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'-P-32-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 operators 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. The operator-binding activity of mutationally altered trp aporepressor proteins when interpreted in terms of the relevant crystal structures showed the following: (1) The small side chain of alanine at position 77, in the turn of the helix-turn-helix, helps the unliganded aporepressor's DNA-interacting domain to collapse into an inactive conformation. (2) Unlike the terminal segments of some related repressors, the extended N-terminal arms of the trp repressor do not interact with DNA in a sequence-specific way. The assay demonstrated the preferential affinity of the gal repressor for a 20 base pair gal operator, suggesting that alkaline phosphatase protection may be generally useful in measuring the affinity of DNA-binding proteins for their target DNA sequences.