General Transcription Factors

1
Chapter 11
General Transcription Factors
False-color transmission electron micrograph of
RNAs being synthesized on a DNA template, forming
a feather-like structure.
2
Table of contents

• Class II Factors
• Class I Factors
• UBF
• Class III Factors

3
11.1 Class II Factors

• The class II preinitiation complex
• Structure and function of TFIID
• Structure and function of TFIIA and TFIIB
• Structure and function of TFIIF
• Structure and function of TFIIE and TFIIH
• Elongation Factors
• The polymerase II holoenzyme

4
11.1.1 The class II preinitiation complex

• The general transcription factors combine with
  RNA polymerase to form a preinitiation complex
• Six general transcription factors named TFIIA,
  TFIIB, TFIID, TFIIE, TFIIF, and TFIIH
• The factors and poly II bind in a specific order
  to growing preinitiation complex.

5
Formation of a complex involving TFIID,TFIIA,and
a promoter-bearing DNA
Figure 11.1 Formation of a complex involving
TFIID, TFIIA, and a promoter-bearing DNA.
Sharp and coworkers mixed a labeled DNA fragment
containing the adenovirus major late promoter
with TFIIA and TFIID separately and together,
then electrophoresed the products. Lane A, with
DNA and TFIIA alone, showed only free DNA, which
migrated rapidly, almost to the bottom of the
gel. Lane D, with DNA and TFIID alone, showed
free DNA plus a non-specific complex (NS). Lane
AD, with both transcription factors, showed a
larger complex with both factors (AD, later
named DA).
6
DNase footprinting the DA complex
Figure 11.2 DNase footprinting the DA complex.
Sharp and colleagues performed DNase
footprinting with TFIIA, TFIID, and a labeled
fragment of DNA containing a TATA box. Lanes 1
and 2 contained sequencing ladders (GA and G,
respectively) obtained by Maxam-Gilbert
sequencing of the same DNA fragment. Lane 3 (also
denoted F, for "free DNA") was a control with DNA
but with no protein added. Lane 4 contained DNA
plus TFIID, which presumably formed a
non-specific complex (NS). Lane 5 contained DNA
plus TFIID and TFIIA (AD). The footprint in lane
5, indicated with a bracket at right, encompasses
the TATA box, which is centered around position
-25. The arrow at the top of the bracket denotes
a site of enhanced DNase sensitivity adjacent to
the protected region.
7
Building the preinitiation complex
Figure 11.3 Building the preinitiation complex.
8
Figure 11.3 Building the preinitiation complex.
(a) the DABPolF complex. Reinberg and colleagues
performed gel mobility shift assays with TFIID,
A, B, and F, and RNA polymerase II, along with
labeled DNA containing the adenovirus major late
promoter Lane 1 shows the familiar DA complex,
formed with TFIID and A Lane 2 demonstrates that
adding TFIIB caused a new complex, DAB, to form
Lane 3 contained TFIID, A, B, and F, but it looks
identical to lane 2. Thus, TFIlF did not seem to
bind in the absence of polymerase II Lanes 4-7
show what happened when the invesbgators added
more and more polymerase II in addibon to the
four transcription factors More and more of the
large complexes, DABPolF and DBPolF, appeared.
Lanes 8-11 contained less and less TFIIF, and we
see less and less of the large complexes.
Finally, lane 12 shows that essentially no
DABPolF or DBPolF complexes formed when TFIIF was
absent, Thus, TFIIF appears to bring polymerase
II to the complex. The lanes on the right show
what happened when Reinberg and colleagues left
out one factor at a time. In lane 13, without
TFIID, no complexes formed at all Lane 14 shows
that the DA complex, but no tubers, formed in the
absence of TFIIB Lane 15 demonstrates that DBPolF
could still develop without TFIIA. Finally, all
the large complexes appeared in the presence of
all the factors (lane 16). (b) The DBPolFEHJA
complex Reinberg and colleagues started with the
DBPolF complex (lacking TFIIA, lane 1 ) assembled
on a labeled DNA containing the adenovirus major
late prommer Next, they added TFIIE, then TFIIH,
then TFIIJ, then TFIIA, in turn, and performed
gel mobility shift assays. With each new
transcription factor, the complex grew larger and
its mobility decreased further. The mobilities of
all the complexes are indicated at right. Lanes 5
-7 show the result of adding more and more TFIIA
to the DBPolFEHJ complex, but most of the
DBPolFEHJA complex had already formed, even at
the lowest TFIIA concentration Lanes 8 -11 show
again the resud of leaving out radons factors,
denoted at the top of each lane At best, only the
DB complex forms At worst, in the absence of
TFIID, no complex at all forms.
9
Footprinting the DA and DAB complexes
Figure 11.4 Footprinting the DA and DAB
complexes. Reinberg and coworkers
performed fooprinting on the DA and DAB complexes
with both DNase and another DNA strand breaker a
1 ,10 phenanthroline-copper ion complex (OP-Cu).
(a) Footprinting on the nontemplate strand. The
DA and DAB complexes formed right over the TATA
box (TATAAA, indicated at right, top to besom)
(b) Footgrinting on the template strand. Again,
the protected region in beth the DA and DAB
complexes was centered on the TATA box (TATAAA,
indicated at right, bottom to top) The arrow near
the top at right denotes a site of enhanced DNA
cleavage at position 10.
10
Footprinting the DABPolF complex
Figure 11.5 Footprinting the DABPolF complex.
Reinberg and colleagues performed DNase
footprinting with TFIID, A, and B (lane 2) and
with TFIID, A, B, and F, and RNA polymerase II
(lane 3). When RNA polymerase and TFIIF joined
the complex, they caused a huge extension of the
footprint, to about position 17. This is
consistent with the large size of RNA polymerase
II
11
Model for formation of the DABPolF complex
Figure 11.6 Model for formation of the DABPolF
complex. TFIIF (green) binds to polymerase
II (Pol II, red) and carries it to the DAB
complex. The result is the DABPolF complex. This
model conveys the conclusion that polymerase II
extends the DAB footprint in the downstream
direction, and therefore binds to DNA downstream
of the binding site for TFIID, A, and B, which
centers on the TATA box.
12
11.1.2 Structure and Function of TFIID

