REPRESSOR FORMS OF THE ENHANCER-BINDING PROTEIN NTRC - SOME FAIL IN COUPLING ATP HYDROLYSIS TO OPEN COMPLEX-FORMATION BY SIGMA(54)-HOLOENZYME

NtrC (nitrogen regulatory protein C) is a bacterial enhancer-binding p rotein that activates transcription by catalyzing isomerization of clo sed complexes between sigma(54)-holoenzyme and a promoter to open comp lexes. To catalyze this reaction, NtrC must be phosphorylated and form an appropriate oligomer so that it can hydrolyze ATP. NtrC can also r epress transcription by sigma(70)-holoenzyme. In this paper we charact erize ''repressor'' mutant forms of NtrC from Salmonella typhimurium, forms that have lost the ability to activate transcription by sigma(54 )-holoenzyme (in vitro activity at least 1000-fold lower than wild-typ e) but retain the ability to repress transcription by sigma(70)-holoen zyme. The amino acid substitutions in NtrC(repressor) proteins that we re obtained by classical genetic techniques alter residues in the cent ral domain of the protein, the domain directly responsible for transcr iptional activation. Commensurate with this, phosphorylation and the a utophosphatase activities of NtrC(repressor) proteins, which are funct ions of the amino-terminal regulatory domain of NtrC, are normal. In a ddition, these proteins have essentially normal DNA-binding, which is a function of the C-terminal region of NtrC and bind cooperatively to enhancers. (The NtrC(G219K) protein has ''improved'' DNA-binding, whic h is discussed.) We previously presented evidence that several NtrC(re pressor) proteins have impaired ATPase activity. We now show that two other repressor proteins, NtrC(A216V) and NtrC(A220T), have as much AT Pase activity as wild-type NtrC when they are phosphorylated and bound to an enhancer and that they have considerably more activity than an unphosphorylated NtrC(constitutive) protein, which is capable of activ ating transcription. These results demonstrate that NtrC(A216V) and Nt rC(A220T) fail in a function of the central domain other than ATPase a ctivity. Although they may fail in contact with sigma(54)-holoenzyme p er se, the fact that alanine is the amino acid normally found at these positions leads us to speculate that these proteins fail in coupling energy to a change in conformation of the polymerase. (C) 1996 Academi c Press Limited