Transcript Document

Chapter 11
General Transcription
Factors
False-color transmission
electron micrograph of RNAs
being synthesized on a DNA
template, forming a feather-like
structure.
Table of contents
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Class II Factors
Class I Factors
UBF
Class III Factors
11.1 Class II Factors
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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
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.
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 A+D, with both transcription
factors, showed a larger complex with both factors
(A+D, later named DA).
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 (G+A 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 nonspecific complex (NS). Lane 5 contained DNA plus TFIID
and TFIIA (A+D). 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.
Building the preinitiation complex
Figure 11.3 Building the preinitiation complex.
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.
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.
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
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.
11.1.2 Structure and Function
of TFIID
• TATA Box binding Protein (TBP)
• TBP-associated factors(TAF)
Methylation interference at the TATA
box
Figure 11.8 Methylation interference at the TATA box.
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
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.
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 proteinDNA 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 TFIIDTATA 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.
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.
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 comple~es, 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.
Structure of the TBP-TATA box
complex
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.
Figure 11.7 Effects of mutations in TBP on transcription by all three RNA polymerases.
Figure 11.7 Effects of mutations in TBP on transcription by all three RNA polymerases.
(a)
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 heatshocked 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:
(b) 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 heatshocked. The P65 →S extract was deficient in transcribing from polymerase II and III
promoters, but could recognize the polymerase promoter, even after heat shock.
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 80°curve.
Structure of a Drosophila TFIID
assembled in vitro from the products of
cloned genes
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.
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).
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.
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 G+A 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.
Model for the interaction between TBP and
TATA-containing or TATA-less promoters
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 [α- 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.
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.
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.
Whole genome analysis of
transcription requirements in yeast
General transcription
Factor (subunit)
TFIID (TAFII145)
TFIID (TFII17)
TFIIE (Tfa1)
TFIIH (Kin28)
Fraction of genes
Dependent on
Subunit function(%)
16
67
54
87
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.
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
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
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 ."
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.
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.
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.
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.
11.1.5 Structure and function of
TFIIE and TFIIH
Formation of the DABPoIFE complex
Figure 11.23 Formation of the DABPolFE complex.
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.
Dependence of transcription on both
subunits of TFIIE
Figure 11.24 Dependence of transcription on both subunits of TFIIE.
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.
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.
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.
TFIIH phosphorylates RNA polymerase II
Figure 11.21 TFIIH phosphorylates RNA polymerase II.
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 [γ-32P]ATP 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.
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.
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.
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 temperaturesensitive mutant (rad25-ts24) cells for transcription of a G-less cassette template at the
permissive (a) and nonpermissive (b) temperatures. After allowing transcription for 010 minutes in the presence of ATP, CTP, and UTP (but no GTP), with one 32Plabeled 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.
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.
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.
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.
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.
• 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.
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.
• TFIIS stimulates proofreading—the
correction of mis-incorporated
nucleotide—presumably 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.
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.
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.
11.2 Class I Factors
The preinitiation complex that
forms at rRNA promoters.
• SL1
• Upstream binding factor (UBF)
11.2.1 SL1
SL1 plays a role in assembling
the polymerase I preinitiation
factor.
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.
Footprinting the rRNA promoter
with SL1 and RNA polymerase
Figure 11.36 Footprinting the rRNA promoter with SL1 and RNA polymerase I.
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.
The core promoter element determines species specificity
Figure 11.37 The core promoter element determines species
specificity.
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.
11.2.2 UBF
• Stimulates transcription by
polymerase I;
• Actives the intact promoter or the
core element;
• Mediates activation by UCE
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).
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.
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 sitedirected 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.
(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 (ψWT) 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.
11.2.3 The Universality of TBP
Effect of mutations in TBP on transcription
by all three RNA polymerases
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.
Co-purification of SL1 and TBP
Figure11.42 Co-purification of SLl and TBP.
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.
Immunodepletion of TBP inhibits
SL1 activity
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 guanidineHCl 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).
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.
11.3.1 TFIIIA
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.
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.
11.3.2 TFIIIB and C
Effect of transcription on DNA binding between a
tRNA gene and trranscription factors
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.
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.
11.3.3 The Role of TBF
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 promoter’s 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.
Transcription of polymerase III
genes complexed only with TFIIIB
Figure 11.51 Transcription of polymerase III genes complexed only with TFIIIB.
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 singleround 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.
Model of preinitiation complexes on TATAless 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.