Open AccessMutational analysis of the DNA-binding domain of the CYS3 regulatory protein of Neurospora crassa.
Author(s) -
Moien Kanaan,
George A. Marzluf
Publication year - 1991
Publication title -
molecular and cellular biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.14
H-Index - 327
eISSN - 1067-8824
pISSN - 0270-7306
DOI - 10.1128/mcb.11.9.4356
Subject(s) - biology , neurospora crassa , leucine zipper , biochemistry , permease , gene , dna , binding site , mutagenesis , b3 domain , dna binding domain , binding domain , dna binding protein , bzip domain , microbiology and biotechnology , mutant , peptide sequence , transcription factor
cys-3, the major sulfur regulatory gene of Neurospora crassa, activates the expression of a set of unlinked structural genes which encode sulfur catabolic-related enzymes during conditions of sulfur limitation. The cys-3 gene encodes a regulatory protein of 236 amino acid residues with a leucine zipper and an upstream basic region (the b-zip region) which together may constitute a DNA-binding domain. The b-zip region was expressed in Escherichia coli to examine its DNA-binding activity. The b-zip domain protein binds to the promoter region of the cys-3 gene itself and of cys-14, the sulfate permease II structural gene. A series of CYS3 mutant proteins obtained by site-directed mutagenesis were expressed and tested for function, dimer formation, and DNA-binding activity. The results demonstrate that the b-zip region of cys-3 is critical for both its function in vivo and specific DNA-binding in vitro.