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Repressor Forms of the Enhancer-binding Protein NtrC: Some Fail in Coupling ATP Hydrolysis to Open Complex Formation by σ54-Holoenzyme

1996; Elsevier BV; Volume: 260; Issue: 3 Linguagem: Inglês

10.1006/jmbi.1996.0403

1089-8638

Anne North, David S. Weiss, Hideyuki Suzuki, Yehuda Flashner, Sydney Kustu,

RNA and protein synthesis mechanisms

NtrC (nitrogen regulatory protein C) is a bacterial enhancer-binding protein that activates transcription by catalyzing isomerization of closed complexes between σ54-holoenzyme and a promoter to open complexes. To catalyze this reaction, NtrC must be phosphorylated and form an appropriate oligomer so that it can hydrolyze ATP. NtrC can also repress transcription by σ70-holoenzyme. In this paper we characterize “repressor” mutant forms of NtrC fromSalmonella typhimurium, forms that have lost the ability to activate transcription by σ54-holoenzyme (in vitroactivity at least 1000-fold lower than wild-type) but retain the ability to repress transcription by σ70-holoenzyme. The amino acid substitutions in NtrCrepressorproteins that were obtained by classical genetic techniques alter residues in the central domain of the protein, the domain directly responsible for transcriptional activation. Commensurate with this, phosphorylation and the autophosphatase activities of NtrCrepressorproteins, which are functions of the amino-terminal regulatory domain of NtrC, are normal. In addition, these proteins have essentially normal DNA-binding, which is a function of the C-terminal region of NtrC and bind cooperatively to enhancers. (The NtrCG219Kprotein has “improved” DNA-binding, which is discussed.) We previously presented evidence that several NtrCrepressorproteins have impaired ATPase activity. We now show that two other repressor proteins, NtrCA216Vand NtrCA220T, have as much ATPase activity as wild-type NtrC when they are phosphorylated and bound to an enhancer and that they have considerably more activity than an unphosphorylated NtrCconstitutiveprotein, which is capable of activating transcription. These results demonstrate that NtrCA216Vand NtrCA220Tfail in a function of the central domain other than ATPase activity. Although they may fail in contact with σ54-holoenzymeper 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.