Solvent-exposed serines in the Gal4 DNA-binding domain are required for promoter occupancy and transcriptional activationin 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.

Transcription factor clusters enable target search but do not contribute to target gene activation

“…This reduction in clustering was not caused by lower Gal4 levels, as V5-tagged Gal4∆DBD levels by western blot were modestly increased compared to Gal4 WT (Figure S4B). In addition, to distinguish between the effect of DNA binding and dimerization, we mutated a single amino acid, S41D, which abolishes Gal4 binding to the endogenous GAL1/10 promoter in vivo and to DNA in vitro but still contains intact dimerization 53 . Similar to Gal4∆DBD, Gal4(S41D) clusters were less abundant and less dense than WT (Figure S4D-G).…”

RuX: A Novel, Flexible, and Sensitive Mifepristone-Induced Transcriptional Regulation System

“…To generate Gal4 DBDs with reduced nucleophilicity without affecting the DNA-binding capability, DNA-binding and nuclear localization regions were identified and modified. The following regions were considered based on data in the literature: The bipartite NLS (R15-K23 and K43-R46) [ 47 ] The DNA interaction surfaces (overlapping with the NLS) within the DBD (C11-K18 and C28-N35) [ 32 ] The upstream DNA interaction surface, defining a stretch of 10 amino acid residues (C11-K20, overlapping with the DNA interaction surface) in which mutations disrupt Gal4 DNA-binding activity (termed as subregion A) [ 34 ] Another stretch of 6 amino acid residues (C21-P26) (subregion B) [ 34 ] Further mutations that affect nuclear localization but retain DNA-binding function: Y40H and K43E [ 35 ] as well as S6A, S22A, and S22D [ 33 ] …”

“…Further mutations that affect nuclear localization but retain DNA-binding function: Y40H and K43E [ 35 ] as well as S6A, S22A, and S22D [ 33 ]…”

“…A broad range of appropriate domains can be considered as a DNA-binding module. One such option, Gal4 DBD, is successfully established in different available systems [ 32 , 33 ]. However, Gal4 DBD is also known to be nucleophilic, possibly causing noise levels in a chimeric transcription factor.…”