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Fakultät für Chemie und Pharmazie - Digitale Hochschulschriften der LMU - Teil 02/06 Podcast
Structure of the MED7/MED21 heterodimer and reconstitution of a recombinant Mediator middle module complex

Structure of the MED7/MED21 heterodimer and reconstitution of a recombinant Mediator middle module complex

Fakultät für Chemie und Pharmazie - Digitale Hochschulschriften der LMU - Teil 02/06 vor 20 Jahren

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The Mediator of transcriptional regulation is the central
coactivator that enables a response of RNA polymerase II (Pol II)
to activators and repressors. Yeast Mediator has a size of more
than one MDa and consists of 25 different polypeptides. Biochemical
studies defined three Mediator modules in yeast, the head (MED17)
the middle (MED9/MED10) and the tail (MED15) modules. During this
work, an E.coli coexpression-copurification system was developed,
which allowed to study pairwise interactions of Mediator middle
module subunits. With the help of this system I reconstituted a
complex of two essential and conserved yeast Mediator middle module
proteins, the MED7/MED21 heterodimer, and solved its crystal
structure. The heterodimer forms an extended structure, which spans
one third of the Mediator length, and almost the diameter of Pol
II. It shows a four helix bundle and a coiled-coil protrusion
connected by a flexible hinge. Multiple conserved patches can be
identified on the surface, which allow for assembly of the middle
module. A combination of the coexpression-copurification system and
assembly of subcomplexes allowed the reconstitution of a
five-subunit Mediator middle module subcomplex. The reconstituted
subcomplex is able to bind Pol II in vitro. MED6 associates with
the middle module and forms a bridge to the head module. The
potential flexibility of this bridge and the MED7/MED21 hinge can
account for changes in Mediator structure upon its binding to Pol
II or to activators.