Characterization of a second MetR-binding site in the metE metR regulatory region of Salmonella typhimurium.

Transcription of the metE gene in Salmonella typhimurium and Escherichia coli is positively regulated by the MetR protein, with homocysteine serving as a coactivator. It was shown previously that MetR binds to and protects from DNase I digestion a 24-bp sequence in the metE metR regulatory region from nucleotides -48 to -71 relative to the metE transcription initiation site (designated as site 1). In this study, we show that purified MetR protein also binds to and protects a second 24-bp sequence adjacent to the original site, from nucleotides -24 to -47 relative to the metE transcription initiation site (designated as site 2). Single and multiple base changes were introduced into sites 1 and 2 in a metE-lacZ fusion. Base pair changes in site 1 or site 2 away from the MetR consensus binding sequence resulted in decreased metE-lacZ expression, suggesting that both sites are necessary for expression. DNase I footprint analysis showed that MetR bound at the high-affinity site 1 enhances MetR binding at the low-affinity site 2. A 2-bp change in site 2 toward the MetR consensus binding sequence resulted in high metE-lacZ expression; the increased expression was MetR dependent but homocysteine independent.