• TATA Box binding Protein (TBP)
• TBP-associated factors(TAF)

13
Methylation interference at the TATA box
14
Figure 11.8 Methylation interference at the TATA
box.
15
Figure 11.8 Methylation interference at the TATA
box. Roeder and colleagues end-labeled DNA
containing the adenovirus major late promoter on
either the template (a) or nontemplate strand
(b), then methylated the DNA under conditions in
which As were preferentially methylated. Then
they added TFIID and filtered the protein-DNA
complexes. DNAs that could still bind TFIID were
retained, while free DNA flowed through. Finally,
they cleaved the filter-bound and free DNAs at
methylated sites with NaOH and subjected the
fragments to gel electrophoresis. The
autoradiographs in (a) and (b) show that the
bound DNA did not cleave in the TATA box, so it
was not methylated there. On the other hand, the
free DNA was cleaved in the TATA box, showing
that it had been methylated there. That is why it
no longer bound TFIID. (c) Summary of methylated
bases in the free DNA fractions. The lengths of
the bars show the intensities of the bands in the
"free" lanes in parts (a) and (b), which indicate
the degree of methylation. Most of the
methylation occurred on As, rather than Gs. These
methyl groups are in the minor groove since this
methylated DNA was incapable of binding TFIID,
these results suggest that TFItD binds in the
minor groove. In reading the sequences in this
and the next figure, remember that the
nontemplate strand contains the TATA sequence.
16
Effect of substituting dU for dT on TFIID binding
to the TATA box
Figure 11.9 Effect of substituting dU for dT on
TFIID binding to the TATA box.
17
Figure 11.9 Effect of substituting dU for dT on
TFIID binding to the TATA box. Roeder and
coworkers bound TWIID to labeled DNA containing
TATA boxes with the sequences given at top. They
did the binding in the presence of excess
unlabeled competitor DNA containing either
wild-type or mutant TATA boxes (mutant sequence
TAGAGAA). To assay for TFIID-TATA box binding,
they electrophoresed the protein-DNA complexes
under non-denaturing conditions which separate
free DNA from protein-bound DNA. In all cases,
the wild-type TATA box was able to compete, so
only free DNA was observed (even-numbered lanes).
However, in all cases, the mutant TATA box was
unable to compete, even when the labeled TATA box
contained a dU instead of a dT. In fact, lane 7
shows that substitution of a dU for a dT in
position 2 of the template strand of the TATA box
(sequence AdUATTTT) actually seemed to enhance
TFIID-TATA box binding compared to the
unsubstitued TATA box (lane 1 ). Since dU and dT
differ in the major groove, but not the minor
groove, and the substitution of dU for dT did not
inhibit binding, this suggests that TFIID binds
in the minor groove.
18
Effect of substituting C for T and I for A on
TFIID binding to the TATA box
Figure 11.10 Effect of substituting C for T and I
for A on TFIlD binding to the TATA box.
19
Figure 11.10 Effect of substituting C for T and I
for A on TFIlD binding to the TATA box.
(a) Appearance of nueleosides as viewed from the
major and minor grooves. Notice that thymine and
cytidine look identical from the minor groove
(green, below), but quite different from the
major groove (red, above) Similarly. adenosine
and inosine look the same from the minor groove,
but very different from the major groove.
(b) Sequence of the adenovirus major late
promoter (MLP) TATA box with Cs substituted for
Ts and Is substituted for AS, yielding a CICI box
. (c) Binding TBP to the CICI box. Start
and Hawley performed gel mobility shift assays
using DNA fragments containing the MLP with a
CICI box (lanes 1-3) or the normal TATA box
(lanes 4-6), or a non-specific DNA (NS) with no
promoter elements (lanes 7-9) The first lane in
each set (1,4, and 7) contained yeast TBP the
second lane in each set (2, 5, and 8) contained
human TSP and the third lane in each set
contained just buffer The yeast and human TBPs
gave rise to slightly different size brotein-DNA
complees, but substituting a CICI box for the
TATA box had little effect on the yield of the
complexes. Thus, TBP binding to the TATA box was
not significantly diminished by the substitutions.
20
Structure of the TBP-TATA box complex
21
Figure 11.6 Structure of the TBP-TATA box
complex. This diagram, based on Sigler and
colleagues' crystal structure of the TBP-TATA box
complex, shows the backbone of the TBP in olive
at top. The long axis of the "saddle" is in the
plane of the page. The DNA below the protein is
in multiple colors. The backbones in the region
that interacts with the protein are in orange,
with the base pairs in red. Notice how the
protein has opened up the narrow groove and
almost straightened the helical twist in that
region. One stirrup of the TBP is seen as an
olive loop at right center, inserting into the
minor groove. The other stirrup performs the same
function, but it is out of view in back of the
DNA. The two ends of the DNA, which do not
interact with the TBP, are in blue and gray blue
for the backbones, and gray for the base pairs.
The left end of the DNA sticks about 25 degrees
out of the plane of the page, and the right end
points inward by the same angle. The overall bend
of about 80 degrees in the DNA, caused by TBP, is
also apparent.
22
Figure 11.7 Effects of mutations in TBP on
transcription by all three RNA polymerases.
23

• Figure 11.7 Effects of mutations in TBP on
  transcription by all three RNA polymerases.
• Locations of the mutations. The boxed region
  indicates the conserved C-terminal domain of the
  TBP red areas denote two repeated elements
  involved in DNA binding. The two mutations are
  P65 ?S, in which proline 65 is changed to a
  serine and 1143 ? N, in which isoleucine 143 is
  changed to asparagine. (b-e) Effects of the
  mutations. Reeder and Hahn made extracts from
  wild-type or mutant yeasts, as indicated at
  bottom, and either heat-shocked them at 37 ?or
  left them at 24?, again as indicated at bottom.
  Then they tested these extracts by S1 analysis
  for ability to start transcription at promoters
  recognized by all three nuclear RNA polymerases
• The rRNA promoter (polymerase I) (c) the CYC1
  (polymerase II) promoter (d) the 5S rRNA
  promoter (polymerase III) and (e) the tRNA
  promoter (also polymerase III). The 1143 ?N
  extract was deficient in transcribing from all
  four promoters even when not heat-shocked. The
  P65 ?S extract was deficient in transcribing from
  polymerase II and III promoters, but could
  recognize the polymerase promoter, even after
  heat shock.

