Importance of Tetramer Formation by the Nitrogen Assimilation Control Protein for Strong Repression of Glutamate Dehydrogenase Formation in Klebsiella pneumoniae

The nitrogen assimilation control protein (NAC) fromKlebsiella pneumoniae is a very versatile regulatory protein. NAC activates transcription of operons such ashut (histidine utilization) andure (urea utilization), whose products generate ammonia. NAC also represses the transcription of genes such asgdhA , whose products use ammonia. NAC exerts a weak repression atgdhA by competing with the binding of a lysine-sensitive activator. NAC also strongly represses transcription ofgdhA (about 20-fold) by binding to two separated sites, suggesting a model involving DNA looping. We have identified negative control mutants that are unable to exert this strong repression ofgdhA expression but still activatehut andure expression normally. Some of these negative control mutants (e.g., NAC86ter and NAC132ter ) delete the C-terminal domain, thought to be required for tetramerization. Other negative control mutants (e.g., NACL111K and NACL125R ) alter single amino acids involved in tetramerization. In this work we used gel filtration to show that NAC86ter and NACL111K are dimers in solution, even at high concentration (NACWT is a tetramer). Moreover, using a combination of DNase I footprints and gel mobility shifts assays, we showed that when NACWT binds to two adjacent sites on a DNA fragment, NACWT binds as a tetramer that bends the DNA fragment significantly. NACL111K binds to such a fragment as two independent dimers without inducing the strong bend. Thus, NACL111K is a dimer in solution or when bound to DNA. NACL111K (typical of the negative control mutants) is wild type for every other property tested: (i) it activates transcription athut andure ; (ii) it competes with the lysine-sensitive activator for binding atgdhA ; (iii) it binds to the same sites at thehut ,ure ,nac , andgdhA promoters as NACWT ; (iv) the relative affinity of NACL111K for these sites follows the same order as NACWT (ure >gdhA >nac >hut ); (v) it induces the same slight bend as dimers of NACWT ; and (vi) its DNase I footprints at these sites are indistinguishable from those of NACWT (except for features ascribed to tetramer formation). The only two phenotypes we know for negative control mutants of NAC are their inability to tetramerize and their inability to cause the strong repression ofgdhA . Thus, we propose that in order for NACWT to exert the strong repression, it must form a tetramer that bridges the two sites atgdhA (similar to other DNA looping models) and that the negative control mutants of NAC, which fail to tetramerize, cannot form this loop and thus fail to exert the strong repression atgdhA .