The Escherichia coli regulatory protein MetJ binds to a tandemly repeated 8 bp palindrome.
Site-directed oligonucleotide mutagenesis has been used to isolate thirty four new mutants in the regulatory region of the Escherichia coli K12 gene, metF. The mutants include single base pair (bp) substitutions and insertions, double bp substitutions and one 7bp deletion. The effects of these and another five previously described mutants on the transcriptional regulation of metF have been analysed by using a metF'-lac'Z fusion in a low copy-number plasmid. These data, and those obtained from DNAse protection studies using pure MetJ with wild-type and mutant metF operator DNA, show that the metF operator is comprised of five tandem 8 bp repeat units that overlap the -10 region of the metF promoter. In the presence of the co-repressor S-adenosylmethionine, the DNAse protection studies yielded dissociation constants of 150 nM and 300 nM for the interaction of MetJ with repeat units 1 to 4 and repeat unit 5, respectively. In the absence of co-repressor, the dissociation constants obtained for these interactions are four to five times greater. It is proposed that regulation at the metF operator requires four molecules of MetJ dimer to bind to the five 8 bp repeat units to form a tandem, overlapping array. Interactions between MetJ molecules make an essential contribution to the stability of this protein-DNA complex.
Davidson
B E
BE
Department of Biochemistry, University of Melbourne, Parkville, Victoria, Australia.
Saint Girons
I
I
D001483:Base Sequence; D004269:DNA, Bacterial; D003851:Deoxyribonucleases; D004926:Escherichia coli / Q000235:genetics*; D008715:Methionine / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D009875:Operator Regions, Genetic; D012091:Repetitive Sequences, Nucleic Acid; D012097:Repressor Proteins / Q000235:genetics*; D012436:S-Adenosylmethionine / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics*
D004269:DNA, Bacterial; D012097:Repressor Proteins; D014157:Transcription Factors; D012436:S-Adenosylmethionine; D008715:Methionine; D003851:Deoxyribonucleases