The Mediator kinase module: structural and functional studies in transcription regulation

Abstract

Transcription of protein-coding genes occurs when RNA polymerase II (RNAPII) and general transcription factors (GTFs) assemble at genomic promoter regions, forming a transcription pre-initiation complex (PIC), which initiates DNA opening and DNAdirected RNA synthesis. Upstream of promoters, gene-specific transcription activators bind upstream activation sequences (UASs), and they relay their activation signal to the RNAPII transcription initiation machinery through an intermediary complex of proteins known as the Mediator complex, which is found universally on all protein-coding genes in eukaryotes. The Mediator complex is a large multisubunit complex of 25 subunits in yeast, and 30 in human, amounting to a total of near 1.5 MDa, and 2 MDa in molecular mass, respectively. Mediator is divided into four structural and functional modules: the head, the middle, the tail, and the Cdk8 kinase modules. The head and middle modules, together known as the core Mediator (cMed), are essential for viability in yeast, and act as a GTF, engaging directly with the PIC and supporting its assembly and function. The tail module is responsible for the majority of interactions with activators, thereby connecting the bridge that allows communication between UASs and promoters. Disorder in the tail accommodates the versatility of Mediator-activator interactions. The Cdk8 kinase module (CKM) is dissociable, and occupies only a subpopulation of Mediator complexes in the cell. It contains the Cdk/cyclin pair Cdk8/cyclin C, which is the only catalytic activity within the Mediator complex. Generally thought to regulate Mediator function by repressing transcription, the exact role of the CKM and its mode of action remain enigmatic. Here, we aimed to investigate the role of the CKM in transcription initiation by a combination of biochemical and structural analyses. We established a new method for recombinant expression and purification of the complete S.cerevisiae CKM. We investigated CKM binding to the PIC and cMed in an in vitro reconstituted system with purified transcription initiation components, and found that CKM is excluded from the PIC, but binds cMed. Using crosslinking-mass spectrometry, we determined that the reason for that was an overlapping binding interface on cMed between the CKM and RNAPII. Furthermore, we explored CKM phosphorylation targets, and found novel targets within the PIC. We modeled the UAS-activator-Mediator interaction in vitro, and found that CKM phosphorylation of activators disrupts both UAS-activator and activator-Mediator interactions and attenuates their phase separated droplets. Taking our findings together with knowledge from published in vivo studies, we propose a hypothetical model for CKM function, which we believe can guide future experiments. Finally, to illuminate the CKM-Mediator interaction, we tried to solve the structures of recombinant CKM and CKM-cMed complexes by cryoelectron microscopy, but were unsuccessful despite extensive trials.