Abstract
Gene activator proteins comprise distinct DNA-binding and transcriptional activation domains (ADs). Because few ADs have been described, we tested domains tiling all yeast transcription factors for activation in vivo and identified 150 ADs. By mRNA display, we showed that 73% of ADs bound the Med15 subunit of Mediator, and that binding strength was correlated with activation. AD-Mediator interaction in vitro was unaffected by a large excess of free activator protein, pointing to a dynamic mechanism of interaction. Structural modeling showed that ADs interact with Med15 without shape complementarity ('fuzzy' binding). ADs shared no sequence motifs, but mutagenesis revealed biochemical and structural constraints. Finally, a neural network trained on AD sequences accurately predicted ADs in human proteins and in other yeast proteins, including chromosomal proteins and chromatin remodeling complexes. These findings solve the longstanding enigma of AD structure and function and provide a rationale for their role in biology.
Highlights
Transcription factors (TFs) perform the last step in signal transduction pathways
Variability due to protein expression, activation duration, and secondary genetic effects was minimized by fusing domains of interest to a three-part artificial TF that (1) is tracked by an mCherry tag, (2) localizes to the nucleus only upon induction with estrogen, and (3) binds uniquely through its mouse DNA-binding domain (DBD) in the promoter of a chromosomally-integrated GFP reporter gene (Figure 1A) (McIsaac et al, 2013; Staller et al, 2018)
Outgrowth of transformed yeast for five days ensured that each cell contained a unique artificial TF (aTF) expression plasmid due to mechanisms maintaining it at single-copy levels (Methods) (Scanlon et al, 2009)
Summary
Transcription factors (TFs) perform the last step in signal transduction pathways. They serve key roles in central processes such as growth, stress response, and development, and their mutation or misregulation underlies many human diseases (Spitz and Furlong, 2012). A TF includes a sequence-specific DNA-binding domain (DBD) and an effector domain that regulates nearby gene transcription. Activation domains (ADs) – effector domains that increase transcription – have long been of particular interest due to their roles as oncogenic drivers and use as scientific tools (Bradner et al, 2017; Brückner et al, 2009; Dominguez et al, 2016). ADs were discovered as regions that could independently stimulate transcription when ectopically recruited to a gene promoter (Brent and Ptashne, 1985). ADs were classified based on their enrichment of certain residues, whether acidic, glutamine-rich, or proline-rich
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
