Constitutive forms of the enhancer-binding protein NtrC: evidence that essential oligomerization determinants lie in...

Nitrogen regulatory protein C (NtrC) is a bacterial enhancer-binding protein that activates transcription by the sigma 54-holoenzyme. To activate transcription, NtrC must hydrolyze ATP, a reaction that depends upon its being phosphorylated and forming an appropriate oligomer. In this paper we characterize "constitutive" mutant forms of the NtrC protein from Salmonella typhimurium; unlike wild-type NtrC, these forms are able to hydrolyze ATP and activate transcription in vitro without being phosphorylated. The amino acids altered in NtrCconstitutive proteins are located in both the N-terminal regulatory domain and the central domain, which is directly responsible for transcriptional activation. The residues that are altered are not conserved among activators of the sigma 54-holoenzyme, and are not identical even among NtrC proteins from members of different subgroups of the proteobacteria (purple bacteria). NtrCconstitutive proteins are phosphorylated normally; phosphorylation increases their ability to hydrolyze ATP and activate transcription. Moreover, the oligomerization of these proteins that occurs when they bind to an enhancer also increases the ATPase activity of both unmodified and phosphorylated forms. Removal of the N-terminal regulatory domain from two NtrCconstitutive proteins with amino acid substitutions in the central domain (NtrCS160F and NtrCV2881) leaves them active, indicating that essential oligomerization determinants lie outside the regulatory domain. This conclusion is confirmed by the observation that the ATPase activity of delta N-NtrCS160F is greatly stimulated when it binds to an enhancer, and by the ability of this protein to activate transcription synergistically with a form of NtrC incapable of DNA-binding. Together with previous results indicating that oligomerization determinants do not lie in the C-terminal DNA-binding domain of NtrC; these results provide evidence that they lie in the central domain.