Purification and cloning of a DNA binding protein from yeast that binds to both silencer and activator elements.
A DNA binding protein (RAP1, previously called SBF-E) has been shown to bind to putative regulatory sites at both yeast mating-type silencers, yet is not the product of genetically identified regulators of the silent loci. Here, we report the purification of RAP1 by DNA affinity chromatography, and the isolation of its gene from a lambda gt11 genomic library using antibodies raised against the protein. Disruption of the chromosomal copy of this gene is lethal. We show that RAP1 protein also binds in vitro to the upstream activation site (UAS) of MAT alpha and ribosomal protein genes. In addition, we show that two different UAS-associated RAP1 binding sites can substitute in vivo for a silencer binding site. Our results suggest that RAP1 may be a transcriptional regulator that can play a role in either repression or activation of transcription, depending upon the context of its binding site.
Shore
D
D
MRC Laboratory of Molecular Biology, Cambridge, England.
Nasmyth
K
K
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification*; D005656:Fungal Proteins / Q000235:genetics / Q000302:isolation & purification*; D005786:Gene Expression Regulation; D005800:Genes, Fungal; D049770:Genes, Mating Type, Fungal; D005809:Genes, Regulator; D008969:Molecular Sequence Data; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics; D014158:Transcription, Genetic
D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D014157:Transcription Factors