Site-directed mutagenesis of the 'zinc finger' DNA-binding domain of the nitrogen-regulatory protein NIT2 of Neurospora.
The major nitrogen-regulatory gene nit-2 of Neurospora crassa activates the expression of numerous unlinked structural genes which specify nitrogen-catabolic enzymes during conditions of nitrogen limitation. The nit-2 gene encodes a regulatory protein of 1036 amino acid residues with a single 'zinc finger' and a downstream basic region, which together may constitute a DNA-binding domain. The zinc finger domain of the NIT2 protein was synthesized in vitro and also expressed as a fusion protein in Escherichia coli to examine its DNA-binding activity. The wild-type NIT2 finger domain protein binds to the promoter region of nit-3, the nitrate reductase structural gene. A series of NIT2 mutant proteins obtained by site-directed mutagenesis was expressed and tested for functional activity. The results demonstrate that both the single zinc-finger motif and the downstream basic region of NIT2 are critical for its trans-activating function in vivo and specific DNA-binding in vitro.
Fu
Y H
YH
Department of Biochemistry, Ohio State University, Columbus 43210.
Marzluf
G A
GA
D000595:Amino Acid Sequence; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D005656:Fungal Proteins; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D005809:Genes, Regulator; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009492:Neurospora crassa / Q000235:genetics*; D014176:Protein Biosynthesis; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D014158:Transcription, Genetic; D016335:Zinc Fingers / Q000235:genetics*
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C068100:NIT2 protein, Neurospora crassa; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors