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

Abstract

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.

Aspects of the Neurospora crassa Sulfur Starvation Response Are Revealed by Transcriptional Profiling and DNA Affinity Purification Sequencing

“…2 and 4A ; Data Set S2 ). We confirmed CYS-3 binding 1.4 kbp upstream of the cys-14 translation start site ( 35 ) ( Data Set S2 ), which is necessary and sufficient for full, regulated expression of cys-14 ( 44 ). The direct regulation of so many transporters by CYS-3 suggests sulfur import is an important aspect of the sulfur starvation response.…”

Sulfate Transport in Penicillium chrysogenum : Cloning and Characterization of the sutA and sutB Genes

“…2 and 4). In N. crassa, S regulation is positively mediated by the DNA-binding protein CYS-3p (22,23,26,27,32). Recently, a positively acting CYS-3p homologue has been found in A. nidulans (37).…”

“…No CYS-3p homologue has been reported for P. chrysogenum. Sequences that weakly resemble the CYS-3p binding-site consensus ATGR YRYCAT (26,27) Hydropathy analysis (28,29,47,58) of SutA and SutB shows a pattern typical for a polytopic membrane protein, with 14 putative hydrophobic transmembrane (TM) helices in the Nterminal part of the protein followed by a long C-terminal extension (Fig. 5A).…”

“…Sulfate uptake is an important point of regulation of the sulfur metabolism in fungi. In Neurospora crassa, sulfate uptake is subject to a mechanism called sulfur (metabolite) repression or regulation, involving the action of positively and negatively acting regulatory proteins on the expression of sulfate permease-encoding genes (22,27,32). A similar situation holds for Aspergillus nidulans (30,35,36) and Saccharomyces cerevisiae (9,55).…”

Sulfate permeasesphylogenetic diversity of sulfate transport.

“…Mutations which abolish sulfate uptake lead to resistance to toxic analogs of sulfate, like selenate, chromate, tungstate and molybdate. The permease-encoding genes were found to be strongly regulated at the transcriptional level (Li et al, 1996;Cherest et al, 1997;van de Kamp et al, 1999;Paszewski et al, 2000;Natorff et al, 2003).…”