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
