Promoter architecture and transcriptional regulation by the NAC protein of Klebsiella aerogenes.

Abstract

The NAC protein of Klebsiella aerogenes is involved in a regulatory cascade that allows the cell to adapt to changes in the quality and quantity of available nitrogen-containing compounds in the medium. NAC is a DNA-binding protein that activates transcription of genes whose products degrade nitrogen-containing compounds, and represses transcription of genes whose products use nitrogen for biosynthesis. Previous work showed that transcriptional activation by NAC requires protein binding to regions of DNA that contain the partially palindromic site ATA-N9-TAT centered at position $-$64. The NAC binding site from the histidine utilization promoter (hutUp) was sufficient to confer regulation by NAC to three semi-synthetic promoters. The displacement of the NAC site relative to these promoters showed that NAC activates transcription from positions $-$64, $-$53 and $-$43, but fails to activate from positions $-$74, $-$69, $-$59 and $-$49. Random and site-directed mutagenesis of the NAC binding site from hutUp revealed that substitution of the conserved nucleotides in the palindromic ends of the binding site, ATA and TAT, reduced protein binding and transcriptional activation. Mutations in between these ends stimulated or decreased protein binding and transcriptional activation, while mutations outside these ends had only marginal effects on transcriptional activation. The replacement of the NAC binding site from hutUp in a semi-synthetic promoter by the NAC binding site from the glutamate dehydrogenase gene resulted in the failure of NAC to activate transcription, despite similar binding affinity and protein-DNA complex structure. In addition, two mutations in the promoter-proximal half site of the NAC binding increased protein binding affinity, but decreased activation. These results suggest two roles for the NAC binding site: the binding of the protein at the right distance and face of the DNA helix, and the presentation of the protein in a functional configuration.