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Molecular and Cellular Biology, July 2000, p. 5269-5275, Vol. 20, No. 14
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
TATA Binding Protein Can Stimulate Core-Directed
Transcription by Yeast RNA Polymerase I
Pavel
Aprikian,
Beth
Moorefield, and
Ronald H.
Reeder*
Hutchinson Cancer Research Center, Seattle,
Washington 98109
Received 10 December 1999/Returned for modification 21 January
2000/Accepted 25 April 2000
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ABSTRACT |
The TATA binding protein (TBP) interacts with two transcription
factor complexes, upstream activating factor (UAF) and core factor
(CF), to direct transcription by RNA polymerase I (polI) in the yeast
Saccharomyces cerevisiae. Previous work indicates that one
function of TBP is to serve as a bridge, ennabling UAF to recruit and
stabilize the binding of CF (23, 24). In this work we show
that, in addition to aiding recruitment, TBP also directly aids CF
function. Overexpression of TBP in strains with UAF components deleted
will stimulate CF-directed transcription nearly to wild-type levels in
vivo. In vitro, increasing the concentration of TBP stimulates
CF-directed transcription in the absence of either UAF or its DNA
binding site. This dual function of TBP, serving as a critical member
of a core promoter complex as well as a contact point for upstream
activators, appears similar to the dual roles that TBP also plays in
transcription by RNA polII.
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INTRODUCTION |
The promoter which directs rRNA
synthesis in the yeast Saccharomyces cerevisiae consists of
two functionally distinct sequence elements (2, 14, 18). One
of these elements is the core domain, which overlaps the site of
transcription initiation. Both in vitro and in vivo the core domain is
capable of directing a low level of accurate initiation by RNA
polymerase I (polI). Transcription from the core domain is strongly
stimulated by the presence of the upstream domain, a region whose
upstream boundary is close to position 
150 and whose downstream
boundary has been mapped to approximately 100.
Genetic and biochemical studies have shown that there are distinct
multiprotein complexes which specifically interact with each of these
promoter domains. Core factor (CF) interacts with the core domain and
contains Rrn6p, Rrn7p, and Rrn11p, proteins which are essential for
polI transcription (12, 15, 17). Upstream activating factor
(UAF) interacts with the upstream domain and contains Rrn5p, Rrn9p, and
Rrn10p (11), the two histones H3 and H4 (9), and
an unidentified protein, p30. UAF cannot independently direct
transcription but strongly stimulates polI transcription when CF is
also present.
The TATA binding protein (TBP) is clearly required for activated levels
of polI transcription (5, 22) and its precise interaction
with the UAF and CF complexes has been an ongoing matter of
investigation. It was initially reported that CF did not contain TBP
(12), but subsequent experiments (17, 23) showed
that TBP is associated with this complex. In addition, it has been
shown that TBP binds to UAF and that this association is required for
UAF to stimulate polI transcription (23). One of the UAF
components, Rrn9p, has been shown to be a direct target of TBP binding,
and mutations in Rrn9p that impair TBP binding can be suppressed by
fusing Rrn9p to TBP (24). It has further been shown that
basal levels of in vitro transcription can be obtained with a
reconstituted system apparently lacking TBP (10). These
experiments raise the possibility that in the yeast polI system the
sole function of TBP is to serve as a bridge between UAF and CF and
thereby enable UAF to recruit CF and position it over the core domain.
In this paper we report experiments which indicate that TBP plays an
important role in the functions of the CF complex in addition to any
role it may have in aiding recruitment by UAF. We show that
overexpression of TBP can bypass the requirement for UAF to achieve
activated levels of polI transcription in vivo. In vitro experiments
show that raising the level of TBP stimulates polI transcription in the
absence of UAF subunits or the upstream domain of the promoter. Taken
together, these results support a model in which TBP has at least two
important roles in polI transcription. TBP binding to UAF stimulates CF
recruitment and overall stabilization of the initiation complex. In
addition, TBP interaction with CF facilitates location of the core
domain of the promoter, polI recruitment, and production of high levels of accurate transcription initiation. The last role of TBP appears similar to its function at many other promoters recognized by RNA polII
and polIII.
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MATERIALS AND METHODS |
Yeast strains and plasmids.
Yeast strains and plasmid used
in this paper are described in Table 1.
All strains are derivatives of RLY01. Strains carrying knockouts of
individual RRN genes were constructed by replacing part of
the coding region with the HIS3 gene as described previously (11, 12), except with RLY08, in which the entire coding
region of the RRN9 gene was replaced by a DNA fragment
carrying the TRP1 gene.
