Purification and characterization of the yeast transcriptional activator GAL4.
We have purified extensively the transcriptional activator, GAL4, from a yeast strain overexpressing the gene product from the ADH1 promoter. Our purification followed GAL4 activity by its binding to a specific DNA target sequence, using filter binding assays. No specific binding activity was detected in extracts from a strain containing a disrupted copy of the GAL4 gene. The purification protocol included fractionation of a whole cell extract by ion-exchange and DNA-affinity chromatography on a column containing a 17-base pair oligomer encoding a near consensus GAL4 binding site. Two polypeptides co-eluted with the GAL4 DNA binding activity from the DNA-affinity column. One had an apparent molecular mass of 99 kDa (the predicted size of the GAL4 protein) and cross-reacted with antibodies raised against GAL4 epitopes from fusion proteins expressed in bacterial cells. The second polypeptide did not cross-react with the anti-GAL4 antibody and is presumed to be the GAL80 transcriptional repressor based on its size (48 kDa) and known physical association with the GAL4 protein. GAL4 binding activity elutes from a gel filtration column as a 155-kDa species suggesting that it exists in solution in a heterodimer complex of one GAL4 and one GAL80 molecule. The dissociation constant of the DNA-affinity-purified GAL4-GAL80 complex for a 900-base pair DNA fragment containing the UASGAL element from the GAL1-GAL10 divergent promoter was, Kd(effective) (0.15 M KCl) = 2.4 x 10(-9) M.
Department of Biology, Indiana University, Bloomington 47405.
M R
MR
Parthun
J A
JA
Jaehning
D015153:Blotting, Western; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins; D005656:Fungal Proteins / Q000302:isolation & purification* / Q000378:metabolism; D046911:Macromolecular Substances; D008970:Molecular Weight; D012097:Repressor Proteins; D012441:Saccharomyces cerevisiae / Q000032:analysis*; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors / Q000302:isolation & purification* / Q000378:metabolism
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C052130:GAL4 protein, S cerevisiae; C055002:GAL80 protein, S cerevisiae; D046911:Macromolecular Substances; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D004247:DNA