Binding of E.coli lac repressor to non-operator DNA.
It is shown by melting profile analysis of lac repressor-DNA complexes that repressor binds tightly and preferentially (relative to single-stranded DNA) to double-stranded non-operator DNA. This binding stabilizes the DNA against melting and the repressor against thermal denaturation. Analysis of the extent of stabilization and the rate of dissociation of repressor from non-operator DNA as a function of sodium ion concentration shows, in confirmation of other studies,(3,4) that the binding constant (K(RD)) is very ionic strength dependent; K(RD) increases from approximately 10(6) M(-1) at approximately 0.1 M Na(+) to values in excess of 10(10) M(-1) at 0.002 M Na(+). Repressor bound to non-operator DNA is not further stabilized against thermal denaturation by inducer binding, indicating that the inducer and DNA binding sites probably represent separately stabilized local conformations. Transfer melting experiments are used to measure the rate of dissociation of repressor from operator DNA. These experiments show that most of the ionic strength dependence of the binding constant is in the dissociation process; the estimated dissociation rate constant decreases from greater than 10(-1) sec(-1) at [Na(+)] >/= 0.02 M to less than 10(-4) sec(-1) at [Na(+)] </= 0.002 M. Competition melting experiments are used to show that at 0.02 to 0.002 M Na(+) the affinity of lac repressor for various natural DNAs and synthetic double-stranded polynucleotides (including poly[d(m(6)A-T)], which carries a methyl group in the large groove) are approximately independent of base composition, except that the affinity of repressor for poly[d(A-T)] is approximately 2- to 3-fold greater than for the other DNAs tested. The affinity for single-stranded polynucleotides is atleast 50-fold less than for the doublehelical forms.
D001426:Bacterial Proteins / Q000378:metabolism; D001667:Binding, Competitive; D004269:DNA, Bacterial / Q000378:metabolism; D004355:Drug Stability; D004794:Enzyme Repression; D004926:Escherichia coli / Q000378:metabolism*; D007700:Kinetics; D007785:Lactose / Q000378:metabolism; D009691:Nucleic Acid Denaturation; D011089:Polydeoxyribonucleotides; D011485:Protein Binding; D013696:Temperature; D013816:Thermodynamics
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D011089:Polydeoxyribonucleotides; D007785:Lactose