repressor and operator DNA fragment. However, analysis of the specific bases involved was not possible. No cross-linking can be observed using p-azidophenylglyoxal modified repressor and operator DNA. Using depurinated operator DNA fragments, effects observed appear due largely to nonspecific DNA binding.