hide
Free keywords:
-
Abstract:
Mediator is the central coactivor complex required
for regulated transcription by RNA polymerase
(Pol) II. Mediator consists of 25 subunits arranged
in the head, middle, tail and kinase modules.
Structural and functional studies of Mediator are
limited by the availability of protocols for the preparation
of recombinant modules. Here, we describe
protocols for obtaining pure endogenous and
recombinant complete Mediator middle module
from Saccharomyces cerevisiae that consists of
seven subunits: Med1, 4, 7, 9, 10, 21 and 31. Native
mass spectrometry reveals that all subunits are
present in equimolar stoichiometry. Ion-mobility
mass spectrometry, limited proteolysis, light scattering
and small-angle X-ray scattering all indicate
a high degree of intrinsic flexibility and an elongated
shape of the middle module. Protein–protein interaction
assays combined with previously published
data suggest that the Med7 and Med4 subunits
serve as a binding platform to form the three
heterodimeric subcomplexes, Med7N/21, Med7C/
31 and Med4/9. The subunits, Med1 and Med10,
which bridge to the Mediator tail module, bind to
both Med7 and Med4.
