Structure of the Mediator subunit Cyclin C and subunit interaction studies within the Mediator head module

The Mediator of transcriptional regulation is the central
coactivator that enables a response of RNA polymerase II to
activators and repressors. It is conserved from yeast to human and
consists of 25 subunits in yeast that are organized in four modules
called head, middle, tail, and CDK8/Cyclin C module. Despite its
central role in transcription the functional mechanism remains
enigmatic. To overcome the lack of detailed structural data on the
Mediator a recombinant expression system was established that
allows large-scale purifications of Mediator head module
subcomplexes. It has been shown that via limited proteolysis assays
and multicistronic expression the problems of insolubility and low
expression rates of Mediator subunits can be overcome, paving the
way for structural studies on subcomplexes of the Mediator head
module. First data indicated that a reconstitution of the complete
head module is within close reach. Large-scale copurification data
led to a detailed interaction map of subunits and subcomplexes from
within the head module and towards the middle module. The second
part of this work describes the structure solution of a subunit in
the CDK8/Cyclin C module – Cyclin C. Cyclin C binds the
cyclin-dependent kinases CDK8 and CDK3, which regulate mRNA
transcription and the cell cycle, respectively. The crystal
structure of Cyclin C reveals two canonical five-helix repeats and
a specific N-terminal helix. In contrast to other cyclins, the
N-terminal helix is short, mobile, and in an exposed position that
allows for interactions with proteins other than the CDKs. A model
of the CDK8/Cyclin C pair reveals two regions in the interface with
apparently distinct roles. A conserved region explains promiscuous
binding of cyclin C to CDK8 and CDK3, and a non-conserved region
may be responsible for discrimination of CDK8 against other CDKs
involved in transcription. A conserved and Cyclin C-specific
surface groove may recruit substrates near the CDK8 active site.
Activation of CDKs generally involves phosphorylation of a loop at
a threonine residue. In CDK8, this loop is longer and the threonine
is absent suggesting an alternative mechanism of activation is
discussed based on a CDK8-Cyclin C model.