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J. Bacteriol. doi:10.1128/JB.01238-06
Copyright (c) 2006, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Functional Analysis of the Carboxy-Terminal Region of Bacillus subtilis TnrA, a MerR Family Protein

Department of Microbiology, Boston University School of Medicine, Boston, Massachusetts 02118

^* To whom correspondence should be addressed. Email: shfisher{at}bu.edu.

	Abstract

The Bacillus subtilis TnrA transcription factor belongs to the MerR family of proteins and regulates gene expression during nitrogen-limited growth. When B. subtilis cells are grown with excess nitrogen, feedback-inhibited glutamine synthetase forms a protein-protein complex with TnrA that prevents TnrA from binding to DNA. The C-terminal region of TnrA is required for the interaction with glutamine synthetase. Alanine scanning mutagenesis of the C-terminal region of TnrA identified three classes of mutants that altered the regulation by glutamine synthetase. While expression of the TnrA-regulated amtB gene was expressed constitutively in the class I (M96A, Q100A, A103G) and class II mutants (L97A, L101A, F105A), the class II mutants were unable to grow on minimal medium unless a complex mixture of amino acids was present. The class III tnrA mutants (R93A, G99A, N102A, H104A, Y107A) were partially defective in the regulation of TnrA activity. In vitro experiments showed that feedback-inhibited glutamine synthetase had a significantly reduced ability to inhibit the DNA-binding activity of several class I and class II mutant TnrA proteins. A coiled-coil homology model of the C-terminal region of TnrA is used to explain the properties of the class I and II mutant proteins. The C-terminal region of TnrA corresponds to a dimerization domain in other MerR family proteins. Surprisingly, gel filtration and cross-linking analysis showed that a truncated TnrA protein which only contains the N-terminal DNA binding domain was dimeric. The implications of these results on the structure of TnrA are discussed.

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