DNA-binding properties of the yeast transcriptional activator, Gcr1p.
In Saccharomyces cerevisiae the GCRI gene product is required for high-level expression of genes encoding glycolytic enzymes. In this communication, we extend our analysis of the DNA binding properties of Gcr1p. The DNA-binding domain of Gcr1p binds DNA with high affinity. The apparent dissociation constant of the Gcr1p DNA-binding domain for one of its specific binding sites (TTTCAGCTTCCTCTAT) is 2.9 x 10(-10) M. However, competition experiments showed that Gcr1p binds this site in vitro with a low degree of specificity. We measured a 33-fold difference between the ability of specific competitor and DNA of random sequence to inhibit the formation of nucleoprotein complexes between Gcr1p and a radiolabeled DNA probe containing its binding site. DNA band-shift experiments, utilizing probes of constant length in which the positions of Gcr1p-binding sites are varied relative to the ends, indicated that Gcr1p-DNA nucleoprotein complexes contain bent DNA. The implications of these findings in terms of the combinatorial interactions that occur at the upstream activating sequence elements of genes encoding glycolytic enzymes are discussed.
Huie
M A
MA
Department of Molecular Genetics and Microbiology, University of Florida, College of Medicine, Gainesville 32610-0266, USA.
Baker
H V
HV
D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005656:Fungal Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D012441:Saccharomyces cerevisiae / Q000737:chemistry*; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators / Q000378:metabolism*; D014157:Transcription Factors
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C076227:GCR1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D004247:DNA