Interactions of purified transcription factors: binding of yeast MAT alpha 1 and PRTF to cell type-specific, upstream activating sequences.
Pheromone receptor transcription factor (PRTF) and MAT alpha 1 are protein transcription factors that are involved in the regulation of the alpha-specific genes in Saccharomyces cerevisiae. We have expressed MAT alpha 1 as a fusion protein in Escherichia coli and purified it from inclusion bodies in milligram quantities. The MAT alpha 1 protein was obtained after specific cleavage of the fusion protein. Quantitative band shift electrophoresis was used to determine the equilibrium dissociation constants that describe the multicomponent binding equilibrium between the PRTF and MAT alpha 1 proteins, and alpha-specific STE3 upstream activating sequence (UAS) DNA. The dissociation constant for the complex of PRTF and the a-specific UAS of STE2 was also measured and found to be 5.9 X 10(-11) M, only three times less than that for the PRTF-STE3 UAS complex. Analyses of these complexes by DNase I footprinting demonstrate that the PRTF binding site is confined to the palindromic P-box sequence in the case of the STE3 UAS, but extends symmetrically from this central region to cover 28 bp for the STE2 UAS. When MAT alpha 1 is bound to the PRTF-STE3 complex, the region of DNA protected is enlarged to that seen for the PRTF-STE2 complex. Our results using these two purified factors in vitro suggest that PRTF has nearly the same affinity for a- and alpha-specific UAS elements and that transcriptional activation requires a particular conformational state for the PRTF-DNA complex which occurs in the PRTF-STE2 and MAT alpha 1-PRTF-STE3 complexes, but not in the PRTF-STE3 complex.
Tan
S
S
MRC Laboratory of Molecular Biology, Cambridge UK.
Ammerer
G
G
Richmond
T J
TJ
D001483:Base Sequence; D001665:Binding Sites; D004271:DNA, Fungal / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D000072235:Mating Factor; D008969:Molecular Sequence Data; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism; D051964:Receptors, Mating Factor; D018000:Receptors, Peptide; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D004271:DNA, Fungal; D010455:Peptides; D011956:Receptors, Cell Surface; D051964:Receptors, Mating Factor; D018000:Receptors, Peptide; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D000072235:Mating Factor