The sequence and binding specificity of UaY, the specific regulator of the purine utilization pathway in Aspergillus nidulans, suggest an evolutionary relationship with the PPR1 protein of Saccharomyces cerevisiae.

Abstract

The uaY gene codes for a transcriptional activator mediating the induction of a number of unlinked genes involved in purine utilization in Aspergillus nidulans. Here we present the complete genomic and cDNA nucleotide sequence of this gene. The gene contains two introns. The derived polypeptide of 1060 residues contains a typical zinc binuclear cluster domain and shows a number of similarities with the PPR1 regulatory gene of Saccharomyces cerevisiae. These similarities are most striking in the putative linker and dimerization regions following the zinc cluster. Gel‐shift and DNase I footprinting experiments have been carried out for three genes subject to UaY‐mediated induction. The binding sequence is 5′‐TCGG‐6X‐CCGA, which is identical to the proposed PPR1 binding sites. Nevertheless, the identity of the base immediately 3′ of the 5′‐TCGG sequence clearly affects the affinity of the site. The site upstream of the uapA gene has been shown to be active in vivo. Binding to this site has been analysed by a number of interference techniques. There is an interesting chemical similarity between the co‐inducer of the purine utilization pathway (uric acid) and that of the genes of the pyrimidine biosynthetic pathway (dihydroorotic acid) and we show that dihydroorotic acid can act as a poor inducer of at least one activity under UaY control. These striking similarities, together with the unique pattern of regulation of pyrimidine biosynthesis in S. cerevisiae, suggest that PPR1 evolved through recruitment into the pyrimidine biosynthetic pathway of an ancestral gene related to uaY.

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 uaY gene encodes a 1060-amino-acid protein, containing a zinc binuclear cluster DNA-binding domain, which binds to DNA sequences of the form CCG-6X-CGG (Suárez et al 1995). The uaY gene is transcribed at a very low level, and at variance with previous reports (Suárez et al 1991b(Suárez et al , 1995, it is mildly autoregulated, with an induction ratio of 1.4 (N. Oestreicher, unpublished results).…”

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

“…Moreover, it binds DNA sequences of the form CCG-6X-CGG. These sequences are identical to those recognized by PPR1 (Roy et al, 1990), and we have proposed that a close evolutionary relationship exists between the two proteins (Suá rez et al, 1995).…”

“…It is interesting that the upstream uapA UaY-binding site shows an asymmetrically placed, non-protected base in DNase I footprints. This asymmetry is absent in the promoters of uaZ and hxA (Suá rez et al, 1995). A mutation resulting in constitutivity for all genes under UaY control (S222 →L; Oestreicher and Scazzocchio, 1995), maps in a different domain from the uaY205 revertants.…”

“…Transformation experiments with pBluescript-derived plasmids containing different and overlapping fragments of the uaY gene (as shown in Fig. 1) located the uaY205 mutation in the 936 bp BamHI fragment situated between nucleotides 2688 and 3624 in the uaY sequence (Suá rez et al, 1995). To clone this fragment, a gene library was made from DNA extracted from strain A. nidulans CS979, which was restricted with EcoRI.…”

“…Under the protein sequence, the resulting reading-frame change (wild type, ¹1 or þ1) is shown. Numbering refers to the published nucleotide sequence and protein sequence (Suá rez et al, 1995). New amino acids resulting from the mutations are shown in bold; where these differ in mutants belonging to the same reading-frame class, they are also underlined.…”