Regulation of Microbial Nitrogen Metabolism

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DESCRIPTION (provided by applicant): The Nitrogen Assimilation Control Protein, NAC, is a transcriptional regulator that activates dozens of operons in K. pneumoniae, one of which (hut) is used to degrade histidine as a nitrogen source. NAC also represses several genes, including gdhA (encoding glutamate dehydrogenase) and its own gene, nac. Previous work from Dr. Bender's lab shows that NAC uses several different mechanisms to regulate transcription, some using dimers and some tetramers of NAC. Indeed, NAC is one of the most versatile transcriptional regulators known. Dr. Bender and his colleagues propose a detailed genetic analysis of these modes of regulation. Their overall hypothesis is that the DNA sequence of the NAC-binding site determines the conformation of NAC, at hut tetramer formation is inhibited, at gdhA it is permitted, and at nac it is greatly stimulated. The genetic analysis of activation at hut will include "positive control" mutants (that can repress but not activate), mutants frozen in activating conformation whatever the DNA sequence, mutants that fail to interact with RNAP, and mutants that fail to dimerize. Repression at gdhA will include a further analysis of "negative control" mutants (that can activate but not repress), as well as a test of the DNA looping model to explain the requirement for two well-separated sites for repression. Other models will also be considered. The role of the DNA sequence of the NAC site at the nac gene will be investigated to establish its role in tetramer formation and to contrast it to the site at gdhA. Although NAC-mediated repression of gdhA is the key focus of this proposal, Dr. Bender will also explore the positive effectors that are required for gdhA expression and their interaction with NAC. In particular, a lysine-sensitive positive effector of gdhA expression will be characterized and used as a model for the characterization of other positive effectors of gdhA.