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Site‐directed mutagenesis of the ‘zinc finger’ DNA‐binding domain of the nitrogen‐regulatory protein NIT2 of Neurospora
Author(s) -
Fu Y.H.,
Marzluf G.A.
Publication year - 1990
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.1990.tb02033.x
Subject(s) - zinc finger , neurospora crassa , biology , mutagenesis , biochemistry , gene , nitrate reductase , mutant , site directed mutagenesis , regulator gene , neurospora , regulation of gene expression , enzyme , transcription factor
Summary 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 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.