Global regulation of sulfur assimilation in <i>Neurospora</i>

Marzluf, George A.; Li, Qunhui; Coulter, Kristin R.

doi:10.1139/b95-241

Cited by 6 publications

(2 citation statements)

References 20 publications

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“…Our results indicate that control of cys-74 depends pri- (Marzluf et a/., 1995), which agrees with their relative physiological importance in vivo. It appears that the 1.4kbp upstream region of the cys-74 gene contains all the regulatory elements necessary for sulphur-responsive regulation, and that one strong CYS3binding site suffices for this control.…”

Section: Discussionsupporting

confidence: 84%

“…Site C, 1.4 kb upstream of the transcriptional start site (tss), is actually composed of two adjacent CYSB recognition elements and has the strongest protein-binding ability. Site 6, located 1 kb upstream of the cys-14 gene, binds to CYS3 with a lower affinity than does site C. Site A, located at -150, has only weak CYS3 protein-binding ability (Marzluf et a/., 1995;Li and Marzluf, 1996). It was suggested that these DNA-binding sites might be involved in the transcriptional activation by CYSB protein (Fu and Marzluf, 1990).…”

Section: Introductionmentioning

confidence: 99%

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Functional in vivo studies of the Neurospora crassa cys‐14 gene upstream region: importance of CYS3‐binding sites for regulated expression

Zhou

Marzluf

1996

Molecular Microbiology

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Sulphate transport in Neurospora crassa is achieved by two distinct sulphate permeases, I and II, encoded by the cys-13 and cys-14 genes, respectively. The synthesis of both sulphate permeases is subject to sulphur repression and requires the global positive-acting regulatory protein CYS3, CYS3, a bZIP DNA binding protein, regulates cys-14 expression at the transcriptional level and binds in vitro specifically to three DNA-recognition sites, A, B, and C, in the cys-14 upstream region. In vivo functional analysis of the cys-14 promoter was carried out with 5' deletions and by deletions or mutations of CYS3 DNA-binding sites. The most distal CYS3-binding site, C, located 1.4kb upstream of the transcriptional start site, is necessary and sufficient to mediate strong transcriptional activation by CYS3; moreover, site C was able to function equally well when it was located at variable distances upstream of the cys-14 gene. Site B, located 1 kb upstream, alone is able to support a moderate degree of cys-14 expression. Site A is not required and does not appear to play any functional role in cys-14 expression, even though it is in close proximity to the transcriptional start site. The presence of multiple copies of CYS3-binding elements A or B in the cys-14 promoter results in a parallel increase of regulated gene expression. When a transforming cys-14 gene becomes integrated at ectopic locations in the host genome, it can be expressed in an unregulated fashion, presumably by coming under the control of other promoter elements. Our results also suggested that at least one enzyme in the sulphate catabolic pathway requires a functional CYS3 protein for expression.

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Section: Discussionsupporting

confidence: 84%

Section: Introductionmentioning

confidence: 99%

Functional in vivo studies of the Neurospora crassa cys‐14 gene upstream region: importance of CYS3‐binding sites for regulated expression

Zhou

Marzluf

1996

Molecular Microbiology

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Determination of the Neurospora crassa CYS 3 sulfur regulatory protein consensus DNA-binding site: amino-acid substitutions in the CYS3 bZIP domain that alter DNA-binding specificity

Marzluf

1996

Current Genetics

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CYS3 is the positive-acting global regulatory protein involved in the sulfur control circuit in Neurospora crassa and belongs to the family of bZIP DNA-binding proteins. Here we report a characterization of native DNA-binding sites recognized by CYS3. DNA footprinting experiments and systematic mutational analysis were used to define the consensus CYS3-binding sequence, 5'-ATGPuPyPuPyCAT, a 10-bp palindrome. The sequence 5'-ATGACGTCAT acts as a strong binding site, and all single nucleotide changes within this sequence resulted in a reduction, or even complete loss, of CYS3 DNA-binding. Site-directed mutagenesis was employed to study two uncharged residues, serine 113 and phenylalanine 116, in the basic region of the CYS3 protein bZip DNA-binding domain. Ser113 appears to be directly involved in a specific interaction with nucleotide 2 of the binding site, possibly by making a direct contact with this base, and Phe116 contributes significantly to DNA-binding affinity.

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Functional analysis of different regions of the positive-acting CYS3 regulatory protein of Neurospora crassa

Coulter¹,

Marzluf²

1998

Current Genetics

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In the filamentous fungus Neurospora crassa during conditions of sulfur limitation, CYS3, a major positive-acting regulatory protein, turns on the expression of an entire set of genes which encode permeases and enzymes involved in the acquisition of sulfur from environmental sources. CYS3 functions as a homodimeric protein and possesses a b-Zip domain that confers sequence-specific DNA binding. Expression of various hybrid GAL4-CYS3 fusion proteins in yeast was used to detect regions involved in gene activation. An amino-terminal serine/threonine-rich domain of CYS3 alone strongly activated expression of beta-galactosidase, the yeast reporter. Moreover, mutant CYS3 proteins with amino-acid substitutions in this region that showed increased expression in Neurospora also displayed an enhanced activation potential in yeast. The cys-3 gene of the exotic N. crassa Mauriceville strain and of N. intermedia were cloned and demonstrated to be functional for gene activation and for sulfur-mediated regulation by complementation of a loss-of-function cys-3 mutation. The amino-terminal serine/threonine-rich region is highly conserved in these two CYS3 proteins, in agreement with the possibility that it serves as the activation domain. Surprisingly, an extended promoter region of the cys-3 gene in the Mauriceville strain and in N. intermedia was very well conserved with that of the standard N. crassa gene, including the presence of three CYS3-binding sites possibly involved in autogenous control. Results are presented which indicate that synthesis of the CYS3 regulatory protein is highly regulated and can be detected in the nucleus of cells subjected to sulfur de-repression, but is not found in the nucleus or the cytoplasm of S-repressed cells. The amino-acid substitutions of the CYS3 protein present in a temperature-sensitive cys-3 mutant and in a second-site revertant of a cys-3 null mutation are presented and are shown to affect their DNA-binding activities.

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Global regulation of sulfur assimilation in Neurospora

Cited by 6 publications

References 20 publications

Functional in vivo studies of the Neurospora crassa cys‐14 gene upstream region: importance of CYS3‐binding sites for regulated expression

Functional in vivo studies of the Neurospora crassa cys‐14 gene upstream region: importance of CYS3‐binding sites for regulated expression

Determination of the Neurospora crassa CYS 3 sulfur regulatory protein consensus DNA-binding site: amino-acid substitutions in the CYS3 bZIP domain that alter DNA-binding specificity

Functional analysis of different regions of the positive-acting CYS3 regulatory protein of Neurospora crassa

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