24
SUMMARY
TFIID contains a 38 kDa TATA box-binding
protein (TBP) plus several other polypeptides
known as TBP-associated factors (TAFIIs). The
C-terminal 180 amino acid fragment of the human
TBP is the TATA box-binding domain. The
interaction between a TBP and a TATA box is an
unusual one that takes place in the DNA minor
groove. The saddle-shaped TBP lines up with the
DNA, and the under-side of the saddle forces open
the minor groove and bends the TATA box into an
80curve.
25
Structure of a Drosophila TFIID assembled in
vitro from the products of cloned genes
26
Relationships among the TAFs of fruit
flies,humans,and yeast
Figure 11.13 relationships among the TAFs of
fruit flies (D.melanogaster), humans (H.
sapiens), and yeast (S. cerevisiae). The
horizontal lines link homologous proteins.
27
Activities of TBF and TFIID on four different
promoters
Figure 11.14 Activities of TBP and TFIID on four
different promoters. Tjian and
colleagues tested a reconstituted Drosophila
transcription system containing either TBP or
TFIID (indicated at top) or templates bearing
four different promoters (also as indicated at
top). The promoters were of two types diagrammed
at bottom The first type, represented by the
adenovirus E1B and E4 promoters, contained a TATA
box (red). The second type, represented by the
adenovirus major late promoter (AdML) and the
Drosophila Hsp70 promoter, contained a TATA box
plus an initiator (I, green) and a downstream
element (D, blue). After transcription in vitro,
Tjian and coworkers assayed the RNA products by
primer extension (top). The autoradiographs show
that TBF and TFIID fostered transcription equally
well from the first type of promoter (TATA box
only), but that TFIID worked much better than TBP
in supporting transcription from the second type
of promoter (TATA box plus initiator plus
downstream element).
28
Identifying the TAFIIs that bind to the hsp70
promoter
Figure 11.15 Identifying the TAFIIs that bind to
the hsp70 promoter. Tjian and colleagues
photo-crosslinked TFIID to a 32p-labeled template
containing the hsp70 promoter. This template had
also been substituted with the photo-sensitive
nucleoside bromodeoxyuridine (BrdU). Next, these
workers irradiated the TFIID-DNA complex with
ultraviolet (UV) light to form covalent bonds
between the DNA and any proteins in close contact
with the major groove of the DNA. Next, they
digested the DNA with nuclease and subjected the
proteins to SDS-PAGE. Lane 1 of the
autoradiograph shows the results when TFIID was
the input protein. TAFII250 and TAFII150 became
labeled, implying that these two proteins had
been in close contact with the labeled DNA's
major groove. Lane 2 is a control with no TFIID.
Lane 3 shows the results when a ternary complex
containing TBP, TAFII250, and TAFII150 was the
input protein. Again, the two TAFIIs became
labeled, suggesting that they bound to the DNA.
Lane 4 shows the results when TBP was the input
protein. It did not become labeled, which was
expected since it does not bind in the DNA major
groove.
29
DNase I footprinting the hsp70 promoter with TBP
and the ternary complex
Figure 11.16 DNase I footprinting the hsp70
promoter with TBP and the ternary complex (TBP,
TAFII250, and TAFII150). Lane 1, no
protein lane 2, TBP lane 3, ternary complex. In
both lanes 2 and 3, TFIIA was also added to
stabilize the DNA-protein complexes, but separate
experiments indicated that it did not affect the
extent of the footprints. Lane 4 is a
Maxam-Gilbert GA sequencing lane used as a
marker. The extents of the footprints caused by
TBP and the ternary complex are indicated by
brackets at left. The locations of the TATA box
and initiator are indicated by boxes at right.
30
Model for the interaction between TBP and
TATA-containing or TATA-less promoters
31
Failure of TBP alone to respond to Sp1
Figure 11.18 Failure of TBP alone to respond to
Sp1. (a) Structure of the test promoter. This is
a composite Sp1-responsive promoter containing
six GC boxes (red) from the SV40 early promoter
and the TATA box (blue) and transcription start
site (initiator, green) from the adenovirus major
late promoter. Accurate initiation from this
promoter in the run-off assay described below
should produce a 375 nt transcript. (b) In vitro
transcription assay. Tjian and colleagues mixed
TFIID, or bhTBP, or vhTBP, as shown at top, with
TFIIA, TFIIB, TFIIE, TFIIF, and RNA polymerase
II, then performed a run-off transcription assay
with a- 32p UTP. Lanes 1 and 2 show that
natural TFIID supported a high level of
transcription from this promoter, and this
transcription was significantly enhanced by the
transcription factor Sp1. Lanes 3-6 demonstrate
that any transcription due to recombinant human
TBP was not stimulated by Sp1 in the absence of
TAFIIs.
32
Activation by Sp1 requires TAFII110
Figure 11.19 Activation by Sp1 requires TAFII110.
Tjian and colleagues used a primer
extension assay to measure transcription from a
template containing a TATA box and three upstream
GC boxes. They used either a Drosophlia cell
extract (a) or a human cell extract (b), each of
which had been depleted of TFIID. They replaced
the missing TFIID with any of the three different
complexes, picture at bottom, containing
combinations of TBP, TAFII250, and TAFII110. They
also added no Sp1 (-), or two increasing
concentrations of Sp1, represented by the wedges.
The autoradiographs show the amount of
transcription, and therefore the activation
achieved by Sp1 with each set of TAFIIs.
Activation was observed in each extract only with
all three TAFIIs.
33
A model for transcription enhancement by
activators
Figure 11.20 A model for transcription
enhancement by activators. (a) TAFII250 does not
interact with either Sp1 or Gal4-NTF-1 (a hybrid
activator with the transcription-activating
domain of NTF-1), so no activation takes place.
(b) Gal4-NTF-1 can interact with either TAFII150
or TAFII60 and activate transcription Sp1 cannot
interact with either of these TAFs or with
TAFII250 and does not activate transcription.
(c) Gal4-NTF-1 interacts with TAFII150 and Sp1
interacts with TAFII110,so both factors activate
transcription. (d) Holo TFIID contains the
complete assortment of TAFIIs, so it can respond
to a wide variety of activators, represented here
by Sp1, Gal4-NTF-1, and a generic activator at
top.
34
Whole genome analysis of transcription
requirements in yeast
35
Figure 11.18 Three-dimensional models of TFIID
and TFTC. Schultz and colleagues made
negatively stained electron micrographs (see
Chapter 19, for method) of TFIID and TFTC, then
digitally combined images to arrive at an
average. Then they tilted the grid in the
microscope and analyzed the resulting micrographs
to glean three-dimensional information for both
proteins. The resulting models for TFIID (green)
and TFTC (blue) are shown.
36
SUMMARY
TFIID contains at least eight TAFIIs, in
addition to TBP. Most of these TAFIIs are
evolutionarily conserved in the eukaryotes. The
TAFIIs serves several functions, but two obvious
ones are interacting with core promoter elements
and interacting with gene-specific transcription
factors. TAFII250 and TAFII150 help TFIID bind to
the initiator and downstream elements of
promoters and therefore can enable TBP to bind to
TATA-less promoter that contain such elements.
TAFII250 and TAFII110 help THIID interact with
Sp1 that is bound to GC boxes upstream of the
transcription start site. These TAFIIs therefore
ensure that TBP can bind to TATA-less promoters
that have GC boxes. Different combinations of
TAFIIs are apparently required to respond to
various transcription activators, at least in
higher eukaryotes. TAFII250 also has two
enzymatic activities. It is a histone acetyl trans
37
11.1.3 Structure and function of TFIIA and TFIIB

