In yeast strains bearing the point mutation called GAL11P (for potentiator), certain GAL4 derivatives lacking any classical activating region work as strong activators. The P mutation confers upon GAL11, a component of the RNA polymerase II holoenzyme, the ability to interact with a portion of the dimerization region of GAL4. The region of GAL11 affected by the P mutation is evidently functionally inert in ordinary cells, suggesting that this mutation is of no functional significance beyond creating an artificial target for the GAL4 dimerization fragment. From these observations and further analyses of GAL11, we propose that a single activator-holoenzyme contact can trigger gene activation simply by recruiting the latter to DNA.

; 2Institut ffir Molekularbiologie, Universit/it Zfirich, SwitzerlandThe single amino acid "P" (potentiator) mutation in the holoenzyme component GALl l creates an interaction between that protein and the dimerization region of GAL4. That interaction triggers strong gene activation when the GAL4 fragment is tethered to DNA. Here we show that, among a series of variants of the GAL4 dimerization region and different GALl lP alleles, the strength of the interaction as quantitated in vitro correlates with the degree of activation in vivo; swapping the protein fragments bearing the GAL4 dimerization region and the GALllP mutation such that the latter is tethered to DNA and the former is attached to the holoenzyme does not diminish gene activation; gene activation in this system is squelched by overproduction of either a fragment bearing the GAL4 dimerization region or a fragment of GALl 1 bearing a P mutation; and neither GALl 1 nor GALl 1P is a target of an acidic activating region. These results argue that the GAL4-GALllP interaction triggers gene activation simply by recruiting the holoenzyme to DNA. Consistent with this view, we also show that fusion of LexA to another holoenzyme component, SRB2, creates an activator, and that an SRB2 mutant predicted on genetic grounds to interact especially efficiently with a holoenzyme containing a specific mutant form of polymerase also activates more efficiently when tethered to DNA.

The GAL4 dimerization domain (GAL4-dd) is a powerful transcriptional activator when tethered to DNA in a cell bearing a mutant of the GAL11 protein, named GAL11P. GAL11P (like GAL11) is a component of the RNA-polymerase II holoenzyme. Nuclear magnetic resonance (NMR) studies of GAL4-dd revealed an elongated dimer structure with C 2 symmetry containing three helices that mediate dimerization via coiled-coil contacts. The two loops between the three coiled coils form mobile bulges causing a variation of twist angles between the helix pairs. Chemical shift perturbation analysis mapped the GAL11P-binding site to the C-terminal helix ␣3 and the loop between ␣1 and ␣2. One GAL11P monomer binds to one GAL4-dd dimer rendering the dimer asymmetric and implying an extreme negative cooperativity mechanism. Alanine-scanning mutagenesis of GAL4-dd showed that the NMR-derived GAL11P-binding face is crucial for the novel transcriptional activating function of the GAL4-dd on GAL11P interaction. The binding of GAL4 to GAL11P, although an artificial interaction, represents a unique structural motif for an activating region capable of binding to a single target to effect gene expression. The GAL4 transcriptional activator is required for the regulation of genes involved in galactose and melibiose metabolism in the yeast Saccharomyces cerevisiae (Johnston 1987). GAL4 has a modular structure in which distinct regions of the molecule mediate recognition and binding to DNA, activation of transcription, and dimerization (Fig. 1A;Keegan et al. 1986;Ma and Ptashne 1987;Lin et al. 1988;Carey et al. 1990). Only the structure of the DNA-binding domain (residues 1-66) has been solved alone in solution (Baleja et al. 1992;Kraulis et al. 1992) and in complex with DNA . It contains an N-terminal DNA-recognition domain (residues 7-40), in which six cysteines bind two Zn +2 ions. In the free protein in solution, the region from residue 41-66 is unstructured. In the crystal structure of the complex with DNA, residues 50-63 form a coiled-coil dimerization element that extends perpendicularly away from the DNA helix . Nevertheless, GAL4 1-65 is a monomer in the absence of DNA. Functional characterization of the 881 residue GAL4 protein has shown that a DNA-independent dimerization domain extends beyond residue 63 comprising the sequence from 65 to 94 (Carey et al. 1989); however, no structure of a construct containing the complete dimerization domain has been reported yet. Transcriptional activation function in yeast has been assigned to two regions: the primary activating region comprising residues 768-881 and a weaker one that lies between residues 148-196 (Keegan et al. 1986;Ma and Ptashne 1987; see Fig. 1).GAL4 50-97 ordinarily mediates dimerization (Carey et al. 1989). However, the same fragment manifests a novel transcriptional activating capability in yeast cells carrying a single point mutation in the GAL11 protein. This mutant GAL11P (P standing for transcriptional potentiator) and wild-type GAL11 are located in an ∼2-MD complex with RNA...