 |
INTRODUCTION |
In eukaryotes, transcriptional initiation by RNA polymerase II
requires a set of general transcriptional factors (reviewed in
Refs. 1-3). These factors are assembled in a stepwise manner to form a
preinitiation complex on the core promoter (1) or are recruited as a
few preassembled units (4-6). In either case, the first step in
preinitiation complex assembly is the binding of a protein complex
called TFIID to the core promoter, which in turn provides a structural
platform for the remainder of the general transcriptional factors to be
incorporated (6). Previous studies have shown that TFIID-promoter
interactions are a major rate-limiting step during transcriptional
initiation and therefore are one of the most important molecular
targets for transcriptional activators (7-9).
TFIID is a multiprotein complex composed of the TATA-binding protein
(TBP)1 and ~10-12
phylogenetically conserved TBP-associated factors (TAFs) (reviewed in
Refs. 9 and 10). A number of biochemical studies have revealed
coactivator and core promoter recognition activities to be two
important functions for TAFs (reviewed in Refs. 9-11). Earlier
experiments using in vitro transcription systems
demonstrated that TBP can mediate basal transcription but is unable to
support activated transcription by itself. In contrast, TFIID, even
when reconstituted with recombinant TBP and TAFs (12), mediates both
basal and activated transcription, supporting the idea that TAFs are
essential cofactors for transcriptional activation (reviewed in Refs. 9
and 10). More recent studies have begun to address how core promoters
of eukaryotic genes are recognized by TFIID (reviewed in Refs. 13 and
14). The three classes of core promoter elements that are currently
known are the TATA element, the initiator, and the downstream promoter
element, each of which may serve as a recognition site for distinct
TFIID subunits (reviewed in Refs. 13 and 14). In addition to the extensively characterized TBP-TATA element interactions (15), the
initiator and downstream promoter element have been shown to be
recognized by TAF250-TAF150 and TAF60-TAF40 heterodimers, respectively
(13, 16). These TAF-DNA and TBP-DNA interactions are important for the
ability of TFIID to bind to the core promoter specifically and to
mediate transcription efficiently (reviewed in Refs. 13 and 14). In
addition, other cofactors such as TFIIA (17), TAFII- and
initiator-dependent cofactors (17), and NC2 (18), appear to
modulate the recognition by TFIID of a wide range of core promoter structures.
These principal functions of TAFs have also been evaluated in living
cells (reviewed in Refs. 10 and 11). Genetic depletion or inactivation
analysis of yeast TAF145, a subunit that is orthologous to human
TAF250, revealed that it was not required for transcription generally
but was essential for a subset of genes in vivo (reviewed in
Ref. 11). Promoter swapping experiments demonstrated that TAF145
function is demanded by the core promoter region rather than by
upstream activating sequences (UASs) as examined for the CLN2, RPS5, and TUB2 genes (19, 20).
Thus, it appears that TAF145 function is tightly connected to core
promoter recognition, in accordance with human TAF250, which directly
recognizes an initiator element as described above (13). On the other
hand, TAF145 has been shown to be required for other transcriptional activities, such as activation of the ADH2 gene by Adr1 (21) and derepression of RNR genes by DNA damage, that are
normally repressed by the Crt1 and Tup1-Ssn6 corepressor complex (22). Consistent with such apparently broad roles in transcription, yeast
TAF145 and/or human TAF250 possess multiple activities (e.g. TAF N-terminal domain activity, which inhibits TBP function (23, 24); serine/threonine kinase that autophosphorylates and
transphosphorylates TFIIF (25); histone acetyl transferase, which
acetylates histones and TFIIE (26, 27); two bromodomains, which bind
acetylated histones (28); and a ubiquitin-activating/conjugating
activity for histone H1 (29)). Some of these activities have been shown to be required for gene expression in vivo (29-31).
How broadly TAF functions are required for gene expression has been
extensively studied in yeast (reviewed in Refs. 11, 32, and 33).
