Mutations in a dispensable region of the UaY transcription factor of Aspergillus nidulans differentially affect the expression of structural genes

Abstract

SummaryThe uaY gene encodes a transcriptional activator mediating uric acid induction of at least nine genes of the purine-utilization pathway. In this article, we characterize a loss-of-function mutation, uaY205, as a 16 bp deletion that results in premature translation termination, and substitutes the C-terminal 63 amino acids for 13 amino acid residues. Reversion analysis demonstrates that the C-terminal 63 amino acid residues are unnecessary for UaY function, and that the lossof-function phenotype resulting from the uaY205 mutation is caused by the new amino acid sequence present in the mutant protein. Revertants in two different frames (wild type and þ1) restore function but show subtle differences in the expression of genes controlled by the UaY protein. Two strains showing elevated expression of genes under UaY control were shown to carry, in addition to a mutation leading to the recovery of the wild-type open reading frame, mutations in unlinked genes. Using crude extracts of Aspergillus nidulans, we have been able to detect, for the first time, in transcription factors of this class, specific retardation of a promoter probe. The binding activity is at least partially dependent on the presence of inducer. The gel shift experiments show that the novel inhibitor y sequence present in the UaY205 protein can act either by affecting the stability of the protein, or via an inter-or intramolecular interaction impairing the specific DNA-binding activity.

Phenotypes of Mutations in the 5′-UTR of a Limiting Transcription Factor in Aspergillus nidulans Can Be Accounted For by Translational Inhibition and Leaky Scanning

“…The suppressor carried by 12r122 does not suppress the uaY2 (amber, Suárez et al 1995), uaY205 (frameshift, Oestreicher et al 1997), and uaY808 (deletion, Suárez et al 1991bSuárez et al , 1995 mutations. The suppressor carried by 12r128 does not suppress uaY808 (other alleles not tested).…”

“…Plasmids used in back-transformation experiments: That the revertant alleles: uaY12r3, 12r17, 12r18, 12r113, 12r118, and 12r130 are responsible for the phenotype observed, was confirmed by partial gene replacement (Miller et al 1985;Oestreicher et al 1997) as described below. A 667-bp fragment (from position À294 to 1373) was amplified [see above for PCR conditions, but using pfu polymerase (Promega) rather than Taq polymerase].…”

“…The leaky mutation uaY109 is a T-to-A transversion at position 1334, which results in a Phe-to-Ile change at position 112 of the UaY protein (N. Oestreicher, unpublished results). uaY205 is a 16-bp deletion starting at position 2992, which results in premature translation termination and substitutes the C-terminal 63 amino acids by 13 new residues (Oestreicher et al 1997).…”

The tightly regulated promoter of the xanA gene of Aspergillus nidulans is included in a helitron

“…The specific inducer of the transporters and enzymes of this pathway is uric acid (Scazzocchio and Darlington, 1968; Scazzocchio, 1973; 1994; Sealy Lewis et al ., 1978; Scazzocchio et al ., 1982; Cecchetto et al ., 2004). Induction is mediated by UaY (Scazzocchio et al ., 1982; Suarez et al ., 1991a,b; Oestreicher and Scazzocchio, 1995; Oestreicher et al ., 1997), a protein belonging to the 6‐cysteine zinc binuclear cluster family of transcriptional activators. UaY binds to inverted repeat sites of the form 5′‐CGG‐X 6 ‐CCG‐3′ (Suarez et al ., 1995).…”

The oxpA5 mutation of Aspergillus nidulans is an allele of adB, the gene encoding adenylosuccinate synthetase

“…An ordered cosmid library of chromosome VIII (obtained from the Fungal Genetics Stock Center) was screened with a 2108-bp BamHI fragment internal to the uaY gene (the insert from the plasmid bAN758; Oestreicher et al 1997), yielding four hybridising cosmids. Three of these, L11FO7, W19C03 and W11C10, were used to transform the strain CS977, which carries both the oxpA5 (resulting in resistance to oxypurinol) and uaY12 (resulting in incapacity to grow on hypoxanthine as sole nitrogen source) mutations.…”