The Role of Polyglutamine in Inter- and Intra-molecular Interactions in Med15-dependent Regulation

Med15 is a general transcriptional regulator and member of the tail module of the RNA Pol II Mediator complex. The S. cerevisiae Med15 protein has a well-structured N-terminal KIX domain, three Activator Binding Domains (ABDs) and several naturally variable polyglutamine (poly-Q) tracts (Q1, Q2, Q3) embedded in an intrinsically disordered central region, and a C-terminal Mediator Association Domain (MAD). We investigated how the presence of ABDs and changes in length and composition of poly-Q tracts influences Med15 activity and function using phenotypic, gene expression, and transcription factor interaction assays of truncation, deletion, and synthetic alleles. We found that individual Med15 activities were influenced by the number of activator binding domains (ABDs) and adjacent polyglutamine composition. We also observed that distant glutamine tracts and Med15 phosphorylation affected the activities of the KIX domain, suggesting that intramolecular interactions may be required for KIX domain interactions with transcription factors. We conclude that robust Med15 activity required at least the Q1 tract and that the length of that tract modulates activity in a context-dependent manner. We speculate that the glutamine tract provides a degree of intramolecular flexibility that is needed for Med15 function. Finally, we found that loss of Msn2-dependent transcriptional activation in Med15 Q1 tract variants correlates well with a reduction in Msn2:Med15 interaction strength.