DNA binding sites of the LysR-type regulator GcvA in the gcv and gcvA control regions of Escherichia coli.

The GcvA protein is a LysR family regulatory protein necessary for both activation and repression of the Escherichia coli glycine cleavage enzyme operon (gcv) and negative regulation of gcvA. Gel shift assays indicated that overexpressed GcvA in crude extracts is capable of binding specifically to DNA containing the gcv and gcvA control regions. DNase I footprint analysis of the gcvA control region revealed one region of GcvA-mediated protection overlapping the transcription initiation site and extending from -28 to +20. Three separate GcvA binding sites in gcv were identified by DNase I footprint analysis: a 29-bp region extending from positions -271 to -242, a 28-bp region extending from -242 to -214, and a 35-bp region covering positions -69 to -34 relative to the transcription initiation site. PCR-generated mutations in any of the three GcvA binding sites in gcv decreased GcvA-mediated activation and repression of gcv.