The importance of tetramer formation by the nitrogen assimilation control protein (NAC) for DNA binding and repression at the <italic>gdhA</italic> promoter in <italic>Klebsiella pneumoniae</italic>.

The Nitrogen Assimilation Control protein, NAC, is a LysR-type transcriptional regulator that has been identified in Klebsiella pneumoniae and Escherichia coli. Negative control (NC) mutants of NAC were isolated. These mutants are able to activate transcription at hut and ure, but fail to strongly repress transcription at gdhA, were isolated. Two mutants resulted in a truncation of NAC to the N-terminal 86 amino acids (NAC86ter) and the N-terminal 132 amino acids (NAC132ter). A third mutant resulted in a change at amino acid position 111 from a leucine to a lysine (NAC L111K). The isolation and the properties of these NACNC mutants suggested that (1) the functions for activation and repression are separable, (2) the N-terminal 86 acids contained all the information for activation, and (3) the C-terminus was necessary for the repression at gdhA. The C-terminus of many LysR-type proteins has been implicated as playing a role for tetramerization. Studies with NAC mutants that map to L111 and L125, its an interacting partner in tetramerization, confirmed that these amino acid residues are part of a tetramerization domain and that NAC tetramers are necessary for the strong repression at gdhA. Studies with the NACNC mutants revealed that NAC bound to the hut and ure promoters as a dimer and the nac, cod, and the gdhA promoters as a tetramer. Further analysis of the nac promoter revealed that this promoter contained two NAC-binding sites, a strong site, ATC-N 9-TAT, and a weak site, ATA-N9-GCT. Mutational analysis of the weak site showed that this site was necessary for the formation of the NAC tetramer-DNA complex. These results suggested that NAC tetramers bind by recognizing two adjacent NAC-binding sites which involves the cooperative interactions between the NAC dimers (through the tetramerization domain) and between the protein and DNA. Interestingly, the length of the footprint by NAC at the nac promoter (62 by protection) and cod promoter (52 by protection) was different. Moreover, the cod promoter did not contain a second NAC-binding site in a DNA, but it did contain an ATA half site. This ATA was positioned 3 helical turns from the consensus NAC-binding site (the nac promoter has an ATA which is positioned 4 helical turns from the NAC-binding site), which was important for the formation of the NAC tetramer-DNA complex. In DNA bending assays of the nac and cod promoters, NAC was able to bend the DNA at the nac promoter, but not at the cod promoter. Therefore, the spacing of the ATA half site affected how NAC bends the DNA. In fact, altering the spacing in the nac and cod promoter by 1 helical turn affected the ability of NAC to bend the DNA. Our studies showed that the NAC tetramer is a flexible protein and the conformation that NAC assumes is determined by the DNA.Ph.D.Biological SciencesMicrobiologyMolecular biologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/125213/2/3186750.pd