Stoichiometry of lac repressor binding to nonspecific DNA: three different complexes form

Abstract

The stoichiometry of lac repressor binding to nonspecific DNA was investigated by three different techniques. Four molecules of the fluorescent probe 5,5'-bis(8-anilino-1-naphthalenesulfonate) [bis(ANS)] bind to each repressor subunit with an average dissociation constant of 20 microM. Nonspecific DNA displaces most of this bound bis(ANS), reducing the fluorescence. Titrations of repressor with nonspecific DNA monitored with high [bis(ANS)] (5-15 microM) had end points at 8 base pairs per repressor. Lower [bis(ANS)] (0.1-1 microM) resulted in end points at either 15 or 26 base pairs per repressor, depending on the ionic strength. These end points correspond to complexes containing approximately one, two, or four repressors per 28 base pairs. Boundary sedimentation velocity experiments with saturating amounts of repressor revealed that five repressors can bind to 28 base pairs. By monitoring the circular dichroism as DNA was added to repressor, the sequential appearance of complexes containing approximately four, two, and one repressors per 28 base pairs was observed. The inability of repressor cores or iodinated repressor to bind to complexes containing one or two repressors per 28 base pairs implies that all of the repressors directly contact the DNA in the complex containing four repressors per 28 base pairs. It is proposed that while two subunits of each repressor contact the DNA in complexes containing one or two repressors per 28 base pairs, only one subunit of each repressor contacts the DNA in the complex with four repressors per 28 base pairs. These results suggest a novel mechanism for the one-dimensional diffusion of repressor along DNA.

Protein Conformational Changes Induced by 1,1'-Bis(4-anilino-5-naphthalenesulfonic acid): Preferential Binding to the Molten Globule of DnaK

“…The Xmax of the complex between the DnaK and bis-ANS was observed A maximum blue shift to 480 nm was obtained with dye/ protein ratio <0.01, and this increased to an upper limit of 505 nm at a dye/protein ratio £ 150. Similar fluorescence emission X^i ncreases with dye concentration have been reported when the dye was mixed with E. coli glutaminyl-tRNA synthetase (Bhattacharyya et al, 1991) and lac repressor (Lawson & York, 1987). At fixed bis-ANS concentration (22 µ ), the Xmax changes with the thermal unfolding of DnaK, passing through a minimum at the temperature corresponding to the I state of DnaK, indicating that the I state of DnaK is responsible for the maximum blue shift in bis-ANS fluorescence (Figure 1, inset).…”

“…The data are most easily explained if the first molecule of dye to bind to the intermediate results in a Xmax of 480 nm, and the binding of the second and third molecules of dye leads to a shift in Xmax to 505 nm, due to the different environment in which the last two dyes are found. The hypothesis of two types of bis-ANS binding has also been proposed for binding to E. coli glutaminyl-tRNA synthetase (Bhattacharyya et al, 1991), lac repressor (Lawson & York, 1987), and lactate dehydrogenase (Wu & Wu, 1978), based on the change of bis-ANS Xmax with dye concentration, and for bis-ANS binding to E. coli RN A polymerase (Martin etal., 1991) and tubulin (Prasad et al, 1986), based on the sigmoidal shape of protein titration curves.…”

A fluorescence spectroscopic study of glutaminyl‐tRNA synthetase from Escherichia coli and its implications for the enzyme mechanism

“…In the other case, an excess-drug concentration over protein concentration was used, so as to cause significant binding to lower-affinity sites. If there is only one class of binding site, identical spectra would be obtained in both cases (Lawson and York, 1987). The emission spectra under the two different conditions have substantially different emission maxima, indicating two different types of binding sites are present.…”

Fluorescence studies of a single tyrosine in a type II DNA binding protein

“…For both SPOl and CT DNA we obtain = 5 ± 0.5, where the uncertainty is due to the uncertainty in mentioned above, suggesting that this value is the "packing" limit for TF1 dimers on DNA. It should be noted that other DNA binding proteins exhibit similarly large values: 5 repressor molecules/28 DNA bp for the nonspecific complex between DNA and the lac repressor (Lawson & York, 1987) and «4-6 nucleotides/molecule of protein for the IKe and Ml 3 gene 5 and the RecA single-stranded DNA binding proteins (Cazenave et al, 1984;de Jong et al, 1987).…”