Interaction of GAL4 and GAL80 gene regulatory proteins in vitro.

The GAL80 protein of Saccharomyces cerevisiae, synthesized in vitro, bound tightly to GAL4 protein and to a GAL4 protein-upstream activation sequence DNA complex, as shown by (i) coimmunoprecipitation of GAL4 and GAL80 proteins with anti-GAL4 antiserum, (ii) an electrophoretic mobility shift of a GAL4 protein-upstream activation sequence DNA complex upon the addition of GAL80 protein, and (iii) GAL4-dependent binding of GAL80 protein to upstream activation sequence DNA immobilized on Sepharose beads. Anti-GAL4 antisera were raised against a GAL4-URA3 fusion protein, which could be purified to homogeneity in a single step with the use of an affinity chromatographic procedure for the URA3 gene product.     

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.     

Mechanism of action of a yeast activator: direct effect of GAL4 derivatives on mammalian TFIID-promoter interactions

We have analyzed interactions between the mammalian TATA factor (TFIID) and derivatives of the yeast activator GAL4. The interaction of the TATA factor on the adenovirus E4 promoter with GAL4 binding sites adjacent to the TATA site was qualitatively altered in response to GAL4 binding. Alterations in the TFIID interactions were observed with two GAL4 derivatives that stimulated hybrid E4 promoter activity in vitro but not with a third derivative that bound to DNA but showed no activation. These results indicate that TFIID is a direct target for a GAL4 activation domain and suggest a simple general model for the activation mechanism.     

GAL4 protein: purification, association with GAL80 protein, and conserved domain structure.

Expression of the yeast Saccharomyces cerevisiae GAL4 protein under its own (galactose-inducible) control gave 5 to 10 times the level of protein observed when the GAL4 gene was on a high-copy plasmid. Purification of GAL4 by a procedure including affinity chromatography on a GAL4-binding DNA column yielded not only GAL4 but also a second protein, shown to be GAL80 by its reaction with an antipeptide antibody. Sequence comparisons of GAL4 and other members of a family of proteins sharing homologous cysteine finger motifs identified an additional region of homology in the middle of these proteins shown by genetic analysis to be important for GAL4 function. GAL4 could be cleaved proteolytically at the boundary of the conserved region, defining internal and carboxy-terminal folded domains.     

Carbonic anhydrase activators: synthesis of high affinity isozymes I, II and IV activators, derivatives of 4-(4-tosylureido-amino acyl)ethyl-1H-imidazole (histamine derivatives)

Reaction of histamine (Hst) with tetrabromophthalic anhydride and protection of its imidazole moiety with tritylsulfenyl chloride, followed by hydrazinolysis, afforded N-1-tritylsulfenyl histamine, a key intermediate which was further derivatized at its aminoethyl moiety. Reaction of the key intermediate with 4-tosylureido amino acids/dipeptides (ts-AA) in the presence of carbodiimides, afforded after deprotection of the imidazole moiety, a series of compounds with the general formula ts-AA-Hst (ts=4-MeC(6) H(4) SO(2) NHCO). Some structurally related dipeptide derivatives with the general formula ts-AA1-AA2-Hst, were also prepared, by in a similar way to the amino acyl compounds mentioned above. The new derivatives were examined as activators of three carbonic anhydrase (CA) isozymes, hCA I, hCA II (cytosolic forms) and bCA IV (membrane-bound form). Efficient activation was observed against all three isozymes, but especially against hCA I and bCA IV, with affinities in the 1-10 nanomolar range for the best compounds. hCA II was on the other hand activatable with affinities around 20-50 nM. This new class of CA activators might lead to the development of drugs/diagnostic agents for the CA deficiency syndrome, a genetic disease of bone, brain and kidneys.