Gene 184 (1997) 229–235 Conservation of a putative inhibitory domain in the GAL4 family members Olivier Poch * U.P.R. Me ´canismes Mole ´culaire de la Division Cellulaire et du De ´veloppement, Institut de Biologie Mole ´culaire et Cellulaire, 15 rue Rene ´ Descartes, 67084 Strasbourg, France Received 24 May 1996; revised 11 July 1996; accepted 12 July 1996 Abstract The GAL4 family members are fungal transcriptional activators composed of several functional domains: a characteristic cysteine-rich DNA-binding domain common to all members, a dimerization domain, various transactivation domains generally exhibiting a high acidic content and a highly variable central region supposed to be involved in regulation and in effector recognition. We report here that the central region of the GAL4 family members share eight conserved motifs embedded in a large functional domain of 225 up to 405 residues. This domain may also be present in four proteins belonging to another family of transcriptional activators sharing a C2H2-type zinc finger. Analysis of the biochemical data available on the well-studied GAL4 protein suggests that this domain may be involved in the regulation of the activity of the protein, particularly in an inhibitory function. This hypothesis is further supported by deletion and site-directed mutagenesis experiments on other GAL4 family members. The mean secondary structure prediction performed on the eight motifs strongly suggests that the inhibitory activity may be mediated by hydrophobic interactions linked to the presence of amphipathic a-helices. Keywords: Fungal transcriptional activator; GAL4 family member; GAL4; PUT3; LEU3; PPR1; PDR1; PDR3; ADR1; Inhibitory domain; Sequence conservation; Saccharomyces cerevisiae 1. Introduction intermediate or a drug. In addition, with the totality of S. cerevisiae sequences available, a large set of potential The GAL4 family members (GFM ) are fungal tran- GFM has been determined whose function is generally scriptional regulatory proteins (Dahwale and Lane, unknown (see Table 1). 1993; Svetlov and Cooper, 1995) which share a charac- At the sequence level, GFM are large proteins (rang- teristic cysteine-rich DNA-binding domain. They are ing from 465 up to 1502 residues) with a characteristic involved in the regulation of numerous anabolic and DNA-binding domain composed of a highly conserved catabolic pathways (i.e., galactose, arginine, proline, 28-residue cysteine-rich motif (C6-type zinc finger) maltose and leucine metabolisms) as well as in the (Svetlov and Cooper, 1995) generally located in the regulation of various pathways such as pleiotropic drug most amino-terminal region. The three-dimensional resistance, chromosome segregation or sporulation structure of the cysteine-rich motif of two GFM ( GAL4 processes ( Table 1). Frequently, the activity of these and PPR1 ) revealed an essentially identical structure in transcriptional activators is known to be stimulated by which two Zn2+ions are coordinated to six invariant the presence of a small molecule such as a metabolic cysteine residues, with the rest of the amino acids folded around the Zn2Cys6 (Baleja et al., 1992; Kraulis et al., 1992; Marmorstein et al., 1992; Marmorstein and Harrison, 1994 ). In addition to a common DNA-binding * Corresponding author. domain, GFM share general functional organization Abbreviations: S. cerevisae, Saccharomyces cerivisiae; S. carlbergensis, with (in the immediate carboxy-terminal side of the zinc Saccharomyces carlbergensis; K. lactis, Kluyveromyces lactis; S. pombe, cluster) a coiled-coil dimerization domain with heptad Schizosaccharomyces pombe; A. nidulans, Aspergillus nidulans; A. repeats of hydrophobic residues (Reece and Ptashne, oryzae, Aspergillus oryzae; N. crassa, Neurospora crassa; C. albicans, Candida albicans; L. edodes, Lentinus edodes. 1993; Zhang et al., 1993; Sua `rez et al., 1995). The 0378-1119/97/$17.00 Copyright © 1997 Elsevier Science B.V. All rights reserved PII S0378-1119(96)00602-6