Functional analysis of the PUT3 transcriptional activator of the proline utilization pathway in Saccharomyces cerevisiae.
Proline can serve as a nitrogen source for the yeast Saccharomyces cerevisiae when preferred sources of nitrogen are absent from the growth medium. PUT3, the activator of the proline utilization pathway, is required for the transcription of the genes encoding the enzymes that convert proline to glutamate. PUT3 is a 979 amino acid protein that constitutively binds a short DNA sequence to the promoters of its target genes, but does not activate their expression in the absence of induction by proline and in the presence of preferred sources of nitrogen. To understand how PUT3 is converted from an inactive to an active state, a dissection of its functional domains has been undertaken. Biochemical and molecular tests, domain swapping experiments, and an analysis of activator-constitutive and activator-defective mutant proteins indicate that PUT3 is dimeric and activates transcription with its negatively charged carboxyterminus, which does not appear to contain a proline-responsive domain. A mutation in the conserved central domain found in many fungal activators interferes with activation without affecting DNA binding protein stability. Intragenic suppressors of the central domain mutation have been isolated and analyzed.
des Etages
S A
SA
Department of Microbiology and Molecular Genetics, UMD-New Jersey Medical School, Newark 07013, USA.
Falvey
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DA
Reece
R J
RJ
Brandriss
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MC
D001483:Base Sequence; D001665:Binding Sites; D004271:DNA, Fungal; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D009584:Nitrogen / Q000378:metabolism; D011392:Proline / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D012996:Solutions; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics*; D014157:Transcription Factors
D004271:DNA, Fungal; D005656:Fungal Proteins; C068399:PUT3 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D012996:Solutions; D015534:Trans-Activators; D014157:Transcription Factors; D011392:Proline; D009584:Nitrogen