The positive-acting sulfur regulatory protein CYS3 of Neurospora crassa: nuclear localization, autogenous control, and regions required for transcriptional activation.
The positive-acting global sulfur regulatory protein, CYS3, of Neurospora crassa turns on the expression of a family of unlinked structural genes that encode enzymes of sulfur catabolism. CYS3 contains a leucine zipper and an adjacent basic region (b-zip), which together constitute a bipartite sequence-specific DNA-binding domain. Specific anti-CYS3 antibodies detected a protein of the expected size in nuclear extracts of wild-type Neurospora under conditions in which the sulfur circuit is activated. The CYS3 protein was not observed in cys-3 mutants. Nuclear extracts of wild type, but not cys-3 mutants, also showed specific DNA-binding activity identical to that obtained with a CYS3 protein expressed in Escherichia coli. A truncated CYS3 protein that contains primarily the b-zip domain binds to DNA with high specificity and affinity in vitro, yet fails to activate gene expression in vivo, and instead inhibits the function of the wild-type CYS3 protein. Amino-terminal, carboxyterminal, and internal deletions as well as alanine scanning mutagenesis were employed to identify regions of the CYS3 protein that are required for its trans-activation function. Regions of CYS3 carboxy terminal to the b-zip motif are not completely essential for function although loss of an alanine-rich region results in decreased activity. All deletions amino terminal to the b-zip motif led to a complete loss of CYS3 function. Alanine scanning mutagenesis demonstrated that an unusual prolinerich domain of CYS3 appears to be very important for function and is presumed to constitute an activation domain. It is concluded that CYS3 displays nuclear localization and positive autogenous control in Neurospora and functions as a trans-acting DNA-binding protein.
Kanaan
M N
MN
Department of Biochemistry, Ohio State University, Columbus 43210.
Marzluf
G A
GA
D000409:Alanine / Q000032:analysis; D001192:Arylsulfatases / Q000378:metabolism; D015153:Blotting, Western; D003542:Cystathionine gamma-Lyase; D004271:DNA, Fungal / Q000032:analysis; D004268:DNA-Binding Proteins / Q000032:analysis / Q000737:chemistry / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics*; D005800:Genes, Fungal; D005809:Genes, Regulator; D016350:Leucine Zippers; D016296:Mutagenesis; D009492:Neurospora crassa / Q000235:genetics*; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics; D011392:Proline / Q000032:analysis; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion; D013455:Sulfur / Q000378:metabolism; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics; D014157:Transcription Factors / Q000032:analysis / Q000737:chemistry / Q000235:genetics*; D015533:Transcriptional Activation
D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D013455:Sulfur; D011392:Proline; D001192:Arylsulfatases; C073674:CYS3 protein, S cerevisiae; D003542:Cystathionine gamma-Lyase; D000409:Alanine