Strain RLY4811 (carrying a temperature-sensitive mutation in the
RPA190 subunit of polI) was found in a genetic screen
similar
to that described by Nogi et al. (
19). Strain RLY01
was transformed
with plasmid pNOY103 (which expresses rRNA under the
control of
the
GAL7 promoter) and mutagenized with
ethylmethanesulfonate,
and colonies that could grow on galactose but
were unable to grow
on glucose were selected. Among the
galactose-dependent colonies,
one exhibited a temperature-sensitive
phenotype which was completely
rescued by transformation with a cloned
copy of the
RPA190 gene
and was not rescued by
transformation with genes for several of
the other polI subunits or
polI transcription factors. This strain
was designated
RLY4811.
RNA pulse-labeling in vivo.
Yeast cells were grown at 30°C
in minimal yeast nitrogen base (YNB) medium supplemented with galactose
and required amino acids. At an A600 of 0.5, cells were harvested by centrifugation, washed with water, and
resuspended in minimal YNB medium supplemented with glucose, amino
acids, and uracil decreased to 1/100 of the normal amount (0.2 mg/liter). After 30 min at 30°C, [5,6-3H]uracil (50 Ci/mmol) was added at 100 µCi/ml, and cells were harvested by
centrifugation 15 min later. Total RNA was extracted using TRIzol
(Gibco BRL) according to the manufacturer's instructions. RNA samples
containing the same amount of incorporated radioactive label were
separated on 1.2% agarose-formaldehyde gels. Gels were treated with
Amplify (Amersham) according to the manufacturer's instructions,
dried, and autoradiographed using Kodak Biomax-MR film.
Western blotting of TBP.
Cells were grown as described above
for the pulse-labeling experiment in 10 ml of medium. Equal numbers of
cells were harvested by centrifugation and resuspended in 100 µl of
loading buffer (0.06 M Tris-HCl [pH 6.8], 10% glycerol, 2% sodium
dodecyl sulfate, 5% 2-
-mercaptoethanol, 0.001% bromophenol blue).
After incubation at 100°C for 5 min, samples were resolved by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to a
nitrocellulose membrane, and reacted with anti-TBP antibodies. Signals
were detected as described previously (17).
In vitro transcription assay.
Whole-cell extracts were
prepared from cultures grown to an A600 of 1.0 essentially as described previously (21) except that ground
cells were thawed into 1/10 volume of extraction buffer containing 10%
glycerol. To normalize extracts to each other, each extract was
titrated to determine the amount yielding the highest level of specific
polI transcription activity when it was assayed with 10 µg of a
wild-type promoter template per ml. All transcription experiments shown
in this paper were performed using one-third of the amount of extract
which gives maximal activity. Each experiment has been repeated with at
least two independently prepared whole-cell extracts.
Templates for in vitro transcription contained either a wild-type polI
promoter, a promoter bearing a linker scanner mutation
in the upstream
domain (LS
142/
122), or a promoter bearing a
linker scanner
mutation in the core domain (LS
29/
2). In some
experiments (see
Fig.
5B and
6) upstream regions of the promoter
were deleted down to
122,
42, or
2 and replaced by vector sequence.
The construction
of these linker scanners or deletions has been
described previously
(
2). To reduce background from nonspecific
transcription,
each promoter was excised as an
EcoRI-
XhoI
fragment
and inserted into the polylinker of pUC19. Transcription was
assayed
by primer extension as described previously (
21) and
quantitated
by PhosphorImager
analysis.
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RESULTS |
TBP overexpression rescues deletions of UAF components but not CF
components.
Genes for CF components, RRN6,
RRN7, and RRN11, were individually disrupted in
strain RLY01 carrying plasmid pNOY103 (20). As previously
shown, these disruption strains are completely deficient in rRNA
transcription as shown by their inability to grow on glucose-containing media (12, 15, 17) (see Fig. 1A). In contrast, these strains all grow on galactose due to transcription of rRNA from the
GAL7 promoter on pNOY103. Genes for the UAF components,
RRN5, RRN9, and RRN10, were also
individually disrupted in the same background. As previously reported
(11), disruption of these genes impairs but does not abolish
rRNA production since the strains are capable of very slow growth on
glucose (see Fig. 1B). Results similar to these have previously been
interpreted as evidence that CF is absolutely required for rRNA
transcription. In contrast, a low level of rRNA synthesis, sufficient
to support cell growth at reduced rates, continues in the absence of UAF.
Each of the
RRN gene disruption strains was secondarily
transformed with the 2µm-based expression vector pSH223, which
carries
the gene for TBP under the control of its own promoter, and
plated
on either glucose- or galactose-containing medium. As we
expected,
the TBP expression vector did not rescue any of the strains
containing
disruptions of CF components as these strains remained
galactose
dependent and showed no growth on glucose (Fig.
1A). In contrast,
strains disrupted in
any of the UAF components (
RRN5,
RRN9, or
RRN10) showed nearly wild-type levels of growth on glucose
when
they were transformed with the TBP expression construct (Fig.
1B).
We also verified that the transformed UAF disruption strains
were
capable of growth in the presence of 5-fluoro-orotic acid,
demonstrating that growth on glucose-containing media is completely
independent of pNOY103 (data not shown).
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FIG. 1.
Overexpression of TBP bypasses the requirement for UAF
components in vivo. (A) Strains carrying disruptions of any of the CF
subunits ( RRN6, RRN7, and
RRN11) are able to grow on galactose (left plate) due to
rRNA transcribed from the GAL7 promoter on pNOY103 but are
unable to grow on glucose (right plate). Overexpression of TBP (top
sectors) does not alter this phenotype (compare to bottom sectors). (B)
Strains carrying disruptions of UAF subunits (RRN5,
RRN9, and RRN10) also grow on galactose due
to rRNA transcribed from pNOY103 (left plate). On glucose the UAF
disruption strains grow very slowly (right plate, bottom sectors).
However, overexpression of TBP allows the UAF disruption strains to
grow at near wild-type rates (right plate, top sectors). This result
indicates that TBP overexpression bypasses the requirement for the UAF
complex.
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These results suggest that increasing TBP levels within the cell can
bypass the requirement for the UAF complex and strongly
stimulate rRNA
transcription. To examine this hypothesis, we monitored
both rRNA
synthesis and TBP expression in these strains
directly.
TBP levels are elevated in cells transformed with a TBP expression
vector.
To verify that cells transformed with pSH223 overexpress
TBP, we examined TBP levels in extracts from both wild-type and UAF disruption strains by Western blot analysis. Figure
2 shows that introduction of the 2µm
TBP expression vector elevates TBP levels in both a wild-type strain
(compare lanes 1 and 2) and in a strain where the UAF component
RRN5 has been disrupted (compare lanes 3 and 4). We observed
that TBP levels were reproducibly lower in the RRN5
disruption strain than in the wild-type strain (compare lanes 1 and 3),
possibly reflecting the fact that the RRN5 strain grows
more slowly on galactose than does the wild type. We also observed that
introduction of the TBP expression vector into both the wild-type and
the RRN5 disruption strains reproducibly led to a
substantial increase in TBP levels.
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FIG. 2.
Transformation with a TBP expression vector (pSH223)
results in overexpression of the protein. Protein extracts from various
strains were blotted onto nitrocellulose and probed with a polyclonal
antibody against yeast TBP. As a loading control the same blots were
also probed with an antibody against the polII transcription factor
TFIIH. Lane 1, extract from wild-type (wt) cells (strain RLY01); lane
2, wild type transformed with pSH223; lane 3, strain disrupted in
RRN5 (RLY07); lane 4, RRN5 strain transformed
with pSH223. Transformation with pSH223 increases TBP levels
significantly in either wild-type or RRN5 cells (lanes 3 and 4) without affecting the level of TFIIH.
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TBP overexpression stimulates rRNA transcription in UAF disruption
strains.
We next compared levels of rRNA synthesized in wild-type
and disruption strains transformed with the TBP expression vector. Cells were pulse-labeled with [5,6-3H]uracil for 15 min,
and relative levels of isotope incorporated into rRNA were measured by
autofluorography. As expected, TBP overexpression in a CF disruption
strain (RRN11) did not stimulate rRNA transcription. rRNA
transcripts remained undetectable in the presence or absence of pSH223
(Fig. 3, compare lanes 5 and 6),
consistent with the observation that the TBP expression vector does not
rescue CF disruption strains. In contrast, TBP overexpression in a UAF
disruption strain (RRN5) stimulated polI transcription to
nearly wild-type levels (Fig. 3, lanes 3 and 4). Again, the results of
the pulse-labeling experiment were consistent with the observation that
transformation of UAF disruption strains with pSH223 allows growth on
glucose. As a control we compared the wild-type strain RLY01 (lane 1)
with a RLY01 strain transformed with the TBP expression vector. The
intensities of the bands corresponding to 18S and 25S rRNAs showed that
TBP overexpression in the wild-type strain had no effect on rRNA
synthesis. We conclude that TBP overexpression stimulates rRNA
transcription in the absence of a functional UAF complex.
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FIG. 3.
Overexpression of TBP stimulates rRNA transcription in a
strain with an inactive UAF (RRN5). Newly made rRNAs in
various strains were pulse-labeled and separated by gel
electrophoresis, and the radioactive bands were detected by
fluorography. The positions of mature 18S and 25S rRNAs are indicated.
TBP overexpression has no effect on rRNA synthesis in a wild-type (wt)
strain (RLY01, compare lanes 1 and 2). In contrast, TBP overexpression
in a RRN5 strain stimulates rRNA synthesis (RLY07) (lanes
3 and 4). As expected, no rRNA synthesis is detected in a
RRN11 strain, with or without TBP overexpression (RLY02)
(lanes 5 and 6).
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TBP overexpression stimulates rRNA synthesis by direct interaction
with the polI machinery.
Yeast strains defective for UAF can, with
some frequency, undergo an epigenetic alteration that results in
transcription of the chromosomal ribosomal DNA by polII rather than by
polI (25). UAF-deficient strains which have undergone this
polymerase switch can now grow on glucose in the absence of pNOY103. In
our own hands we have observed that colonies with the polymerase switch phenotype will arise from strain RLY07 (RRN5) with a
frequency of about 10
7 (data not shown). These
observations raise the possibility that TBP overexpression might
stimulate the frequency with which polymerase switch variants arise and
thus account for the ability of UAF-deficient strains to grow on
glucose upon TBP overexpression.
To address this possibility we constructed strain RLY4811 (isogenic to
RLY07), which expresses a temperature-sensitive allele
of the largest
subunit of polI (
rpa190-1ts) in the background
of an
RRN5 disruption. Strains RLY07 and RLY4811 were transformed
with either pSH223 (TBP expression), pRS425RRN5 (Rrn5p expression),
or
pRS425 (an empty vector [Table
1]). We compared the ability
of the
transformants to grow on either glucose or galactose medium
at both
permissive and nonpermissive temperatures. As expected
from previous
results, RLY07 (
RRN5) transformed with either pSH223
or
pRS425RRN5 can grow on either glucose or galactose at either
the
permissive or nonpermissive temperature. Also as expected,
RLY07
transformed with the empty vector can grow only on galactose.
These
results essentially reproduce what is shown in Fig.
1.
The critical result in Fig.
4 was
obtained with strain RLY4811 (which carries a
temperature-sensitive allele of the
RPA190 gene as well as a
disrupted
RRN5 gene) transformed with the same
set of
plasmids. RLY4811 behaves identically to RLY07 at the permissive
temperature in that TBP overexpression restores growth on glucose
(Fig.
4A). However, strain RLY4811 cannot grow on glucose at the
nonpermissive temperature where polI is inactivated (Fig.
4B).
This
result demonstrates that the rescue of UAF disruption strains
by TBP
overexpression is due to a stimulation of polI transcription
and is not
due to transcription of the ribosomal DNA by polII.
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FIG. 4.
TBP overexpression stimulates rRNA production by polI.
Strain RLY07 (RRN5) was secondarily transformed with
either an empty vector (pRS315), a vector expressing RRN5
(pRS315RRN5), or the TBP overexpression vector (pSH223). In parallel,
strain RLY4811 (isogenic to RLY07 but also carrying a
temperature-sensitive [ts] allele of RPA190) was
transformed with the same three vectors. (A) At the permissive
temperature (30°C) strains RLY07 and RLY4811 behave the same. They
grow on galactose due to the presence of pNOY103 and fail to grow on
glucose. Transformation with an expression vector for either RRN5 or
TBP allows both strains to grow on glucose. (B) At the nonpermissive
temperature (35.5°C) strain RLY4811 cannot grow on glucose even when
RRN5 or TBP is expressed (right plate, bottom sectors). This result
indicates that TBP overexpression stimulates rRNA production via polI
and not via some other RNA polymerase.
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TBP overexpression stimulates in vitro transcription from the core
domain of the promoter.
UAF requires the upstream domain of the
polI promoter in order to stimulate transcription (24).
Therefore, if TBP overexpression can stimulate transcription
independently of the UAF complex, it should stimulate transcription in
the absence of the upstream domain. In Fig.
5 we examine this possibility using an in
vitro transcription assay.
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FIG. 5.
Requirement for either the upstream domain of the
polI promoter or the UAF complex can be bypassed in vitro by
overexpression of TBP. (A) Transcription extracts were made from either
wild-type (WT) (RLY01) cells (lanes 1 through 6) or from wild-type
cells overexpressing TBP (lanes 7 through 12). Each extract was used to
transcribe templates bearing either a wild-type polI promoter (lanes 1, 4, 7, and 10), a promoter with an inactivated upstream domain
(LSup; lanes 2, 5, 8, and 11), or a promoter with an
inactivated core domain (LScore; lanes 3, 6, 9, and 12).
Assays were performed at either a high (10-µg/ml) or a low
(0.5-µg/ml) template concentration. Each extract was titrated to
determine the amount which gave the maximum transcription with a
wild-type template at 10 µg of DNA per ml. Thirty percent of the
maximal amount of extract was used for each of the experiments whose
results are shown. In wild-type extracts, the negative effect of
inactivating the upstream domain (LSup) can be overcome by
increasing the template concentration (compare lanes 2 and 5).
Inactivation of the core domain (LScore) eliminates
transcription at all template concentrations (lanes 3 and 6).
Overexpression of TBP strongly stimulates transcription from a template
lacking an upstream domain at a low template concentration (compare
lanes 5 and 11). (B) Extracts were made from a RRN5
strain (RLY07) or from a RRN5 strain overexpressing TBP.
Each extract was used to transcribe either a wild-type template (lanes
1 and 5), a template with a 5' deletion to position 122 of the
promoter (lanes 2 and 6), a template deleted to 42 (lanes 3 and 7),
or a template deleted to 2 (lanes 4 and 8) at a 10-µg/ml template
concentration. Extracts were used at 30% of the amount which gave
maximal activity on a wild-type template. Overexpressing TBP stimulates
transcription from promoters bearing deletions of the UAF binding
region (lanes 6 and 7) to the same level seen with an intact promoter
(lane 5).
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In Fig.
5A, whole-cell extracts prepared from either wild-type cells or
wild-type cells overexpressing TBP were used to transcribe
three
different promoter templates: a wild-type promoter, a promoter
with the
upstream domain inactivated by linker insertion (LS
142/
122),
and a
promoter with a linker insertion in the core domain (LS
29/
2).
Figure
5A, lanes 1 to 6, shows the transcription signal
obtained using
wild-type extract to transcribe each of the three
promoter variants. As
previously described (
2), polI transcription
is unaffected
by mutations in the upstream domain at a high template
concentration
(10 µg/ml; compare lanes 1 and 2) but is abolished
by mutations in
the core domain (compare lanes 1 and 3). At a
low template
concentration (0.5 µg/ml), however, transcription
is strongly
dependent upon the presence of the upstream domain
(compare lanes 4 and
5). Figure
5A, lanes 7 to 12, repeats the
same transcription shown in
lanes 1 to 6 except that the extract
was made from wild-type cells
overexpressing TBP. At low template
levels, the presence of excess TBP
in the extract boosted transcription
from the LS
142/
122 mutant
template to nearly wild-type levels
(compare lanes 10 and 11). As
expected, overexpression of TBP
did not rescue transcription of
templates carrying mutations in
the core domain under any conditions
(lanes 9 and 12). These results
indicate that increasing TBP levels can
stimulate transcription
from the core domain in the absence of the
upstream
domain.
We have also examined transcription of mutant polI promoters using
extracts made from cells lacking Rrn5p (Fig.
5B). In this
experiment
promoter templates were either wild type or contained
deletions that
extended from upstream of the promoter down to
5'
122, 5'
42, or 5'
2. The
122 and
42 deletions are expected
to remove part or all of
the upstream promoter domain, while the
2 deletion removes the core
domain as well (
2,
10,
14).
When the
RRN5
extract was assayed at 30% of maximal activity,
even the wild-type
promoter gave only a weak signal (lane 1),
indicating that UAF activity
was absent in this extract. Overexpression
of TBP strongly stimulated
transcription from the wild-type promoter
(lane 5) and stimulated
transcription from both of the deletion
mutants to the same level as
that of the wild type (lanes 6 and
7). Thus, increasing TBP levels can
stimulate transcription to
wild-type levels in the absence of both the
upstream domain and
the UAF
complex.
Transcription from the core domain is stimulated by addition of
recombinant TBP.
Extract from cells lacking Rrn5p produced a weak
polI transcription signal on either a wild-type, a 122, or a
42 template (Fig. 5B, lanes 1 through 3). Figure
6 shows the results of a further
experiment in which either an LS 142/122 template or a template
that has the upstream domain deleted (42) was transcribed but in
the presence of increasing amounts of recombinant TBP. Recombinant TBP
stimulated polI transcription about threefold (compare lane 1 with lane
3) or fourfold (compare lane 7 with lane 10) in the absence of either
Rrn5p or the upstream domain. As expected, addition of TBP did not
stimulate transcription from a template lacking the core domain (lanes
5 and 6).
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FIG. 6.
Recombinant TBP stimulates transcription from the core
domain in an extract defective for UAF. Extract from strain RLY07 was
used to transcribe templates either mutated in the upstream domain
(LSup, lanes 1 through 4), mutated in the core domain
(LScore, lanes 5 and 6), or having the upstream domain
deleted (42, lanes 7 through 11). Template concentrations were 10 µg/ml, and recombinant TBP (rTBP) was added to the reaction mixtures
as indicated. PhosphorImager quantitation indicates about a threefold
stimulation of transcription between lanes 1 and 3 and about a fourfold
stimulation between lanes 7 and 10.
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DISCUSSION |
Elevating TBP concentration stimulates core-dependent polI
transcription.
In this paper we show that elevating the
intracellular level of TBP can stimulate transcription from the rRNA
promoter and that this stimulation is independent of either UAF
components in vivo or the upstream domain of the promoter in vitro.
These observations support two important conclusions. First, they
demonstrate that TBP operates through the core domain of the
promoter and is therefore functionally a member of the CF complex.
Second, they suggest that the primary role of the UAF complex is to
recruit TBP or to stabilize its interactions within the CF complex.
It has been clearly demonstrated that binding of the UAF complex to the
upstream domain of the promoter strongly stimulates
rRNA transcription
and is required for preinitiation complex stability
in vitro
(
11). At issue has been the role of TBP in this process
and
the mechanism by which UAF stimulates transcription. Although
TBP was
shown to bind to the CF complex by coimmunoprecipitation
(
17) and affinity chromatography (
23) assays, its
requirement
for transcription factor activity has not been established.
In
a previous study, we demonstrated that TBP was specifically
coimmunoprecipitated
with the CF subunit Rrn11p and that all three CF
components (Rrn6p,
Rrn7p, and Rrn11p) were jointly required to restore
transcription
activity to an Rrn11p-depleted extract (
17).
However, we were
unable to address the requirement of TBP for CF
activity since
the CF-depleted extract still contained substantial
amounts of
TBP, presumably sequestered in TFIID and TFIIIB complexes
utilized
for polII and polIII transcription. Steffan and coworkers
circumvented
this problem by preparing TBP-responsive extracts from a
yeast
strain carrying the TBP point mutation I143N (
23).
They showed
that I143N extracts supported low levels of transcription
from
the core promoter but did not support activated transcription
from
promoters with an upstream domain. They also demonstrated
that addition
of recombinant TBP stimulated transcription from
a wild-type promoter
but did not increase basal transcription
from a core promoter template.
These results demonstrate that
TBP is required for UAF-mediated
transcriptional activation but
do not address its possible role in CF
activity since the I143N
point mutation may impair interactions with
UAF subunits without
altering interactions with CF proteins. If this is
the case, TBP
addition in vitro would not be expected to stimulate
transcription
from the core promoter alone since the I143N mutant would
be functionally
wild type for basal transcription. In support of this
scenario,
we note that several TBP point mutations which specifically
impair
activated transcription from polII promoters but support
wild-type
levels of basal transcription in vitro have been reported
(
13).
In this work we have shown that increasing the concentration of TBP in
an in vitro reaction stimulates high levels of polI
transcription from
templates carrying either a block mutation
in the upstream domain (Fig.
5A and
6) or a complete deletion
of the upstream domain (Fig.
5B).
Likewise, increasing TBP concentration
stimulates core-dependent
transcription when TBP is overexpressed
in a wild-type extract (Fig.
5A), when it is overexpressed in
an
RRN5 knockout extract
(Fig.
5B), and when it is added as recombinant
protein to an
RRN5 knockout extract (Fig.
6). This ability of
TBP to
stimulate core-dependent transcription is seemingly in
conflict with a
previous report by Keener et al. (
10) in which
polI
transcription was reconstituted in vitro from highly purified
components. A low level of core-dependent transcription was observed
in
the apparent absence of TBP, and those authors were unable
to stimulate
core-dependent transcription by addition of recombinant
TBP. It is
possible that this discrepancy is due to the use of
whole-cell extracts
for transcription versus the use of highly
purified components. For
example, in the polII system, the basal
factor TFIIA is required for
transcription in vivo and in whole-cell
extracts but is dispensable
when transcription is reconstituted
from purified factors
(
6).
Our in vitro results with whole-cell extracts are strongly supported by
in vivo data. Increasing the TBP level in a UAF disruption
strain leads
to increased rRNA synthesis (
RRN5) (Fig.
3) and
allows
galactose-dependent strains carrying disruptions of each
of the UAF
components (
RRN5,
RRN9, or
RRN10) to grow on glucose
(Fig.
1). Thus, increasing the
concentration of TBP allows the
requirement for UAF activator to be
bypassed in vivo as well as
in vitro. It further implies that TBP plays
an important role
in CF function, in addition to its role in
UAF-dependent
activation.
A simple model for how TBP stimulates core-directed transcription is
that it increases the number of promoters with CF bound
to them. This
may be accomplished by altering the CF complex,
by stabilizing its
association with DNA, or both. It is also possible
that TBP provides
DNA sequence specificity to the binding of CF
in a manner similar to
what has been proposed for the role of
TBP in the binding of the polIII
core complex, TFIIIB (
7,
8).
It will require further
research to decide among these
possibilities.
Yeast CF is functionally homologous with vertebrate SL1.
