GCN4p activation of the yeast TRP3 gene is enhanced by ABF1p and uses a suboptimal TATA element.
Transcription of the TRP3 gene of Saccharomyces cerevisiae is regulated by GCN4p from a position proximal to the transcriptional initiation sites. The promoter's apparent lack of a conventional TATA element sequence has led it to be used as a model for TATA-less promoters. Through mutational analysis of the TRP3 promoter, we have identified two additional regulatory elements required for expression. The first, located 57 base pairs (bp) upstream of the GCN4p binding site, binds ABF1p in vitro. The ABF1p binding site was required for maximal levels of GCN4p-activated transcription in vivo; however, the -fold activation by GCN4p was not altered by ABF1p. The second element, positioned 23 bp downstream of the GCN4p binding site, contained the TATA-like sequence, TATTAA. This element was required for both basal and activated expression and almost certainly functions as a TATA-binding protein interaction site. Mutations that improved its TATA character for native or an altered specificity mutant of TATA-binding protein correspondingly improved its function. Interestingly, basal expression induced by ABF1p was virtually unchanged in the presence of point mutations in the TATTAA element. Furthermore, unlike the case for HIS3 where only a limited subset of TATA-like sequences can activate transcription in conjunction with GCN4p, many divergent TATA-like sequences allowed GCN4p activation of TRP3. We suggest that the apparent promoter specific use of these TATA elements by GCN4p results from ABF1p amplifying the GCN4p-induced expression to a detectable level.
Martens
J A
JA
Department of Biochemistry, University of Western Ontario, London, Canada.
Brandl
C J
CJ
D000878:Anthranilate Synthase / Q000096:biosynthesis* / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D004271:DNA, Fungal / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005656:Fungal Proteins / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D007209:Indole-3-Glycerol-Phosphate Synthase / Q000096:biosynthesis* / Q000235:genetics; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000096:biosynthesis* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D017354:Point Mutation; D011494:Protein Kinases / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D016385:TATA Box; D014157:Transcription Factors; D014158:Transcription, Genetic
C061886:ABF1 protein, S cerevisiae; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D009097:Multienzyme Complexes; D009838:Oligodeoxyribonucleotides; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D011494:Protein Kinases; D007209:Indole-3-Glycerol-Phosphate Synthase; D000878:Anthranilate Synthase; C087439:TRP3 protein, S cerevisiae