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 sequences 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) amplification 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-affinity binding were eluted by addition of the tryptophan analog beta-indole acrylic acid and amplified by PCR. Following iterative cycles of affinity chromatography and PCR, the selected DNAs were cloned and sequenced. The CTAG tetranucleotide, present in the consensus sequence of all natural operators, was found in all selected DNAs. Mapping experiments utilizing the repressor affinity column showed the CTAG motif to be a critical determinant for repressor binding. Quantitative electrophoretic mobility shift assays with purified trp repressor revealed that although some of the DNAs were bound by one repressor dimer, others were 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.
Czernik
P J
PJ
Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock 72205-7199.
Shin
D S
DS
Hurlburt
B K
BK
D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D002846:Chromatography, Affinity; D016384:Consensus Sequence; D004247:DNA / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D017931:DNA Primers / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism*; D007211:Indoles; D007700:Kinetics; D008969:Molecular Sequence Data; D009876:Operon; D016133:Polymerase Chain Reaction; D012097:Repressor Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D014364:Tryptophan / Q000378:metabolism; D029968:Escherichia coli Proteins
D001426:Bacterial Proteins; D004247:DNA; D017931:DNA Primers; D007211:Indoles; D012097:Repressor Proteins; D014364:Tryptophan; C027190:TRPR protein, E coli; D029968:Escherichia coli Proteins; C001446:indoleacrylic acid