• TFIIA 2-3 subunits,
• binds to TBP and stabilizes
• binding between TFIID and
  promoters
• TFIIB a linker between TFIID and
• TFIIF/polymerase

38
Hypothetical structure of a TFIIA-TFIIB-TBP-TATA
box complex
Figure 11.19 Hypothetical structure of a
TFIIA-TFIIB-TBP-TATA box complex. This
is a combination of two structures a human core
TFIIB-plant TBP-adenovirus TATA box structure,
and a yeast TFIIA-TBP-TATA box structure. None of
the proteins is complete. They are all core
regions that have the key elements needed to do
their jobs. The DNA is gray the two halves of
core TBP are light blue (upstream half) and dark
blue (downstream half) the amino terminal domain
of core TFIIB is red and the carboxyl terminal
domain is magenta the core large subunit of
TFIIA is green, and the small subunit is yellow.
The transcription start site is at right, denoted
"1 ."
39
SUMMARY
THIIA contains two subunits (yeast), or
three subunits (fruit flies and humans). This
factor is probably more properly considered a
TAFII since it binds to TBP and stabilizes
binding between TFIID and promoters. TFIIB serves
as a linker between TFIID and TFFIIF/ polymerase
II. It has two domains, one of which is
responsible for binding to TFIID, the other for
continuing the assembly of the preinitiation
complex. A structure for the TFIIA-TFIIB-TBP-TATA
box complex can be imagined, based on the known
structures of the TFIIA-TBP-TATA box and
TFIIB-TBP-TATA box complexes. This structure
shows TFIIA and TFIIB binding to the upstream and
downstream stirrups, respectively, of TBP. This
puts these two factors in advantageous positions
to perform their functions.
40
11.1.4 Structure and function of TFIIF
Binding of the polymerase to the DAB
complex requires prior interaction with TFIIF,
composed of two polypeptides called RAP30 and
RAP70. RAP30 is the protein that ushers
polymerase into the growing complex.
41
Role of TFIIF in binding RNA polymerase II to the
preinitiation complex
Figure 11.22 Role of TFIIF in binding RNA
polymerase II to the preinitiation complex.
42
Figure 11.22 Role of TFIIF in binding RNA
polymerase II to the preinitiation complex.
Greenblatt, Reinberg, and colleagues
performed phenyl-Superose micro column
chromatography on TFIIF and tested fractions for
(a) TFIIF transcription factor activity (b)
preinitiation complex formation with RNA
polymerase II, using a gel mobility shift assay
and (c) content of RAP30, detected by Western
blotting and probing with an anti-RAP30 antibody.
(a) TFIIF activity assay. Lane I, activity of the
protein loaded onto the column (input) lane ,
positive control with known TFIIF activity other
lanes are numbered according to their order of
elution from the column. The great majority of
the TFIIF activity eluted in fractions 16-22. (b)
Gel mobility shift assay. The lanes on the left
show the complexes formed with the TFIIF input
fraction alone (I), and with various combinations
of highly purified TFIID, A, B, polymerase II,
and TFIIF. The numbered lanes show the shifts in
the DAB complex produced by addition of
polymerase II plus the same column fractions as
in part (a). The ability to form the DABPolF
complex resided in the same fractions with TFIIF
activity (16-22). (c) Western blot to detect
RAP30. The labeling of the lanes has the same
meaning as in panel (a). The fractions with RAP30
(16-22) were the same ones with TFIIF activity
and the ability to bring polymerase II into the
preinitiation complex. Thus, RAP30 seems to have
this activity.
43
11.1.5 Structure and function of TFIIE and TFIIH
44
Formation of the DABPoIFE complex
Figure 11.23 Formation of the DABPolFE complex.
45
Figure 11.23 Formation of the DABPolFE complex.
Tjian, Reinberg, and colleagues performed
gel mobility shift assays with various
combinations of transcription factors, polymerase
II, and a DNA fragment containing the adenovirus
major late promoter. The protein components in
each lane are given at top, and the complexes
formed are indicated at left. Note that TFIID, A,
B, F, E, and polymerase II formed the DABPolFE
complex, as expected (lane 4). Lanes 5-8 show
that increasing quantities of the two subunits of
TFIIE, added separately, cannot join the DABPolF
complex. However, lanes 9 and 10 demonstrate that
the two polypeptides can join the complex if they
are added together. Lane 11 is a repeat of lane
10, and lane 12 is identical except that it is
missing TFIID. This is a reminder that everything
depends on TFIID, even with all the other factors
present.
46
Dependence of transcription on both subunits of
TFIIE
Figure 11.24 Dependence of transcription on both
subunits of TFIIE.
47
Figure 11.24 Dependence of transcription on both
subunits of TFIIE. (a) Tjian and Reinberg
performed run-off transcription of a DNA fragment
containing the adenovirus major late promoter in
the presence of all transcription factors except
TFIIE. They added whole TFIIE or the products of
cloned genes encoding the subunits of the
transcription factor in increasing concentration,
as indicated at top. The wedge shapes illustrate
the increase in concentration of each factor from
one lane to another. Lanes 1 and 2 show that
native TFIIE can reconstitute transcription
activity. However, the subunits added separately
cannot do this, as portrayed in lanes 3-10. On
the other hand, the two subunits together can
stimulate transcription. (b) The same kind
of run-off assays, using the TATA-less G61
promoter, showed that the TFIIE produced by
cloned genes stimulates Sp1-dependent
transcription. Lanes 1 and 2 contained native
TFIIE purified from HeLa cells. Lanes 3 and 4
contained TFIIE subunits produced by cloned
genes. Lanes 5 and 6 had no TFIIE. Clearly, TFIIE
is necessary, and the factor made by cloned genes
works as well as the native one. Also, as we have
seen before, transcription of the TATA-less
promoter requires Sp1.
48
The preinitiation complex envisioned by Tjian and
Reinberg
Figure 11.25 The preinitiation complex envisioned
by Tjian and Reinberg. This construct
contains air of the factors in the DABPolFE
complex plus TFIIH (orange), another general
transcription factor we shall discuss next.
49
Phosphorylation of preinitiation complexes
Figure 11.26 Phosphorylation of preinitiation
complexes. Reinberg and colleagues performed
gel mobility shift assays with preinitiation
complexes DAB through DABPolFEH, in the presence
and absence of ATP, as indicated at top Only when
TFIIH was present did ATP shift the mobility of
the complex (compare lanes 7 and 8). The simplest
