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Abstract
Expression of the ompF and ompC genes of Escherichia coli requires the OmpR protein for transcriptional activation. In vivo binding of the OmpR protein to the ompF and ompC promoter regions was observed using an in vivo dimethyl sulfate DNA footprinting technique. Two different sequence motifs were found to be protected by OmpR in both the ompF and ompC promoter regions. This technique was further used to localize the DNA-binding domain of OmpR to be within the C-terminal 117 amino acid residues. Binding of the C-terminal portion OmpR to the ompF and ompC promoter regions, however, did not result in activation of transcription. Our results, together with sequence homologies between OmpR and other regulatory proteins, suggests that OmpR has separable domain structures: the C-terminal portion for binding-specific DNA sequences and the N-terminal portion for interacting with RNA polymerase and/or other transcription factors.
Highlights
Of OmpR recognizesthe specific DNAsequences upstream of the ompF and ompC promoters so that themodulator domain is able to activate transcriptionof the gene
Comparison of sequence similarity between OmpR, PhoB, CheB, and NtrC, as well as other regulatory proteins has suggested a common domain at -terminal portionsof these proteins (Winans et al, 1986).Various techniques have been developed to study protein-DNA interactions in vivo (Church and Gilbert, 1984; Martin et d . , 1986; Gilmour and Lis, 1984; Solomon et al, 1988)
One of these methods is in vivo chemical DNA footprinting either on genomic DNA (Church and Gilbert, 1984) or on plasmid DNA (Martin et al, 1986)
Summary
Pellet was suspended in 0.5ml of 25 mM Tris-HC1 (pH 8.0) containing 10 mM EDTA (pH 8.0), 25% glucose, and 50 mg/ml lysozyme, and. The general method for i n vivo DNA footprinting The linear plasmid DNA was footprinting of the respective promoter region wascarried out purified by electrophoresis on a preparative agarose gel (0.7%, in a wild type (MC4100) (Casadaban, 1976) and an isogenic E buffer). In thepresent in vivo DNA footprinting method, it is most critical how well the dimethyl sulfate-treated plasmid can be separated from other contaminants on the agarose gel.
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