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

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 (NBShutU) 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 NBShutU and NBSureD within the context of ureDp as well as within codBp. The NBShutU functioned at both of these sites. The NBSureD has the same asymmetric core as the NBShutU. Inverting the NBSureD 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 NBSureD. 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 NBSureD, like that of the NBShutU, 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.