explanation is that TFIIH promotes
phosphorylation of the input polymerase
(polymerase IIA) to polymerase IIO.
50
TFIIH phosphorylates RNA polymerase II
Figure 11.21 TFIIH phosphorylates RNA polymerase
II.
51
Figure 11.21 TFIIH phosphorylates RNA polymerase
II. (a) Reinberg and colleagues incubated
polymerase IIA with various mixtures of
transcription factors, as shown at top. They
included ?-32PATP in all reactions to allow
phosphorylation of the polymerase, then
electrophoresed the proteins and performed
autoradiography to visualize the phosphorylated
polymerase. Lane 4 shows that TFIID, B, F, and E,
were insufficient to cause phosphorylation. Lanes
5-10 demonstrate that TFIIH alone is sufficient
to cause some polymerase phosphorylation, but
that the other factors enhance the
phosphorylation. TFIIE provides particularly
strong stimulation of phosphorylation of the
polymerase IIa subunit to IIo. (b) Time
course of polymerase phosphorylation. Reinberg
and colleagues performed the same assay for
polymerase phosphorylation with TFIID, B, F, and
H in the presence or absence of TFIIE, as
indicated at top. They carried out the reactions
for 60 or 90 min, sampling at various
intermediate times, as shown at top. The small
bracket at left indicates the position of the
polymerase IIo subunit, and the larger bracket
shows the locations of IIa and IIo together
(IIa/IIo). Arrows also mark the positions of the
two polymerase subunit forms. Note that
polymerase phosphorylation is more rapid in the
presence of TFIIE. (c) Graphic
presentation of the data from panel (b). Green
and red curves represent phosphorylation in the
presence and absence, respectively, of TFIIE.
Solid lines and dotted lines correspond to
appearance of phosphorylated polymerase subunits
IIa and IIo, or just IIo, respectively.
52
(No Transcript)
53
TFIIH phosphorylates the CTD of polymerase II
Figure 11.28 TFIIH phosphorylates the CTD of
polymerase II. (a) Reinberg phosphorylated
increasing amounts of polymerases IIA, IIB, or
IIO, as indicated at top, with TFIID, B, F, E,
and H and radioactive ATP as described in Figure
11.27. Polymerase liB, lacking the CTD, could not
be phosphorylated. The unphosphorylated
polymerase IIA was a much better phosphorylation
substrate than IIO, as expected. (b)
Purification of the phosphorylated CTD. Reinberg
and colleagues cleaved the CTD from the
phosphorylated polymerase Ila subunit with the
protease chymotrypsin (Chym), electrophoresed the
products, and visualized them by autoradiography.
Lane 1, reaction products before chymotrypsin
cleavage lanes 2 and 3, reaction products after
chymotrypsin cleavage. The position of the CTD
had been identified in a separate experiment.
54
Helicase activity of TFIIH
Figure 11.29 Helicase activity of TFIIH.
(a) The helicase assay. The substrate consisted
of a labeled 41-nt piece of DNA (red) hybridized
to its complementary region in a much larger,
unlabeled, single-stranded M13 phage DNA (blue).
DNA helicase unwinds this short helix and
releases the labeled 41-nt DNA from its larger
partner. The short DNA is easily distinguished
from the hybrid by electrophoresis. (b)
Results of the helicase assay. Lane 1,
heat-denatured substrate lane 2, no protein
lane 3, 20 ng of RAD25 with no ATP lane 4, 10 ng
of RAD25 plus ATP lane 5, 20 ng of RAD25 plus
ATP.
55
The TFIIH DNA helicase gene product(RAD25) is
required for transcription in yeast
Figure 11.30 The TFIIH DNA helicase gene product
(RAD25) is required for transcription in yeast.
Prakash and colleagues tested extracts from
wild-type (RAD25) and temperature-sensitive
mutant (rad25-ts24) cells for transcription of a
G-less cassette template at the permissive (a)
and nonpermissive (b) temperatures. After
allowing transcription for 0-10 minutes in the
presence of ATP, CTP, and UTP (but no GTP), with
one 32P-labeled nucleotide, they electrophoresed
the labeled products and detected the bands by
autoradiography. The origin of the extract (RAD25
or rad25-ts24), as well as the time of incubation
in minutes, is given at top. Arrows at left
denote the positions of the two G-less
transcripts. We can see that transcription is
temperature-sensitive when the TFIIH DNA helicase
(RAD25) is temperature-sensitive.
56
A model for the participation of general
transcription factors in initiation
Figure 11.31 A model for the participation of
general transcription factors in initiation,
promoter clearance, and elongation. (a) TBP (or
TFIID), along with TFIIB, TFIIF, and RNA
polymerase II form a minimal initiation complex
that makes abortive transcripts (magenta) at the
initiator, which is melted. (b) TFIIE and TFIIH
join the complex, converting it to an active
transcription complex. (c) The DNA helicase
activity of TFIIH uses ATP to unwind more of the
DNA double helix at the initiator. Somehow, this
allows promoter clearance. (d) With addition of
NTPs, the elongation complex continues elongating
the RNA. TBP and TFIIB remain at the promoter
TFIIE and TFIIH are not needed for elongation and
dissociate from the elongation complex.
57
SUMMARY
TFIIE, composed of two molecules each of a
34 kDa and a 56 kDa polypeptide, binds after
polymerase and TFIIF. Both subunits are required
for binding and transcription stimulation. A
protein known as MO15/CDK7, which associates
closely with TFIIH, phosphorylates the carboxyl
terminal domain (CTD) of the largest RNA
polymerase 11 subunit. TFIIE greatly stimulates
this process in vitro. TFIIE and TFIIH are not
essential for formation of an open promoter
complex, or for elongation, but they are required
for promoter clearance. TFIIH has a DNA helicase
activity that is essential for transcription, at
least in yeast, presumably because it facilitates
promoter clearance.
58
11.1.6 Elongation factors
Transcription can be controlled at the
elongation level. One factor, TFIIS, stimulates
elongation by limiting long pauses at discreet
sites TFIIF also stimulates elongation,
apparently by limiting transient pausing.
59
Effect of TFIIS on transcription initiation and
elongation combined
Figure 11.33 Effect of TFIIS on transcription
initiation and elongation combined.
Reinberg and Roeder carried out this experiment
in the same manner as in Figure 11.32, except for
the orcer of additions to the reaction. Here,
they added TFIIS (or buffer) at the beginning
instead of last (see time line at bottom). Thus,
TFIIS had the opportunity to .stimulate both
initiation and elongation. The dashed vertical
lines show no more stimulation than in Figure
11.32.
60

