An alkaline phosphatase protection assay to investigate trp repressor/operator interactions.

We have used an alkaline phosphatase protection assay to investigate the interaction of the trp repressor with its operator sequence. The assay is based on the principle that the trp repressor will protect a terminally 5'-32P-labeled operator DNA fragment from attack by alkaline phosphatase. The optimal oligonucleotide for investigating the trp repressor/operator interaction extends two base pairs from each end of the genetically defined target sequence predicted by in vivo studies [Bass et al. (1987) Genes Dev. 1, 565-572]. The assay works well over a 10,000-fold range of protein/DNA affinity and is used to show that the corepressor, L-tryptophan, causes the liganded repressor to bind a 20 base pair trp operator duplex 6400 times more strongly than the unliganded aporepressor. The affinity of the trp repressor for operators containing symmetrical mutations was interpreted in terms of the trp repressor/operator crystal structure as follows: (1) Direct hydrogen bonds with the functional groups of G-9 of the trp operator and the side chain of Arg 69 of the trp repressor contribute to DNA-binding specificity. (2) G-6 of the trp operator is critical for DNA-binding specificity probably because of the two water-mediated hydrogen bonds between its functional groups and the N-terminus of the trp repressor's E-helix. (3) Sequence-dependent aspects of the trp operator's conformation help stabilize the trp repressor/operator complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Stereochemical effects of L-tryptophan and its analogues on trp repressor's affinity for operator-DNA.

We have employed a filter binding assay to help study the mechanism by which bound L-tryptophan enables the Escherichia coli trp repressor to bind its operators. We have prepared variants of the trp repressor using structural analogues of the natural corepressor, L-tryptophan, and measured the affinity of these variants for a 20-base pair oligonucleotide duplex containing a symmetrical idealization of the trp operator from the E. coli trpEDCBA operon. By normalizing for each analogue's previously determined affinity for the trp aporepressor, we have estimated the extent to which each of the functional groups of bound L-tryptophan contributes to operator affinity. We discuss the likely role of these functional groups in the context of the crystal structures of the inactive, unliganded trp aporepressor, the liganded, active repressor, an inactive pseudorepressor (Pseudorepressors are formed by analogues of L-tryptophan that bind at the tryptophan-binding site but form near isomorphs of the repressor that have poor affinity for operator-DNA.) and the trp repressor/operator complex. We find that the alpha-amino group and an unsubstituted amino (-NH-) nitrogen of L-tryptophan's indole ring are essential for operator affinity. The former properly orients the corepressor and the latter interacts directly with the DNA. The alpha-carboxyl group, on the other hand, greatly enhances but is not essential for operator binding. The alpha-carboxylate's role, which is dependent on the corepressor's orientation in the binding pocket, is apparently to position the guanidinium group of Arg-84 for favorable contacts with the operator's sugar-phosphate backbone.

Crystal structure of trp repressor/operator complex at atomic resolution.

The crystal structure of the trp repressor/operator complex shows an extensive contact surface, including 24 direct and 6 solvent-mediated hydrogen bonds to the phosphate groups of the DNA. There are no direct hydrogen bonds or non-polar contacts to the bases that can explain the repressor's specificity for the operator sequence. Rather, the sequence seems to be recognized indirectly through its effects on the geometry of the phosphate backbone, which in turn permits the formation of a stable interface. Water-mediated polar contacts to the bases also appear to contribute part of the specificity.

6-Nitro-L-tryptophan: a novel spectroscopic probe of trp aporepressor and human serum albumin.

The binding of 6-nitro-L-tryptophan to trp aporepressor and human serum albumin has been examined by visible difference spectroscopy and circular dichroism. 6-Nitro-L-tryptophan, prepared by nitration of L-tryptophan with nitric acid in glacial acetic acid, exhibits a visible and near-uv absorption spectrum with lambda max at about 330 nm (epsilon = 7 X 10(3) M-1 cm-1) and a shoulder near 380 nm in H2O. In the presence of trp aporepressor, the visible absorption intensity is sharply diminished. Visible difference spectral titration data give KD = 1.27 X 10(-4) M and n = 0.95 per subunit at 25 degrees C. While 6-nitro-L-tryptophan exhibits no significant circular dichroism between 300 and 500 nm, the complex with trp aporepressor exhibits strong circular dichroism signals, with a negative maximum at 386 nm (delta epsilon = -7.5 M-1 cm-1) and a positive maximum at 310 nm (delta epsilon = +6 M-1 cm-1). Circular dichroism titration data give KD = 1.69 X 10(-4) M and n = 0.90 per subunit at 25 degrees C. The KD values determined spectroscopically are in excellent agreement with that determined by equilibrium dialysis, KD = 1.5 X 10(-4) M at 25 degrees C. In the presence of human serum albumin, the spectrum of 6-nitro-L-tryptophan exhibits a blue shift and an increase in absorption intensity; similar changes are observed in solvents of low dielectric contrast such as 80% aqueous dioxane. Visible difference spectral titration data give KD = 8.0 X 10(-5) M and n = 0.95 for human serum albumin. The complex of 6-nitro-L-tryptophan with human serum albumin exhibits a strong positive circular dichroism maximum at 380 nm (delta epsilon = +9.8 M-1 cm-1) with a shoulder at 310-320 nm. Circular dichroism titration data give KD = 6.4 X 10(-5) M and n = 0.83, in good agreement with the visible difference spectral results. Taken together, our results demonstrate the utility of 6-nitro-L-tryptophan as a spectroscopic probe for tryptophan-binding proteins.

Crystals of the trp repressor-operator complex suitable for X-ray diffraction analysis.

Crystals of a simulated trp repressor-operator complex have been grown that are large enough and are sufficiently well ordered and durable to provide a high quality molecular image of this regulatory protein X DNA complex to better than 3-A resolution. The "operator" consists of a 2-fold rotationally symmetric 18-base pair duplex that is extended by a dT residue at both 5'-termini. This system exhibits extensive crystal polymorphism. The crystal form and diffraction properties are very sensitive to the length and terminal structure of the operator fragment, as well as the type and concentration of multivalent ions. When combined with the experience reported by others, our results do not support a consistent strategy for crystallization of protein X DNA complexes.

The structural basis for the interaction between L-tryptophan and the Escherichia coli trp aporepressor.

We have employed equilibrium dialysis to help study the mechanism by which the unliganded Escherichia coli trp aporepressor is activated by L-tryptophan to the liganded trp repressor. By measuring the relative affinity of L-tryptophan and various tryptophan analogues for the co-repressor's binding site, we have estimated the extent to which each of the functional groups of L-tryptophan contributes to the liganding process and discuss their role in the context of the crystal structures of the trp repressor and aporepressor. We have found that the indole ring and alpha carboxyl group of L-tryptophan are mainly responsible for its affinity to the aporepressor. The alpha amino group, however, has a small negative contribution to the affinity of L-tryptophan for the aporepressor which may be associated with its essential role in operator-specific binding.