Mutant forms of the enhancer-binding protein NtrC can activate transcription from solution.

Activators of the sigma54-holoenzyme catalyze the isomerization of closed complexes between this polymerase and a promotor to open complexes in a reaction that depends upon hydrolysis of a nucleoside triphosphate. The activators normally bind to DNA sites with the properties of transcriptional enhancers and contact the polymerase by means of DNA loop formation. Here, we demonstrate that mutant forms of the activator nitrogen regulatory protein C (NtrC) that lack one helix of the helix-turn-helix (HTH) DNA-binding motif or the entire motif retain residual capacity to activate transcription from solution, despite the fact that they are largely unable to dimerize and have greatly decreased ability to hydrolyze ATP. We show that substitution of alanine for three hydrophilic residues in the second helix of the HTH yields a stable, dimeric form of NtrC defective in DNA-binding. Like mutant forms with deletions of one or both helices, the NtrC3ala protein failed to bind DNA in a sensitive affinity co-electrophoresis assay, indicating that its affinity for a strong enhancer was reduced by at least 5000-fold. (The assay detected enhancer-binding by two mutant forms of NtrC with single amino acid substitutions in the HTH and non-specific DNA-binding by the wild-type protein.) The phosphorylated NtrC3ala protein had normal ATPase activity in solution but, unlike the activity of the phosphorylated wild-type protein, which could be stimulated at least tenfold by an oligonucleotide carrying a strong enhancer, the ATPase activity of the phosphorylated NtrC3ala protein was not stimulated. At concentrations of 100 nM or greater, the phosphorylated NtrC3ala protein activated transcription from the major glnA promoter. In agreement with the fact that it did not show detectable DNA-binding in other assays, its ability to activate transcription was no greater on templates carrying the glnA enhancer than on templates lacking an enhancer. The results indicate that both roles of the glnA enhancer, tethering and facilitation of the formation of an active oligomer of NtrC, can be bypassed if the protein is present at high concentrations in solution.