• Transcription can be controlled at the elongation
  level, TFIIS, stimulates elongation by limiting
  long pauses at discrete sites. TFIIF also
  stimulates elongation, apparently by limiting
  transient pausing.

61
Figure 11.29 A model for proofreading by RNA
polymerase II. (a) The polymerase, transcribing
the DNA from left to right, has just incorporated
an incorrect nucleotide (yellow). (b) The
polymerase backtracks to the left, extruding the
3'-end of the RNA, with its misincorporated
nucleotide, out of the active site of the enzyme.
At this point, the polymerase is irreversibly
arrested unless the extruded RNA can be removed.
(c) The ribonuclease activity of the polymerase
clips off the 3'-end of the RNA, including the
incorrect nucleotide. (d) The polymerase resumes
transcription.
62

• TFIIS stimulates proofreadingthe correction of
  mis-incorporated nucleotidepresumably by
  stimulating the RNase activity of the RNA
  polymerase, allowing it to cleave off a
  mis-incorporated nucleotide (with a few other
  nucleotides) and replace it with the correct one.

63
11.1.7 The polymerase II holoenzyme
Yeast and mammalian cells have an RNA
polymerase II holoenzyme that contains many
polypeptide in addition to the subunits of the
polymerase. The yeast holoenzyme contains a
subset of general transcription factors and at
least some of the SRB proteins. The rat
holoenzyme contains all the general transcription
factors and at least some of the SRB proteins.
The rat holoenzyme contains all the general
transcription factors except TFIIA.
64
Purified yeast RNA polymerase II holoenzyme
Figure 11.34 Purified yeast RNA polymerase II
holoenzyme. Kornberg and colleagues used a
purification scheme that included
immunoprecipitation to isolate a polymerase II
holoenzyme from yeast cells, then subjected the
polypeptide constituents of this holoenzyme to
SDS-PAGE Lane 2 displays these polypeptides (h
-pol II), while lane 1 contains the subunits of
the "core RNA polymerase II" (c- pol II) for
comparison.
65
11.2 Class I Factors

• The preinitiation complex that forms at rRNA
  promoters.
• SL1
• Upstream binding factor (UBF)

66
11.2.1 SL1

• SL1 plays a role in assembling the
  polymerase I preinitiation factor.

