THE LACTOSE REPRESSOR-OPERATOR DNA INTERACTION: CHEMICAL AND PHYSICAL STUDIES OF THE COMPLEX (MODIFICATION, EQUILIBRIUM, PROTEIN, STOPPED-FLOW, KINETICS)
PEGGY ANNETTE WHITSON, WHITSON, PEGGY ANNETTE (Creator)
The role of basic amino acid residues in the lactose repressor protein has been explored by chemical modification. Inducer binding was increased by lysine reaction, while both DNA binding activities were diminished, although to differing degrees. Modification of arginines decreased DNA binding activities. The extent of lysine modification was affected by the presence of both sugar and DNA ligands. IPTG presence protected lysine 290 from modification and enhanced reaction at lysine 327, suggesting a conformational change consequent to inducer binding. The presence of DNA during reaction protected against DNA binding activity loss, presumably due to steric restriction of reagent access to basic residues essential for DNA binding. Examination of dissociation kinetics of repressor- ('32)P operator DNA complex demonstrated that accurate measurement of the kinetic rate constant requires consideration of the observed equilibrium binding. The dissociation rate constant measured for pLA 322-8 (2.4 x 10('-3) s('-1) in 0.15 M KCl) was less than observed for (lamda)plac and pIQ, each of which contain pseudo-operator sequences, while the rate constant for a 40 bp operator-containing DNA fragment was greater than for pLA 322-8. The ability to form a ternary complex of 2 operators/repressor and the diminished dissociation rates for pseudo-operator-containing DNAs suggest a role for the pseudo-operators in the dissociation process. Kinetic and equilibrium constants for lactose repressor-operator DNA interaction have been examined under various conditions. Salt effects on pIQ and pLA 322-8 binding were consistent with a pre-equilibrium model; the 40 bp fragment, however, did not fit either a pre-equilibrium or a screening controlled mechanism. The number of ionic interactions for repressor binding to pIQ and pLA 322-8 was 8 vs 6 for the repressor-40 bp fragment, indicating that residues beyond the central 40 bases influence ionic contacts. Unusual biphasic temperature dependence was
Thesis, Dissertation, English, 1986
Rice University, 1986