Mutational analysis reveals dispensability of the N‐terminal region of the Aspergillus transcription factor mediating nitrogen metabolite repression

Abstract

Mutational analysis has enabled identification and localization of an upstream exon of the areA gene of Aspergillus nidulans mediating nitrogen metabolite repression. A mutation in the initiation codon and frameshift mutations, which revert by restoration of the reading frame, established the coding role of the exon and mutations affecting intron splicing in conjunction with DNA sequencing of reverse transcriptase polymerase chain reaction (RT-PCR) products localized the coding region intron. The resulting AREA translation product would have 876 residues. Deletion of the upstream exon such that translation of the remaining areA coding region would yield a protein containing only the 719 C-terminal residues has only a subtle phenotype, very similar to those resulting from single amino acid replacements in upstream exon-encoded regions of strong sequence similarity to the Neurospora crassa and Penicillium chrysogenum homologues. A number of areA mRNAs of different sizes are synthesised and appear to be functionally redundant. Synthesis of at least the smallest mRNA(s) is probably subject to autogenous activation. Suppression of frameshift mutations by compensating mutations preventing intron splicing suggests that insertion of a markedly hydrophobic sequence can impair AREA function. Finally, translational initiation for areA can occur within a region of at least 123 nucleotides.

Nuclear Gln3 Import Is Regulated by Nitrogen Catabolite Repression Whereas Export Is Specifically Regulated by Glutamine

“…1975; Kudla et al 1990;Langdon et al 1995;Platt et al 1996a,b). We compared growth of wild-type (pRR536), 340ddd Gln3-Myc 13 (pRR1215), 64araa Gln3-Myc 13 (pRR1090), 64drdd Gln3-Myc 13 (pRR752), and 332dddd Gln3-Myc 13 (pRR787) transformants in both repressive (glutamine) and derepressive (proline or allantoin) media (Figure 9A).…”

Role of Glutamine Synthetase in Nitrogen Metabolite Repression in Aspergillus nidulans

“…AreA binds via a single GATA zinc finger to the promoters of nitrogen-responsive genes (25,41). The synthesis and activity of AreA are modulated through changes in areA mRNA transcription and stability, as well as interaction with the negatively acting NmrA protein in response to changes in the nitrogen status of the cell (2,26,36). The phenotypes of mutants affected in ammonium assimilation indicate that ammonium alone is not the key effector for the modulation of AreA function.…”

Molecular characterisation of meaB, a novel gene affecting nitrogen metabolite repression in Aspergillus nidulans

“…Null mutants of areA are characterised by their inability to grow on nitrogen sources other than ammonium and L-glutamine [2,6]. The product of this gene, AREA, is a member of the GATA family of DNA binding proteins [8][9][10], capable of binding at promoter sequences containing the core motif -GATA-to direct gene expression [11,12]. areA activity is regulated, in response to the nitrogen state of the cell, by two independent mechanisms.…”