CHARACTERIZATION OF A 2ND METR-BINDING SITE IN THE METE METR REGULATORY REGION OF SALMONELLA-TYPHIMURIUM

Transcription of the metE gene in Salmonella typhimurium and Escherich ia coil 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 tra nscription initiation site (designated as site 1). In this study, we s how 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 sit e 2). Single and multiple base changes were introduced into sites 1 an d 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-la cZ expression, suggesting that both sites are necessary for expression . DNase I footprint analysis showed that MetR bound at the high-affini ty site 1 enhances MetR binding at the low-affinity site 2. A 2-bp cha nge in site 2 toward the MetR consensus binding sequence resulted in h igh metE-lacZ expression; the increased expression was MetR dependent but homocysteine independent.