67
SL1 is a species-specific transcription factor
Figure 11.35 SL1 is a species-specific
transcription factor. Tjian and colleagues
performed a run-off assay with a mouse cell-free
extract and two templates, one containing a mouse
rRNA promoter, the other containing a human rRNA
promoter. The mouse and human templates gave rise
to run-off transcripts of 2400 and 1500 nt,
respectively. As shown at bottom, lane 1
contained no human SL1, and essentially only the
mouse template was transcribed. As Tjian and
colleagues added more and more human SL1, they
observed more and more transcription of the human
template, and less transcription of the mouse
template. In lane 5, transcription of both
templates seems to be suppressed.
68
Footprinting the rRNA promoter with SL1 and RNA
polymerase
Figure 11.36 Footprinting the rRNA promoter with
SL1 and RNA polymerase I.
69
Figure 11.36 Footprinting the rRNA promoter with
SL1 and RNA polymerase I. Tjian and
colleagues performed DNase I footprinting with
either the nontemplate strand (a), or the
template strand (b) of the human rRNA promoter
They added SL1 and/or RNA polymerase, as
indicated at bottom. Brackets indicate footprint
regions, while stars designate sites of enhanced
DNase sensitivity Polymerase I by itself can
protect a region (A) of the UCE polymerase and
SL1 together extend the protection into another
region (B) of the UCE. Binding of SL1 by itself
is not detectable by this assay (c) Summary of
footprints. Bars above and below the UCE region
represent the footprints on the template and
nontemplate strands. respectively, with the A and
B sections delineated. Again, stars represent the
sites of enhanced cleavage.
70
The core promoter element determines species
specificity
Figure 11.37 The core promoter element
determines species specificity.
71
Figure 11.37 The core promoter element
determines species specificity. Tjian and
colleagues constructed human, mouse,and hybrid
human/mouse rRNA premofers and tested them for
promoter activily by a run-off transcription
assay with partially purified human RNA
polymerase I and highly purified human SL1. All
reactions contained a control template, ?5'/-83,
which had a human rRNA promoter lacking the UCE.
This gave a basal level of transcription in all
cases and could be used to normalize the
reactions. The expected position of each run-off
transcript is indicated at left with an arrow.
The first two lanes in each set of three
contained increasing quantities of human SL1. as
indicated by the "wedges" the third lane in each
set had no SL1. Diagrams of each construct are
given at right. Human promoter elements are
rendered in green, and mouse elements in pink
Only when the construct contained a human core
element did transcription occur. The nature of
the UCE was irrelevant. Human SL1 was also
required. Thus, the core element determines the
species-specificity of the rRNA promoter.
72
11.2.2 UBF

• Stimulates transcription by polymerase I
• Actives the intact promoter or the core element
• Mediates activation by UCE

73
Interaction of UBF and SL1 with the rRNA promoter
Figure 11.38 Interaction of UBF and SL1 with the
rRNA promoter. Tjian and colleagues
performed DNase I footprinting with the human
rRNA promoter and various combinations of
polymerase I UBF and SL1 (a), or UBF and SL1
(b) The proteins used in each lane are indicated
at bottom. The positions of the UCE and core
elements are shown at left, and the locations of
the A and B sites are illustrated with brackets
at right Stars mark the positions of enhanced
DNase sensitivity SL1 caused no footprint on its
own, but enhanced and extended the footprints of
UBF in both the UCE and the core element This
enhancement is especially evident in the absence
of polymerase I (panel b).
74
Activation of transcription from the rRNA
promoter by SL1 and UBF
Figure 11.39 Activation of transcription from the
rRNA promoter by SL1 and UBF. Tjian and
colleagues used an S1 assay to measure
transcription from the human rRNA promoter in the
presence of RNA polymerase I and various
combinations of UBF and SL1, as indicated at top,
The top panel shows transcription from the
wild-type promoter the bottom panel shows
transcription from a mutant promoter (?5' -57)
lacking UCE function. SL1 was required for at
least basal activity, but UBF enhanced this
activity on both templates.
75
Effect of mutations in the UCE on UBF activation
(a)Description of mutants and effects on binding
Inserted linkers are represented by boxes,
deletions by spaces, and bases altered following
site-directed mutagenesis by Xs. The positions of
sites A and B of the UCE, relative to the
mutations, are given at bottom. Binding of UBF,
or UBF/SL1. to each mutant promoter is reported
at right. Tjian and colleagues measured the
binding by footprinting the criterion for
UBF/Shl binding was extension of the footprint
into site B.
76
(b)Effect on transcription
(b) Effect on transcription. Tjian and Coworkers
measured transcription by 81 analysis as in
Figure 11.39, in the presence (right panel) or
absence (left panel) of UBF. They included SL1
and polymerase I in all cases. They also added a
pseudo wild-type template (?WT) as an internal
control in all cases. The nature of the test
template (wild-type or mutant) is given at the
top of each lane. Mutant-186/-163 behaved like
the wild-type template in that it supported
stimulation by UBF. By contrast, all the other
mutant templates were considerably impaired in
ability to respond to UBF.
77
11.2.3 The Universality of TBP
78
Effect of mutations in TBP on transcription by
all three RNA polymerases
79
11.2.4 Structure and function of SL1
SL1 is composed of TBP and three
TAFs, TAFI110, TAFI63, and TAFI48. Fully
functional and species-specific SL1 can be
reconstituted from these purified components, and
binding of TBP to the TAFIs precludes binding to
the TAFIIS.
80
Co-purification of SL1 and TBP
Figure11.42 Co-purification of SLl and TBP.
81
Figure11.42 Co-purification of SLl and TBP.
(a) Heparin agarose column chromatography Top
Pattern of elution from the column of total
protein (red) and salt concentration (blue), as
well as three specific proteins (brackets).
Middle SL1 activity, measured by S1 analysis, in
selected tractions Bottom TBP protein, detected
by Western blotting, in selected fractions Both
SL1 and TBP were centered around fraction 56
(b) Glycerol gradient ultracentrifugation Top
Sedimentation profile of TBP Two other proteins,
catalase and aldolase, with sedimentation
coefficients of 11.3 S and 73 S, respectively,
were run in a parallel centrifuge tube as markers
Middle and bottom panels, as in pad (a) Both SL1
and TBP sedimented to a position centered around
fraction 16.
82
Immunodepletion of TBP inhibits SL1 activity
83
The TAFs in SL1
Figure 11.44 The TAFs in SL1. Tjian
and colleagues immunoprecipitated SL1 with an
anti-TBP antibody and subjected the polypeptides
in the immunoprecipitate to SDS-PAGE. Lane 1,
molecular weight markers lane 2,
immunoprecipitate (IP) lane 3, purified TBP for
comparison lane 4, another sample of
immunoprecipitate lane 5, TFIID TAFs
(PolII-TAFs) for comparison lane 6, pellet after
treating immunoprecipitate with 1 M guanidine-HCl
and re-precipitating, showing TBP and antibody
lane 7, supernatant after treating
immunoprecipitate with 1 M guanidine-HCl and
reprecipitating, showing the three TAFs (labeled
at right).
84
11.3 Class III Factors

• TFIIIA
• TFIIIB and C
• The role of TBP
• Transcription of all class III genes requires
  TFIIIB and C, and Transcription of the 5S rRNA
  genes requires these two plus TFIIIA.

