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Binding of synthetic lactose operator DNAs to lactose repressors

1977; National Academy of Sciences; Volume: 74; Issue: 8 Linguagem: Inglês

10.1073/pnas.74.8.3292

1091-6490

David V. Goeddel, Daniel G. Yansura, Marvin H. Caruthers,

Advanced biosensing and bioanalysis techniques

The nitrocellulose filter assay was used to study the interactions of wild-type (SQ) and tight-binding (QX86) lac repressors with synthetic lac operators 21 and 26 base pairs long. The repressor binding properties of both operators were very similar, indicating that both contain the same specific repressor recognition sites. The repressor-operator association rate constants ( k a ) were more sensitive than dissociation rate constants ( k d ) to changes in ionic strength. The responses of both k a and k d to ionic strength were relatively small compared to the effects previously observed with λh80 dlac as operator DNA. These results suggest that under natural conditions there are electrostatic interactions between lac repressor and DNA regions outside of the 26 base pair operator sequence. Association rate constants for SQ repressor with either operator are higher than have been predicted for diffusion-limited reactions. We postulate that long-range electrostatic attractions between repressor and operator accelerate the association reaction. The presence of nonoperator DNA decreased association rate constants, the effect being more noticeable at an ionic strength of 0.05 M than at 0.20 M. Nonoperator DNA reduced k a values for associations involving QX86 repressor to a greater extent than for those with SQ repressor. The two types of repressors also had different rate constants for interactions with synthetic operators. The values for k a and k d were both higher with SQ repressor than with QX86 repressor. However, the rate constants were more sensitive to ionic strength when the repressor used was QX86.