CONSTITUTIVE FORMS OF THE ENHANCER-BINDING PROTEIN NTRC - EVIDENCE THAT ESSENTIAL OLIGOMERIZATION DETERMINANTS LIE IN THE CENTRAL ACTIVATION DOMAIN

Nitrogen regulatory protein C (NtrC) is a bacterial enhancer-binding p rotein that activates transcription by the sigma(54)-holoenzyme. To ac tivate 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 form s are able to hydrolyze ATP and activate transcription in vitro withou t being phosphorylated. The amino acids altered in NtrC(constitutive) proteins are located in both the N-terminal regulatory domain and the central domain, which is directly responsible for transcriptional acti vation. The residues that are altered are not conserved among activato rs of the sigma(54)-holoenzyme, and are not identical even among Nt C proteins from members of different subgroups of the proteobacteria (pu rple bacteria). NtrC(constitutive) proteins are phosphorylated normall y; phosphorylation increases their ability to hydrolyze ATP and activa te transcription. Moreover, the oligomerization of these proteins that occurs when they bind to an enhancer also increases the ATPase activi ty of both unmodified and phosphorylated forms. Removal of the N-termi nal regulatory domain from two NtrC(constitutive) proteins with amino acid substitutions in the central domain (NtrC(S160F) and NtrC(V2881)) leaves them active, indicating that essential oligomerization determi nants lie outside the regulatory domain. This conclusion is confirmed by the observation that the ATPase activity of Delta N-NtrC(S160F) 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 indicat ing 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.