ACTIVATION OF TRANSCRIPTION INITIATION FROM THE NAC PROMOTER OF KLEBSIELLA-AEROGENES

The nac gene of Klebsiella aerogenes encodes a bifunctional transcription factor that activates or represses the expression of several operons under conditions of nitrogen limitation. In experiments with purified components, transcription from the nac promoter was initiated by sigma(54) RNA polymerase and was activated by the phosphorylated form of nitrogen regulator I (NRI) (NtrC). The activation of the nac promoter required a higher concentration of NRT similar to P than did the activation of the Escherichia coli glnAp(2), promoter, and both the promoter and upstream enhancer element contributed to this difference. The nac promoter had a lower affinity for sigma(54) RNA polymerase than did glnAp,, and uninitiated competitor-resistant transcription complexes formed at the nac promoter decayed to competitor-sensitive complexes at a greater rate than did similar complexes formed at the glnAp, promoter. The nac enhancer, consisting of a single high-affinity NRI-binding site and an adjacent site with low affinity for NRI, was less efficient in stimulating transcription than was the glnA enhancer, which consists of two adjacent high-affinity NRI-binding sites. When these binding sites were exchanged, transcription from the nac promoter aas increased and transcription from the glnAp(2) promoter was decreased at low concentrations of NRI similar to P. Another indication of the difference in the efficiency of these enhancers is that although activation of a nac promoter construct containing the glnA enhancer was relatively insensitive to subtle alterations in the position of these sites relative to the position of the promoter, activation of the natural nac promoter or a nac promoter construct containing only a single high-affinity NRI similar to P binding site was strongly affected by subtle alterations in the position of the NRI similar to P binding site(s), indicating a face-of-the-helix dependency for activation.