Recruitment of the transcriptional machinery through GAL11P: structure and interactions of the GAL4 dimerization domain

Abstract

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...

Transcriptional Regulation inSaccharomyces cerevisiae: Transcription Factor Regulation and Function, Mechanisms of Initiation, and Roles of Activators and Coactivators

“…Taken together, the artificial recruitment studies show that, under some circumstances, transcription can be enhanced by direct recruitment of the transcription machinery, but do not by themselves prove that natural activators work by recruitment. Similar conclusions were reached in a genetic study where a mutation (Gal11p) in the Mediator subunit Gal11/ Med15 was found that created a new protein-protein interaction with the Gal4 dimerization domain and stimulated transcription in the absence of a Gal4 activation domain (Barberis et al 1995;Hidalgo et al 2001).…”

Design of temperature-sensitive mutants solely from amino acid sequence

“…Both binding and activation are only seen in cells bearing the Gal11P mutation and not in cells with normal Gal11 protein. Ala scanning mutagenesis over the entire 50-97 regions revealed that predominantly hydrophobic residues (L69, L70, I71, F72, L86, L89, L92, L93, L96, and F97) were important for both transcriptional activation and Gal11P binding (34). It is very unlikely that so many hydrophobic residues could be part of a binding site, and we suggest instead that these residues are buried in intact Gal4.…”

“…The maximum number of Ts mutants was obtained at position 68. While this work was in progress, the NMR structure of the isolated Gal4 dimerization domain (residues 50-106) was reported (34). NMR spectra as a function of pH and temperature revealed the presence of several temperature-dependent, noncooperative changes, indicating a flexible and dynamic structure.…”

Self-association of the Amino-terminal Domain of the Yeast TATA-binding Protein

“…The resulting complex is quite stable (K d Յ 3 ϫ 10 Ϫ7 M) under representative in vitro conditions (50). As summarized in Fig.…”