Purification and binding properties of the Mal63p activator of Saccharomyces cerevisiae.
Mal63p is a transcriptional activator for maltose fermentation in Saccharomyces cerevisiae. We have purified it to homogeneity from a yeast strain in which the MAL63 gene is under the control of the GAL1-GAL10 promoter. Purification included fractionation of a whole-cell extract by ion-exchange chromatography, chromatography using both non-specific DNA-affinity (calf thymus), and sequence-specific DNA-affinity chromatography. Mal63p activity was assayed by its binding to a fragment of the MAL61-MAL62 promoter, using both filter-binding and electrophoretic-mobility shift assays. DNase-I footprinting identified a new binding site (site 3) between the two previously known sites (sites 1 and 2). Mal63p is a dimer, and methylation-protection experiments identify the recognition motif as: c/a GC N9 c/a GC/g.
Sirenko
O I
OI
Wayne State University School of Medicine, Department of Biochemistry, Detroit, MI 48201, USA.
Ni
B
B
Needleman
R B
RB
D001483:Base Sequence; D001665:Binding Sites; D004271:DNA, Fungal / Q000235:genetics / Q000378:metabolism; D005285:Fermentation; D005656:Fungal Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D005800:Genes, Fungal; D008320:Maltose / Q000378:metabolism; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D015534:Trans-Activators / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
D004271:DNA, Fungal; D005656:Fungal Proteins; D015534:Trans-Activators; D008320:Maltose