Architecture of the Mediator Head Module

Abstract

Mediator is a key regulator of eukaryotic transcription, connecting activators and repressors bound to regulatory DNA elements with RNA polymerase II (Pol II). In the yeast S. cerevisiae, Mediator is organized into three modules (Head, Middle/Arm and Tail) that together comprise 21 subunits with a total mass over 1 MDa. Our understanding of Mediator assembly, and of its role in regulating transcription, has so far been hampered by limited structural information. By using state-of-the-art protein complex engineering and phasing methods, we solved the structure of the Mediator Head module (7 subunits, 223 kDa) at 4.3 angstrom resolution. Our structure reveals three distinct domains (Fixed jaw, Movable jaw, and Neck) that come together to form a flexible central joint region. Strikingly, we discovered a novel and elaborate structure in the Neck domain, in which a total of ten helices from five different subunits form a large multi-helical bundle as a single structure unit. An intricate pattern of interactions within this helical bundle ensures stable assembly of the Head subunits and provides binding sites for general transcription factors (GTFs) and Pol II. Mapping previously identified genetic mutations onto our Head module X-ray structure suggests that the Head module may juxtapose TFIIH and the carboxyl-terminal domain (CTD) of the largest subunit of Pol II (Rpb1) to facilitate CTD phosphorylation. These results reveal architectural principles underlying the role of Mediator in the regulation of gene expression.