A simple in vivo footprinting method to examine DNA-protein interactions over the yeast PYK UAS element.
In this report a modification to the in vivo footprinting assay is described. The method includes dimethyl sulfate treatment of whole yeast cells, followed by reiterative primer extension of the methylated genomic DNA using Taq DNA polymerase. Under appropriate reaction conditions chain extension terminates opposite a methylated purine when Taq DNA polymerase encounters a modified adenine or guanine. The procedure was used to examine, in vivo DNA-protein contacts over the upstream activation site (UAS) of the Saccharomyces cerevisiae PYK gene. In vivo analysis, using isogenic strains of yeast and Escherichia coli transformed with plasmid DNAs, confirmed the binding of both the trans-acting factor RAP1 and the transcriptional activator GCR1 to cis-acting recognition sites located within the PYK UAS element.
Dumitru
I
I
Department of Microbiology, University of Toronto, Ontario, Canada.
McNeil
J B
JB
D001483:Base Sequence; D004271:DNA, Fungal / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics; D005821:Genetic Techniques; D008745:Methylation; D008969:Molecular Sequence Data; D011770:Pyruvate Kinase / Q000235:genetics*; D012045:Regulatory Sequences, Nucleic Acid; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D013463:Sulfuric Acid Esters
D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D013463:Sulfuric Acid Esters; D011770:Pyruvate Kinase; C007482:dimethyl sulfate