Structure of the Complete RNA Polymerase II Elongation Complex and its Interaction with the Elongation Factor TFIIS

This thesis describes crystal structures of complete, 12-subunit
yeast RNA polymerase II (Pol II) in complex with a synthetic
transcription bubble and product RNA, with an NTP substrate
analogue, and in complex with the transcription elongation factor
TFIIS. The structure of the Pol II-transcription bubble-RNA complex
reveals incoming template and non-template DNA, a seven base-pair
DNA-RNA hybrid, and three nucleotides each of separating DNA and
RNA. Based on this structure, those parts of Pol II were identified
which are involved in separating template DNA from non-template DNA
before the active site, and DNA from product RNA at the upstream
end of the DNA-RNA hybrid. In both instances, strand separation can
be explained by Pol II-induced duplex distortions. Only parts of
the complete transcription bubble present in the complexes are
ordered in the crystal structure, explaining the way in which high
processivity of Pol II is reconciled with rapid translocation along
the DNA template. The presence of an NTP substrate analogue in a
conserved putative pre-insertion site was unveiled in a Pol
II-transcription bubble-RNA complex crystal soaked with the
substrate analogue GMPCPP. The structure of the Pol II-TFIIS
complex was obtained from Pol II crystals soaked with TFIIS. TFIIS
extends from the Pol II surface to the active site and complements
the active site with two essential and invariant acidic residues
for hydrolytic RNA cleavage. TFIIS also induces extensive
structural changes in Pol II that reposition nucleic acids, in
particular RNA, near the active centre. These results support the
idea that Pol II contains a single tuneable active site for RNA
polymerisation and cleavage. The technical obstacles imposed by
crystal structure determination of large, transient protein-DNA-RNA
complexes were overcome by two novel, fluorescence-based assays to
monitor and optimise the composition of the crystals. Both assays
are not limited to Pol II complexes, but can serve as a general
tool for the crystallographic community.