Evidence for a second interaction between the regulatory amino-terminal and central output domains of the response regulator NtrC (nitrogen regulator I) in Escherichia coli

Nitrogen limitation in Escherichia coli activates about 100 genes. Their expression requires the response regulator NtrC ( also called nitrogen regulator I or NRI). Phosphorylation of the amino-terminal domain (NTD) of NtrC activates the neighboring central domain and leads to transcriptional activation from promoters that require sigma(54)-containing RNA polymerase. The NTD has five beta strands alternating with five alpha helices. Phosphorylation of aspartate 54 has been shown to reposition alpha helix 3 to beta strand 5 (the "3445 face") within the NTD. To further study the interactions between the amino-terminal and central domains, we isolated strains with alterations in the NTD that were able to grow on a poor nitrogen source in the absence of phosphorylation by the cognate sensor kinase. We identified strains with alterations located in the 3445 face and alpha helix 5. Both types of alterations stimulated central domain activities. The alpha helix 5 alterations differed from those in the 3445 face. They did not cause a large scale conformational change in the NTD, which is not necessary for transcriptional activation in these mutants. Yeast two-hybrid analysis indicated that substitutions in both alpha helix 5 and the 3445 face diminish the interaction between the NTD and the central domain. Our results suggest that alpha helix 5 of the NTD, in addition to the 3445 face, interacts with the central domain. We present a model of interdomain signal transduction that proposes different functions for alpha helix 5 and the 3445 face.