Properties of the NAC (Nitrogen Assimilation Control Protein)-Binding Site within the ureD Promoter of Klebsiella pneumoniae

Abstract

ABSTRACT The nitrogen assimilation control protein (NAC) of Klebsiella pneumoniae is a LysR-type transcriptional regulator that activates transcription when bound to a DNA site (ATAA-N5-TnGTAT) centered at a variety of distances from the start of transcription. The NAC-binding site from the hutU promoter (NBS hutU ) is centered at −64 relative to the start of transcription but can activate the lacZ promoter from sites at −64, −54, −52, and −42 but not from sites at −47 or −59. However, the NBSs from the ureD promoter ( ureDp ) and codB promoter ( codBp ) are centered at −47 and −59, respectively, and NAC is fully functional at these promoters. Therefore, we compared the activities of the NBS hutU and NBS ureD within the context of ureDp as well as within codBp . The NBS hutU functioned at both of these sites. The NBS ureD has the same asymmetric core as the NBS hutU . Inverting the NBS ureD abolished more than 99% of NAC's ability to activate ureDp . The key to the activation lies in the TnG segment of the TnGTAT half of the NBS ureD . Changing TnG to GnT, TnT, or GnG drastically reduced ureDp activation (to 0.5%, 6%, or 15% of wild-type activation, respectively). The function of the NBS ureD , like that of the NBS hutU , requires that the TnGTAT half of the NBS be on the promoter-proximal (downstream) side of the NBS. Taken together, our data suggest that the positional specificity of an NBS is dependent on the promoter in question and is more flexible than previously thought, allowing considerable latitude both in distance and on the face of the DNA helix for the NBS relative to that of RNA polymerase.