The Saccharomyces cerevisiae PUT3 Activator Protein Associates with Proline-Specific Upstream Activation Sequences

Abstract

The PUT1 and PUT2 genes encoding the enzymes of the proline utilization pathway of Saccharomyces cerevisiae are induced by proline and activated by the product of the PUT3 gene. Two upstream activation sequences (UASs) in the PUT1 promoter were identified by homology to the PUT2 UAS. Deletion analysis of the two PUT1 UASs showed that they were functionally independent and additive in producing maximal levels of gene expression. The consensus PUT UAS is a 21-base-pair partially palindromic sequence required in vivo for induction of both genes. The results of a gel mobility shift assay demonstrated that the proline-specific UAS is the binding site of a protein factor. In vitro complex formation was observed in crude extracts of yeast strains carrying either a single genomic copy of the PUT3 gene or the cloned PUT3 gene on a 2 microns plasmid, and the binding was dosage dependent. DNA-binding activity was not observed in extracts of strains carrying either a put3 mutation that caused a noninducible (Put-) phenotype or a deletion of the gene. Wild-type levels of complex formation were observed in an extract of a strain carrying an allele of PUT3 that resulted in a constitutive (Put+) phenotype. Extracts from a strain carrying a PUT3-lacZ gene fusion formed two complexes of slower mobility than the wild-type complex. We conclude that the PUT3 product is either a DNA-binding protein or part of a DNA-binding complex that recognizes the UASs of both PUT1 and PUT2. Binding was observed in extracts of a strain grown in the presence or absence of proline, demonstrating the constitutive nature of the DNA-protein interaction.

Proline catabolism is a key factor facilitating Candida albicans pathogenicity

“…Put3 is a transcription factor in S . cerevisiae that contains an N-terminal Zn (II) 2 Cys 6 binuclear cluster DNA binding domain and a C-terminal domain that undergoes a proline-dependent conformational change that activates PUT gene expression [ 34 , 35 ]. As in S .…”

“…Put3 is a transcription factor in S. cerevisiae that contains an N-terminal Zn (II) 2 Cys 6 binuclear cluster DNA binding domain and a C-terminal domain that undergoes a proline-dependent conformational change that activates PUT gene expression [34,35]. As in S. cerevisiae, (CFG154), put2-/-(CFG318) and put3-/-(CFG156); put1-/-put2-/-(put1 put2, CFG159) derived using both pV1524 and pV1093; and control strains pV1093 (CFG181) and pV1524 (CFG182) carrying the vector without guide RNA.…”

FacB, the Aspergillus nidulans activator of acetate utilization genes, binds dissimilar DNA sequences

“…Zn(II)2Cys6 DNA-binding domains interact with DNA-binding sites which are similar in both structure and sequence, consisting of conserved terminal trinucleotides, usually in a symmetrical configuration, spaced by an internal variable sequence of defined length, e.g. GAL4 and LAC9 bind to CGGN 11 CCG, PPR1 and UaY bind to CGGN 6 CCG, and PUT3 binds to CGGN 10 CCG (Carey et al, 1989;Siddiqui and Brandriss, 1989;Halvorsen et al, 1990Halvorsen et al, , 1991Roy et al, 1990;Go ¨decke et al, 1991;Marmorstein et al, 1992;Sua ´rez et al, 1995; for a review, see Todd and Andrianopoulos, 1997).…”