85
11.3.1 TFIIIA
86
Effect of anti-TFIIIA on transcription by
polymerase III
Figure 11.45 Effect of anti-TFIIIA on
transcription by polymerase III.
Brown and colleagues added cloned 5S rRNA and
tRNA genes to an oocyte extract (a), or a somatic
cell extract (b) in the presence of labeled
nucleotide and no antibody (lanes 1), an
irrelevant antibody (lanes 2), or an anti-TFIIIA
antibody (lanes 3). After transcription, these
workers electrophoresed the labeled RNAs. The
anti-TFIIIA antibody blocked 5S rRNA gene
transcription in both extracts, but did not
inhibit tRNA gene transcription in either
extract. The oocyte extract could process the
pre-tRNA product to the mature tRNA form, while
the somatic cell extract could not. Nevertheless,
transcription occurred in both cases.
87
Schematic representation of two of the zinc
fingers in TFIIIA
Figure 11.46 Schematic representation of two of
the zinc ringers in TFIIIA. The zinc
(cyan) in each finger is bound to four amino
acids two cysteines (yellow) and two histidines
(blue), holding the finger in the proper shape
for DNA binding.
88
11.3.2 TFIIIB and C
89
Effect of transcription on DNA binding between a
tRNA gene and trranscription factors
90
Binding of TFIIIB and C to a tRNA gene
Figure 11.48 Binding of TFIIIB and C to a tRNA
gene. Geiduschek and coworkers performed
DNase footprinting with a labeled tRNA gene (all
lanes), and combinations of purified TFIIIB and C
Lane a, negative control with no factors lane b,
TFIIIC only lane c, TFIIlB plus TFIIIC lane d,
TFIIIB plus TFIIIC added, then heparin added to
strip off any loosely bound protein Note the
added protection in the upstream region afforded
by TFIIIB in addition to TFIIIC (lane c) Note
also that this upstream protection provided by
TFIIIB survives heparin treatment, while the
protection of boxes A and B does not Yellow boxes
represent coding regions for mature tRNA Boxes A
and B within these regions are indicated in blue.
91
Order of binding of transcription factors to a 5S
rRNA gene
Figure 11.49 Order of binding of transcription
factors to a 5S rRNA gene. Setzer and
Brown added factors TFIIIA, B, and C, one at a
time to a cloned 5S rRNA gene bound to cellulose.
After each addition, they washed away any unbound
factor before incubation with the next factor.
Finally, they added polymerase Ill and
nucleotides, one of which was labeled, and
assayed 5S rRNA synthesis by electrophoresing the
products. The order of addition of factors is
indicated at the top of each lane. Only when
TFIIIB was added last did accurate 5S rRNA gene
transcription occur. Thus, TFIIIB appears to need
the help of the other factors to bind to the gene.
92
11.3.3 The Role of TBF
93
Figure 11.50 Hypothetical scheme for assembly of
the preinitiation complex on a classical
polymerase III promoter (tRNA), and start of
transcription. (a) TFIIIC (light green) binds to
the internal promoters A and B blocks (green).
(b) TFIIIC promoters binding of TFIIIB (yellow),
with its TBP (blue) to the region upstream of the
transcription start site. (c) TFIIIB promoters
polymerase III (red) binding at the start site,
ready to begin transcribing. (d) Transcription
begins. As the polymerase moves to the right,
making RNA (not shown), it presumably removes
TFIIIC from the internal promoter. But TFIIIB
remains in place, ready to sponsor a new round of
polymerase binding and transcription.
94
Transcription of polymerase III genes complexed
only with TFIIIB
Figure 11.51 Transcription of polymerase III
genes complexed only with TFIIIB.
95
Figure 11.51 Transcription of polymerase III
genes complexed only with TFIIIB. Geiduschek
and coworkers made complexes containing a tRNA
gene and TFIIIB and C (two panels at left), or a
5S rRNA gene and TFIIIA, B, and C (two panels at
right), then removed TFIIIC with heparin (lanes
e-h), or TFIIIA and C with a high ionic strength
butter (lanes l-n). They passed the stripped
templates through gel filtration columns to
remove any unbound factors, and demonstrated by
gel retardation and DNase footpdnting (not shown)
that the purified complexes contained only TFIIIB
bound to the upstream regions el the respective
genes. Next, thsy tested these stripped complexes
alongside unstripped complexes for ability to
support single-round transcription (S lanes a,
e, i, and l), or multiple-round transcription (M
all other lanes) for the times indicated at
bottom. They added extra TFIIIC in lanes c and g,
and extra TFIIIB in lanes d and h as indicated at
top. They confined transcription to a single
round in lanes a, e, i, and I by including a
relatively low concentration of heparin, which
allowed elongation of RNA to be completed, but
then bound up the released polymerase so it could
not re-initiate. Notice that the stripped
template, containing only TFIIIB, supported just
as much transcription as the unstripped template
in both single-roued and multiple-round
experiments, even when the experimenters added
extra TFIIIC (compare lanes c and g, and lanes k
and n). The only case in which the unstripped
template performed better was in lane d, which
was the result of adding extra TFIIIB. This
presumably resulted from some remaining free
TFIIIC that helped the extra TFIIIB bind, thus
allowing more preinitiation complexes to form.
96
Model of preinitiation complexes on TATA-less
promoters recognized by all three polymerases
Figure 11.52 Model of preinitiation complexes on
TATA-less promoters recognized by all three
polymerases. In each case, an assembly factor
(green) binds first (UBF, Spl, and TFIIIC in
class I, II, and III promoters, respectively),
This in turn attracts another factor (yellow),
which contains TBP (blue) this second factor is
SL1, TFIID, or TFIIIB in class I, II, or III
promoters, respectively. These complexes are
sufficient to recruit polymerase for
transcription of class I and III promoters, but
in class II promoters more basal factors (purple)
besides polymerase II must bind before
transcription can begin.
97
THE END!