MUTANT FORMS OF THE ENHANCER-BINDING PROTEIN NTRC CAN ACTIVATE TRANSCRIPTION FROM SOLUTION

Activators of the sigma(54)-holoenzyme catalyze the isomerization of c losed complexes between this polymerase and a promotor to open complex es in a reaction that depends upon hydrolysis of a nucleoside triphosp hate. 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 activato r nitrogen regulatory protein C (NtrC) that lack one helix of the heli x-turn-helix (HTH) DNA-binding motif or the entire motif retain residu al capacity to activate transcription from solution, despite the fact that they are largely unable to dimerize and have greatly decreased ab ility 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 NtrC(3ala) protein failed to bin d DNA in a sensitive affinity co-electrophoresis assay, indicating tha t its affinity for a strong enhancer was reduced by at least 5000-fold . (The assay detected enhancer-binding by two mutant forms of NtrC wit h single amino acid substitutions in the HTH and non-specific DNA-bind ing by the wild-type protein.) The phosphorylated NtrC(3ala) protein h ad normal ATPase activity in solution but, unlike the activity of the phosphorylated wild-type protein, which could be stimulated at least t enfold by an oligonucleotide carrying a strong enhancer, the ATPase ac tivity of the phosphorylated NtrC(3ala) protein was not stimulated. At concentrations of 100 nM or greater, the phosphorylated NtrC(3ala) pr otein activated transcription from the major glnA promoter. In agreeme nt with the fact that it did not show detectable DNA-binding in other assays, its ability to activate transcription was no greater on templa tes carrying the glnA enhancer than on templates lacking an enhancer. The results indicate that both roles of the glnA enhancer, tethering a nd facilitation of the formation of an active oligomer of NtrC, can be bypassed if the protein is present at high concentrations in solution . (C) 1997 Academic Press Limited.