"Switch I" mutant forms of the bacterial enhancer-binding protein NtrC that perturb the response to DNA

NtrC (nitrogen regulatory protein C) is a bacterial enhancer-binding protei n of 469 residues that activates transcription by sigma(54)-holoenzyme. A r egion of its transcriptional activation (central) domain that is highly con served among homologous activators of sigma(54)-holoenzyme-residues 206-220 -is essential for interaction with this RNA polymerase: it is required for contact with the polymerase and/or for coupling the energy from ATP hydroly sis to a change in the conformation of the polymerase that allows it to for m transcriptionally productive open complexes. Several mutant NtrC proteins with amino acid substitutions in this region, including NtrC(A216V) and Nt rC(G219K), have normal ATPase activity but fail in transcriptional activati on. We now report that other mutant forms carrying amino acid substitutions at these same positions, NtrC(A216C) and NtrC(G219C), are capable of activ ating transcription when they are not bound to a DNA template (non-DNA-bind ing derivatives with an altered helix-turn-helix DNA-binding motif at the C terminus of the protein) but are unable to do so when they are bound to a DNA template, whether or not it carries a specific enhancer. Enhancer DNA r emains a positive allosteric effector of ATP hydrolysis. as it is for wild- type NtrC but, surprisingly, appears to have become a negative allosteric e ffector for some aspect of interaction with sigma(54)-holoenzyme. The conse rved region in which these amino acid substitutions occur (206-220) is equi valent to the Switch I region of a large group of purine nucleotide-binding proteins. Interesting analogies can be drawn between the Switch I region o f NtrC and that of p21(ras)