Journal article
Constitutive Forms of the Enhancer-binding Protein NtrC: Evidence that Essential Oligomerization Determinants Lie in the Central Activation Domain
Journal of molecular biology, Vol.249(4), pp.700-713
06/16/1995
DOI: 10.1006/jmbi.1995.0330
PMID: 7602583
Abstract
Nitrogen regulatory protein C(NtrC) is a bacterial enhancer-binding protein that activates transcription by the σ54-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 fromSalmonella typhimurium; unlike wild-type NtrC, these forms are able to hydrolyze ATP and activate transcriptionin vitrowithout being phosphorylated. The amino acids altered in NtrCconstitutiveproteins 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 σ54-holoenzyme, and are not identical even among NtrC proteins from members of different subgroups of the proteobacteria (purple bacteria). NtrCconstitutiveproteins 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 NtrCconstitutiveproteins with amino acid substitutions in the central domain (NtrCS160Fand NtrCV288I) 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 ΔN-NtrCS160Fis 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.f2f2Present addresses: Yehuda Flashner, Israel Institute for Biological Research. P.O. Box 19, 70450 Ness-Ziona, Israel; David S. Weiss, Department of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA; John Keener, Department of Biological Chemistry, University of California at Irvine, Irvine, CA 92717, USAAbbreviations used: NtrC, nitrogen regulatory protein C; NtrB, histidine autokinase; NB, nutrient broth.
Details
- Title: Subtitle
- Constitutive Forms of the Enhancer-binding Protein NtrC: Evidence that Essential Oligomerization Determinants Lie in the Central Activation Domain
- Creators
- Yehuda FlashnerDavid S WeissJohn KeenerSydney Kustu
- Resource Type
- Journal article
- Publication Details
- Journal of molecular biology, Vol.249(4), pp.700-713
- Publisher
- Elsevier Ltd
- DOI
- 10.1006/jmbi.1995.0330
- PMID
- 7602583
- ISSN
- 0022-2836
- eISSN
- 1089-8638
- Language
- English
- Date published
- 06/16/1995
- Academic Unit
- Microbiology and Immunology
- Record Identifier
- 9984002395302771
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