| dc.description.abstracteng | The transcription of protein coding genes is a key step in the conversion of genomic information
into the content of a cell’s proteome. In eukaryotes, this process is performed by the multi-subunit
DNA-dependent RNA polymerase (Pol) II and subject to intricate regulatory systems. During the
consecutive phases of transcription, initiation, elongation and termination, Pol II associates with
various factors that modulate its activity and thereby aid in the coordination of gene expression.
Transcriptional control at the level of initiation is dominated by the general transcription factors
(GTFs) TFIIA, TFIIB, TFIID, TFIIE, TFIIF and TFIIH. At the core-promoter Pol II assembles
with the GTFs in an ordered manner to form the pre-initiation complex (PIC), which is competent
to induce the opening of promoter DNA and to commence RNA synthesis. The subsequent
transition from initiation to the elongation phase is preluded by poorly understood events that
result in detachment of Pol II from the promoter (‘promoter escape’) and disassembly of the
initiation machinery. Both promoter opening and escape mark pivotal initiation checkpoints that
are susceptible to regulation, and for both processes an ATP-dependent involvement of the TFIIH
complex has been demonstrated. TFIIH equals Pol II in size and intricacy, consists of two
dissociable ‘core’ and ‘kinase’ modules, and comprises three subunits with conserved enzymatic
function. Two of these, a DNA-translocase and a kinase, act at the distinct stages of initiation,
respectively. In addition to the GTFs, promoter-located Pol II associates with the global coactivator
Mediator. This multi-protein complex bridges between the initiation machinery and
transcription factors bound at distant enhancer DNA elements and can adapt transcription in
response to environmental and developmental stimuli. The ‘head’ and ‘middle’ module segments
of Mediator (core Mediator, cMed) are essential and directly contact the initiation complex.
Mediator promotes initiation by cooperatively enhancing the recruitment of Pol II and the GTFs
to the core-promoter, stabilizing the assembled PIC and stimulating TFIIH kinase activity. Owing
to the sheer size and complexity of Mediator and TFIIH, however, the underlying molecular
mechanisms that govern the dynamic progression of the initiation machinery through the stages of
PIC formation, promoter opening and promoter escape remain largely elusive. An extensive
structural characterization of initiation complexes containing these factors may contribute to
deduce their essential functions in detail but to date has been impeded by their flexibility and poor
biochemical stability, as well as by the lack of highly pure samples for analysis.
In an effort to overcome such limitations, the first protocol for the large-scale preparation
of recombinant full-length TFIIH from the yeast Saccharomyces cerevisiae was established. This
work describes effective co-expression approaches in Escherichia coli and insect cell systems that
yielded various TFIIH subcomplexes. Using purified TFIIH core and kinase modules, the
assembly of complete 10-subunit TFIIH was demonstrated. In addition, this work reports the in
vitro reconstitution and cryo-electron microscopy (EM) analysis of the yeast PIC-cMed complex,
a macromolecular 46-subunit assembly of ~2 MDa, which encompasses all initiation-related
proteins essential for cell viability in yeast. Reconstructions of the TFIIH-comprising PIC and
PIC-cMed complex were derived at nominal resolutions of 4.7 Å and 5.8 Å, respectively. The obtained cryo-EM maps reveal secondary structure throughout and provide the first visualization
of TFIIH within the initiation machinery at high resolution. To facilitate model building and
placement into the density, crosslinking experiments were conducted, which in particular aided to
identify interactions between TFIIE and TFIIH. Based on previously determined models for the
cMed complex and the PIC without TFIIH (core PIC, cPIC), on newly generated homology and
ab initio models for multiple domains in TFIIE and TFIIH, and on de-novo built segments, the to
date most complete and accurate structure of a transcription initiation complex was compiled. The
remaining, unassigned protein sequences largely comprise regions that are predicted to be
disordered and thus likely adopt flexible conformations within the structure.
The PIC and PIC-cMed models reveal interactions between cMed, the cPIC and TFIIH,
and in particular demonstrate how TFIIE anchors TFIIH to the cPIC. The subunit arrangement
within the cPIC and cMed, as well as previously proposed interfaces between cMed, the Rpb4/7
stalk and the foot domain of Pol II, and TFIIB were confirmed and further explicated. Upon
binding to the PIC, the cMed middle module undergoes significant rearrangements, which result
in partial loss of its contacts to the head module and in ‘opening’ of the cMed structure. The
previously observed ‘cradle’ that is formed by the cPIC and cMed and may accommodate the Cterminal
domain (CTD) of Pol II is further defined by TFIIH. The core-TFIIH module emanates
from the cPIC in a crescent-like shape, with its ATPases Ssl2 (XPB in human) and Rad3 (XPD) at
the ends of the lobes and in proximity to the cPIC. Rad3 is anchored to the cPIC by a TFIIH
kinase module subunit, Tfb3 (MAT1), and Ssl2 engages with downstream promoter DNA. The
ATPases are connected via the scaffolding subunits Ssl1 (p44), Tfb2 (p52), Tfb4 (p34) and Tb5
(p8). The previously uncharacterized subunit Tfb1 (p62) serves as a flexible tether between
various parts of core-TFIIH. Tfb3 directly contacts Pol II by binding in a groove between the Pol
II stalk and the large TFIIE subunit Tfa1 (TFIIEα). To accommodate Tfb3, the stalk of Pol II is
shifted in both structures. Additional interactions between TFIIH and the cPIC involve the Cterminal
section of Tfa1. Several novel Tfa1 elements that bind the TFIIH subunits Tfb1 and Ssl2
were detected, thus indicating a mechanism for TFIIE-mediated TFIIH recruitment to the PIC.
The structures also provide further information on the function of the catalytic TFIIH subunits
during promoter opening and escape. The ATPase Ssl2, which is implicated in the unwinding of
promoter DNA, is contacted by the newly assigned ‘E-bridge’ helix in Tfa1, suggesting a concept
for TFIIE-stimulated DNA opening. Comparison of the Ssl2 ATPase domains to those of
enzymes, which translocate on double-stranded DNA (dsDNA), revealed striking similarity and a
respective Ssl2 translocation mechanism was proposed, consistent with biochemical evidence.
The kinase module of TFIIH comprises the kinase Kin28 (CDK7), which phosphorylates the Pol
II CTD and stimulates promoter escape. Except for the N-terminus of Tfb3, which anchors it to
Rad3 and the cPIC, this module is flexible in the PIC structure. However, it adopts a preferred
position outside of the cradle and in proximity to Mediator in the PIC-cMed complex. Moreover,
it is located adjacently to one of two openings in the cradle that emerge after conformational
rearrangement of cMed upon its PIC incorporation, thereby implying how Kin28 may access the
CTD and how Mediator may enhance its kinase activity. | de |