Amino-acid substitutions in the zinc finger of NIT2, the nitrogen regulatory protein of Neurospora crassa, alter promoter element recognition

Abstract

NIT2, the major nitrogen regulatory protein of Neurospora crassa mediates nitrogen catabolite derepression of the structural genes which specify enzymes of nitrogen catabolism. The promoter of the structural gene for L-amino acid oxidase, a nitrogen-regulated enzyme, was found to contain two NIT2 binding sites, each with two copies of a GATA core consensus sequence. Site-directed mutagenesis was employed to create amino-acid substitutions within the single zinc-finger region of NIT2, which serves as the DNA-binding domain. The affect of those mutations upon NIT2 function in vivo in the activation of three separate structural genes was examined by transformation assays and relevant enzyme activities, and DNA-binding activity in vitro was determined by gel band mobility-shift assays. It was shown that specific amino-acid residues within the zinc-finger loop region of NIT2 are important for DNA-binding activity, whereas other residues influence the specificity of DNA binding. Mutant NIT2 proteins were obtained which retain DNA-binding activity and alter the specificity of DNA recognition, thus allowing a distinction between related DNA elements.