The amino-terminal 100 residues of the nitrogen assimilation control protein (NAC) encode all known properties of NAC from Klebsiella aerogenes and Escherichia coli

Abstract

The nitrogen assimilation control protein (NAC) from Klebsiella aerogenes or Escherichia coli (NAC(K) or NAC(E), respectively) is a transcriptional regulator that is both necessary and sufficient to activate transcription of the histidine utilization (hut) operon and to repress transcription of the glutamate dehydrogenase (gdh) operon in K. aerogenes. Truncated NAC polypeptides, generated by the introduction of stop codons within the nac open reading frame, were tested for the ability to activate hut and repress gdh in vivo. Most of the NAC(K) and NAC(E) fragments with 100 or more amino acids (wild-type NAC(K) and NAC(E) both have 305 amino acids) were functional in activating hut and repressing gdh expression in vivo. Full-length NAC(K) and NAC(E) were isolated as chimeric proteins with the maltose-binding protein (MBP). NAC(K) and NAC(E) released from such chimeras were able to activate hut transcription in a purified system in vitro, as were NAC(K)129 and NAC(E)100 (a NAC(K) fragment of 129 amino acids and a NAC(E) fragment of 100 amino acids) released from comparable chimeras. A set of NAC(E) and NAC(K) fragments carrying nickel-binding histidine tags (his6) at their C termini were also generated. All such constructs derived from NAC(E) were insoluble, as was NAC(E) itself. Of the his6-tagged constructs derived from NAC(K), NAC(K)100 was inactive, but NAC(K)120 was active. Several NAC fragments were tested for dimerization. NAC(K)120-his6 and NAC(K)100-his6 were dimers in solution. MBP-NAC(K) and MBP-NAC(K)129 were monomers in solution but dimerized when the MBP was released by cleavage with factor Xa. MBP-NAC(E) was readily cleaved by factor Xa, but the resulting NAC(E) was also degraded by the protease. However, MBP-NAC(E)-his6 was completely resistant to cleavage by factor Xa, suggesting an interaction between the C and N termini of this protein.