Biochemical and genetic characterization of the dominant positive element driving transcription ofthe yeast TBP-encoding gene, SPT15.
We previously demonstrated that a combination of both positive and negative cis -acting upstream elements control the transcription of the gene encoding TBP ( SPT15 ) in Saccharomyces cerevisiae . One of these elements found in that study, resident between 5' flanking sequences -147 and -128 , and termed PED (for positive element distal), was found to play an essential positive role in driving transcription of the gene encoding TBP. In this report, we map at nucleotide-level resolution, the critical residues which comprise PED, purify and sequence the protein that binds to it and determine that this PED binding factor is Abf1p, an abundant yeast protein previously broadly implicated in both gene regulation and DNA replication. In the case of the TBP-encoding gene, however, Abf1p works through the PED element which is a non-consensus binding site. Based upon the work of others, the PED-variant ABF1 site would be predicted to be a very poor binding site for this factor yet Abf1p binds PED and a consensus ABF1 site with comparable affinity. These results are discussed in light of the broader context of Abf1p-mediated gene regulation.
Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville,TN 37232-0615, USA.
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D001483:Base Sequence; D018797:Cell Cycle Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D002846:Chromatography, Affinity; D015342:DNA Probes; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D016385:TATA Box; D035181:TATA-Box Binding Protein / Q000031:analogs & derivatives; D014157:Transcription Factors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014158:Transcription, Genetic; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics
C061886:ABF1 protein, S cerevisiae; D018797:Cell Cycle Proteins; D015342:DNA Probes; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; C115017:SPT15 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D035181:TATA-Box Binding Protein; D014157:Transcription Factors; D012321:DNA-Directed RNA Polymerases; D001616:beta-Galactosidase