Startseite
Podcasts
Fakultät für Chemie und Pharmazie - Digitale Hochschulschriften der LMU - Teil 02/06 Podcast
Structure and mechanism of the RNA polymerase II CTD phosphatase Scp1 and large-scale preparation of the RNA polymerase II-TFIIF complex

Structure and mechanism of the RNA polymerase II CTD phosphatase Scp1 and large-scale preparation of the RNA polymerase II-TFIIF complex

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

0:00

TFIIF is the only general transcription factor that has been
implicated in the preinitiation complex assembly, open complex
formation, initiation and transcription elongation. In addition,
TFIIF stimulates Fcp1, a central phosphatase needed for recycling
of RNA polymerase II (Pol II) after transcription by
dephosphorylation of the Pol II C-terminal domain (CTD). This
thesis reports the X-ray structure of the small CTD phosphatase
Scp1, which is homologous to the Fcp1 catalytic domain. The
structure shows a core fold and an active center similar to
phosphotransferases and –hydrolases that solely share a DXDX(V/T)
signature motif with Fcp1/Scp1. It was further demonstrated that
the first aspartate in the signature motif undergoes metalassisted
phosphorylation during catalysis, resulting in a phosphoaspartate
intermediate that was structurally mimicked with the inhibitor
beryllofluoride. Specificity may result from CTD binding to a
conserved hydrophobic pocket between the active site and an
insertion domain that is unique to Fcp1/Scp1. Fcp1 specificity may
additionally arise from phosphatase recruitment near the CTD via
the Pol II subcomplex Rpb4/7, which is shown to be required for
Fcp1 binding to the polymerase in vitro. Until now, the main
impediment in the high resolution crystallographic studies of TFIIF
in complex with Pol II and other members of transcription machinery
was unavailability of soluble, stoichiometric TFIIF complex in
sufficient amounts. This thesis reports on the development of the
overexpression system in yeast and a purification protocol that
enabled for the first time to isolate milligram amounts of a pure
and soluble, 15-subunit (~0,7 MDa) stoichiometric Pol IITFIIF
complex. Such complex together with the promoter DNA, RNA, TBP and
TFIIB assembles in vitro into the yeast initially transcribing
complex, which can now be studied structurally.