FUNCTIONAL SELECTION AND CHARACTERIZATION OF DNA-BINDING SITES FOR TRP REPRESSOR OF ESCHERICHIA-COLI

trp repressor of Escherichia coli controls transcription initiation in operons involved in tryptophan biosynthesis by binding to operator se quences within the regulated promoters. Naturally occurring operators are homologous over an 18-base pair region and display dyad symmetry. We have examined the sequence determinants of a repressor binding site using a functional selection/polymerase chain reaction (PCR) amplific ation strategy. A trp repressor affinity column was generated and used to select binding-competent DNAs from a randomized pool of synthetic double-stranded DNA. DNAs that showed tryptophan-dependent high-affini ty binding were eluted by addition of the tryptophan analog beta-indol e acrylic acid and amplified by PCR. Following iterative cycles of aff inity chromatography and PCR, the selected DNAs were cloned and sequen ced. The CTAG tetranucleotide, present in the consensus sequence of al l natural operators, was found in all selected DNAs. Mapping experimen ts utilizing the repressor affinity column showed the CTAG motif to be a critical determinant for repressor binding. Quantitative electropho retic mobility shift assays with purified trp repressor revealed that although some of the DNAs were bound by one repressor dimer, others we re bound by two repressor dimers with cooperativity. Measured binding constants ranged from 0.035 to 0.5 nM for the selected DNAs, compared with 0.1 nM for the trp operator.