GAL11P: a yeast mutation that potentiates the effect of weak GAL4-derived activators

A mutant yeast in which a weak GAL4-derived activator functions as a strong activator bears a single mis-sense mutation in GAL11 (a.k.a. SPT13). The first 74 amino acids of GAL4, including the zinc-dependent DNA binding region, attached to an acidic activating sequence, are sufficient to respond both to GAL11 and to our mutant GAL11P (potentiator). PPR1, a yeast activator with a similar zinc finger sequence, also responds to GAL11 and to GAL11P, whereas regulators bearing unrelated DNA binding motifs do not. GAL11 itself works as a strong activator when tethered to DNA by fusion to the bacterial LexA protein, and deletion of GAL11 is known to cause a 5- to 10-fold reduction in GAL4 activity. We suggest that a complex of GAL4 and GAL11 constitutes a particularly strong activator; evidence that the putative GAL4-GAL11 complex ordinarily forms preferentially on DNA suggests a biological rationale for GAL11 action.     

Fluorescence based assay of GAL system in yeast Saccharomyces cerevisiae

The GAL1 promoter is one of the strongest inducible promoters in the yeast Saccharomyces cerevisiae. In order to improve recombinant protein production we have developed a fluorescence based method for screening and evaluating the contribution of various gene deletions to protein expression from the GAL1 promoter. The level of protein synthesis was determined in 28 selected mutant strains simultaneously, by direct measurement of fluorescence in living cells using a microplate reader. The highest, 2.4-fold increase in GFP production was observed in a gal1 mutant strain. Deletion of GAL80 caused a 1.3-fold increase in fluorescence relative to the isogenic strain. GAL3, GAL4 and MTH1 gene deletion completely abrogated GFP synthesis. Growth of gal7, gal10 and gal3 also exhibited reduced fitness in galactose medium. Other genetic perturbations affected the GFP expression level only moderately. The fluorescence based method proved to be useful for screening genes involved in GAL1 promoter regulation and provides insight into more efficient manipulation of the GAL system.