title: Solvent-exposed serines in the Gal4 DNA-binding domain are required for promoter occupancy and transcriptional activation in vivo.
abstract: The yeast transcriptional activator Gal4 has long been the prototype for studies of eukaryotic transcription. Gal4 is phosphorylated in the DNA-binding domain (DBD); however, the molecular details and functional significance of this remain unknown. We mutagenized seven potential phosphoserines that lie on the solvent-exposed face of the DBD structure and assessed them for transcriptional activity and DNA binding in vivo. Serine to alanine mutants at positions 22, 47, and 85 show the greatest reduction in promoter occupancy and transcriptional activity at the MEL1 promoter containing a single UASGAL . Substitutions with the phosphomimetic aspartate restored DNA-binding and transcriptional activity at serines 22 and 85, suggesting that they are potential sites of Gal4 phosphorylation in vivo. In contrast, the serine to alanine mutants, except serine 22, were fully proficient for binding to the GAL1-10 promoter, containing multiple UASGAL sites, although they had a reduced ability to activate transcription. Collectively, these data show that at the GAL1-10 promoter, functions of the DBD in transcriptional activation can be uncoupled from roles in promoter binding. We suggest that the serines in the DBD mediate protein-protein contacts with the transcription machinery, leading to stabilization of Gal4 at promoters.
authors.affiliation: Department of Molecular Biology, Ruđer Bošković Institute, Zagreb, Croatia.
authors.forename: Branka
authors.initials: B
authors.lastname: Jeličić
authors.forename: Josipa
authors.initials: J
authors.lastname: Nemet
authors.forename: Ana
authors.initials: A
authors.lastname: Traven
authors.forename: Mary
authors.initials: M
authors.lastname: Sopta
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D040081:Protein Array Analysis; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs / Q000502:physiology*; D012694:Serine / Q000378:metabolism*; D012997:Solvents; D015533:Transcriptional Activation; D015003:Yeasts / Q000235:genetics / Q000378:metabolism
chemical_list: D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D012997:Solvents; D012694:Serine
keywords: Gal4; transcription; yeast