The
vertebrate SL1 complex was first identified by its ability to direct
accurate initiation in transcription systems primarily dependent on the
core domain of the human polI promoter (16). Subsequently it
was shown that SL1 consists of three polypeptides tightly associated
with TBP (4) and that TBP is essential for SL1 activity
(3). Thus, vertebrate SL1 shares significant functional homologies with yeast CF. Both complexes recruit polI, help it to
select the start site, and utilize TBP. It is interesting that SL1 and
CF perform similar transcriptional functions despite the fact that the
three TBP-associated factors present in SL1 (TAF110, TAF63, and TAF48)
share no obvious amino acid sequence similarity with those in CF
(Rrn6p, Rrn7p, and Rrn11p). Activation mechanisms seem to have diverged
even more strongly from yeast to vertebrates. Even though vertebrate
polI promoters have both core and upstream domains, analogous to those
of yeast, nothing resembling the yeast UAF activation complex has been
found in vertebrates. Instead, SL1 interacts with upstream binding
factor (1), a high-mobility-group box protein which
stabilizes and stimulates SL1-directed transcription. Nothing related
to UBF has been observed in the yeast polI system. At this point it
appears that polI transcription systems have retained a TBP-containing
core complex from fungi to humans which performs similar functions. In
contrast, the activation mechanism seems to have diverged considerably.
| |
ACKNOWLEDGMENTS |
We thank Judy Roan for expert technical assistance.
This work was partially supported by NIH grant GM26624 awarded to
R.H.R. B.M. was supported by NIH training grant CA09657.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Hutchinson
Cancer Research Center, 1100 Fairview Ave. North, Seattle, WA 98109. Phone: (206) 667-4513. Fax: (206) 667-4082. E-mail:
rreeder{at}fhcrc.org.
| |
REFERENCES |
| 1.
|
Bell, S. P.,
R. M. Learned,
H.-M. Jantzen, and R. Tjian.
1988.
Functional cooperativity between transcription factors UBF1 and SL1 mediates human ribosomal RNA synthesis.
Science
241:1192-1197[Abstract/Free Full Text].
|
| 2.
|
Choe, S. Y.,
M. C. Schultz, and R. H. Reeder.
1992.
In vitro definition of the yeast RNA polymerase I promoter.
Nucleic Acids Res.
20:279-285[Abstract/Free Full Text].
|
| 3.
|
Comai, L.,
N. Tanese, and R. Tjian.
1992.
The TATA-binding protein and associated factors are integral components of the RNA polymerase I transcription factor, SL1.
Cell
68:965-976[CrossRef][Medline].
|
| 4.
|
Comai, L.,
J. C. B. M. Zomerdijk,
H. Beckman,
S. Zhou,
A. Admon, and R. Tjian.
1994.
Reconstitution of transcription factor SL1: exclusive binding of TBP by SL1 or TFIID subunits.
Science
266:1966-1972[Abstract/Free Full Text].
|
| 5.
|
Cormack, B. P., and K. Struhl.
1992.
The TATA-binding protein is required for transcription by all three nuclear RNA polymerases in yeast cells.
Cell
69:685-696[CrossRef][Medline].
|
| 6.
|
Hampsey, M.
1998.
Molecular genetics of the RNA polymerase II general transcriptional machinery.
Microbiol. Mol. Biol. Rev.
62:465-503[Abstract/Free Full Text].
|
| 7.
|
Joazeiro, C. A. P.,
G. A. Kassavetis, and E. P. Geiduschek.
1996.
Alternative outcomes in assembly of promoter complexes: the roles of TBP and a flexible linker in placing TFIIIB on tRNA genes.
Genes Dev.
10:725-739[Abstract/Free Full Text].
|
| 8.
|
Kassavetis, G. A.,
C. A. Joazeiro,
M. Pisano,
E. P. Geiduschek,
T. Colbert,
S. Hahn, and J. A. Blanco.
1992.
The role of the TATA-binding protein in the assembly and function of the multisubunit yeast RNA polymerase III transcription factor, TFIIIB.
Cell
71:1055-1064[CrossRef][Medline].
|
| 9.
|
Keener, J.,
J. A. Dodd,
D. Lalo, and M. Nomura.
1997.
Histones H3 and H4 are components of upstream activation factor required for the high-level transcription of yeast rDNA by RNA polymerase I.
Proc. Natl. Acad. Sci. USA
94:13458-13462[Abstract/Free Full Text].
|
| 10.
|
Keener, J.,
C. A. Josaitis,
J. A. Dodd, and M. Nomura.
1998.
Reconstitution of yeast RNA polymerase I transcription in vitro from purified components.
J. Biol. Chem.
173:33795-33802.
|
| 11.
|
Keys, D. A.,
B.-S. Lee,
J. A. Dodd,
T. T. Nguyen,
L. Vu,
E. Fantino,
L. M. Burson,
Y. Nogi, and M. Nomura.
1996.
Multiprotein transcription factor UAF interacts with the upstream element of the yeast RNA polymerase I promoter and forms a stable preinitiation complex.
Genes Dev.
10:887-903[Abstract/Free Full Text].
|
| 12.
|
Keys, D. A.,
J. S. Steffan,
J. A. Dodd,
R. T. Yamamoto,
Y. Nogi, and M. Nomura.
1994.
