Abstract
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.