RRN6 and RRN7 encode subunits of a multiprotein complex essential for the initiation of rDNA transcription by RNA polymerase I in Saccharomyces cerevisiae.
Genes Dev.
8:2349-2362[Abstract/Free Full Text].
|
| 13.
|
Kim, T. K.,
S. Hashimoto,
R. J. Kelleher,
P. M. Flanagan,
R. D. Kornberg,
M. Horikoshi, and R. G. Roeder.
1994.
Effects of activation-defective TBP mutations on transcription initiation in yeast.
Nature
369:252-255[CrossRef][Medline].
|
| 14.
|
Kulkens, T.,
D. L. Riggs,
J. D. Heck,
R. J. Planta, and M. Nomura.
1991.
The yeast RNA polymerase I promoter: ribosomal DNA sequences involved in transcription initiation and complex formation in vivo.
Nucleic Acids Res.
19:5363-5370[Abstract/Free Full Text].
|
| 15.
|
Lalo, D.,
J. S. Steffan,
J. A. Dodd, and M. Nomura.
1996.
RRN11 encodes the third subunit of the complex containing Rrn6p and Rrn7p that is essential for the initiation of rDNA transcription by yeast RNA polymerase I.
J. Biol. Chem.
271:21062-21067[Abstract/Free Full Text].
|
| 16.
|
Learned, R. M.,
S. Cordes, and R. Tjian.
1985.
Purification and characterization of a transcription factor that confers promoter specificity to human RNA polymerase I.
Mol. Cell. Biol.
5:1358-1369[Abstract/Free Full Text].
|
| 17.
|
Lin, C.-W.,
B. Moorefield,
J. Payne,
P. Aprikian,
K. Mitomo, and R. H. Reeder.
1996.
A novel 66-kilodalton protein complexes with Rrn6, Rrn7, and TATA-binding protein to promote polymerase I transcription initiation in Saccharomyces cerevisiae.
Mol. Cell. Biol.
16:6436-6443[Abstract].
|
| 18.
|
Musters, W.,
J. Knol,
P. Maas,
A. F. Dekker,
H. van Heerikhuizen, and R. J. Planta.
1989.
Linker scanning of the yeast RNA polymerase I promoter.
Nucleic Acids Res.
17:9661-9678[Abstract/Free Full Text].
|
| 19.
|
Nogi, Y.,
L. Vu, and M. Nomura.
1991.
An approach for isolation of mutants defective in 35S ribosomal RNA synthesis in Saccharomyces cerevisiae.
Proc. Natl. Acad. Sci. USA
88:7026-7030[Abstract/Free Full Text].
|
| 20.
|
Nogi, Y.,
R. Yano, and M. Nomura.
1991.
Synthesis of large rRNAs by RNA polymerase II in mutants of Saccharomyces cerevisiae defective in RNA polymerase I.
Proc. Natl. Acad. Sci. USA
88:3962-3966[Abstract/Free Full Text].
|
| 21.
|
Schultz, M. C.,
S. Y. Choe, and R. H. Reeder.
1991.
Specific initiation by RNA polymerase I in a whole-cell extract from yeast.
Proc. Natl. Acad. Sci. USA
88:1004-1008[Abstract/Free Full Text].
|
| 22.
|
Schultz, M. C.,
R. H. Reeder, and S. Hahn.
1992.
Variants of the TATA binding protein can distinguish subsets of RNA polymerase I, II, and III promoters.
Cell
69:697-702[CrossRef][Medline].
|
| 22a.
|
Sikorski, R. S., and P. Hieter.
1989.
A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in S. cerevisiae.
Genetics
122:19-27[Abstract/Free Full Text].
|
| 23.
|
Steffan, J. S.,
D. A. Keys,
J. A. Dodd, and M. Nomura.
1996.
The role of TBP in rDNA transcription by RNA polymerase I in Saccharomyces cerevisiae: TBP is required for upstream activation factor-dependent recruitment of core factor.
Genes Dev.
10:2551-2563[Abstract/Free Full Text].
|
| 24.
|
Steffan, J. S.,
D. A. Keys,
L. Vu, and M. Nomura.
1998.
Interaction of TATA-binding protein with upstream activation factor is required for activated transcription of ribosomal DNA by RNA polymerase I in Saccharomyces cerevisiae in vivo.
Mol. Cell. Biol.
18:3752-3761[Abstract/Free Full Text].
|
| 25.
|
Vu, L.,
I. Siddiqui,
B.-S. Lee,
C. A. Josaitis, and M. Nomura.
1999.
RNA polymerase switch in transcription of yeast rDNA: role of transcription factor UAF (upstream activation factor) in silencing rDNA transcription by RNA polymerase II.
Proc. Natl. Acad. Sci. USA
96:4390-4395[Abstract/Free Full Text].
|
Molecular and Cellular Biology, July 2000, p. 5269-5275, Vol. 20, No. 14
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