Genome-wide expression analysis suggests that any TAFs thus far
examined are not universally required for transcription, in contrast to
other general transcriptional factors, such as Srb4, Kin28, and the
largest subunit of RNA polymerase II, which are required for almost all
genes (reviewed in Refs. 11, 32, and 33). More puzzlingly, mutations in
different TAFs affect different sets of genes, ranging from 3% (tsm1)
to 67% (taf17) of the whole genome (33). It has been proposed that
TAFs shared by TFIID and SAGA, another crucial histone acetyl
transferase complex regulating a subset of genes distinct from TFIID
(34), tend to affect a broader range of genes. This is probably because mutations in the TAFs that they have in common should decrease the
activities of TFIID and SAGA simultaneously (reviewed in Ref. 35).
However, this supposition has not yet been firmly established, because
TFIID-specific TAFs such as TAF40 (36) and TAF48/TSG2 (37, 38) have
been shown to be generally required for transcription. The wide range
of affected genes (3-67% of the genome) may be partly explained by
the allele specificities because, for example, the tighter allele of
TAF17 caused much more dramatic loss of transcription than the milder
one (39). The recent finding that chick TAF31 can be deleted
genetically without affecting the expression of most genes, although it
is an ortholog of the TAF (TAF17) that has the most universal function
among yeast TAFs, indicates that the requirement for TAFs is
evolutionarily divergent (40).
To further clarify how TAF function is involved in gene expression
in vivo, we believe it is important to isolate a wide range of conditional taf alleles and to inspect the
transcriptional defect at the molecular level in each taf
mutant. We recently isolated two novel temperature-sensitive
taf145 mutants in which the expression profiles of some
genes were not identical to those in previously reported
taf145 mutants (20). In our mutants, the core promoter of
theTUB2 gene failed to mediate basal transcription, but the
function was restored by inserting a consensus TATA element (20).
Consistent with this, we show here that the TATA element is important
for transcription from the CLN2 and CYC1
promoters. Interestingly, however, the creation of a consensus TATA
element cannot restore transcription from the RPS5 promoter.
We demonstrate that the RPS5 promoter is mostly impaired in
activated transcription and only slightly impaired in basal
transcription and that the creation of a consensus TATA element was
able to rescue the latter defect. Most importantly, we find that the
requirement for TAF145 function in activated transcription of the
RPS5 promoter depends on both core promoter structure and
UASs. These results imply that a specific function of TAF145 is
involved in both core promoter recognition and core promoter and
UAS-specific activated transcription.
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EXPERIMENTAL PROCEDURES |
Yeast Strains and Media--
Standard techniques were used for
yeast growth and transformation (41, 42). The yeast strains used in
this study, YTK3010 (wild type), YTK3002 (Y570N), YTK3003 (N568
),
and YTK3005 (T657K) were generated by plasmid shuffle techniques from
the parental strain Y22.1 (20). They carry a deletion of the
chromosomal TAF145 coding region and the wild type or mutant
TAF145 gene on a TRP1-based low copy number
vector (20).
Construction of Mini-CLN2 Hybrid Gene Reporters--
pM1452
(shown as
CLN2TATA in Fig. 1)
and pM1591 (shown as
UASGAL+CYC1TATA/-174
in Fig. 1 and UASGAL+CYC1/-174 in Fig.
5) were described previously (20). pM1452 and pM1591 were subjected to
site-specific mutagenesis (43) to create pM3226 (shown as
CLN2GAGA in Fig. 1) and pM3227 (shown
as UASGAL+CYC1GAGA/-174 in Fig.
1) using oligonucleotides TK1289 and TK1290, respectively. The
oligonucleotides used in this study are listed in Table
I. pM3228 (shown as RPS5/-593
TAAAAT in Fig. 1) was constructed by replacing the 765-bp
SphI/XhoI fragment of pM1452 (encompassing the
CLN2 promoter) with a 732-bp DNA fragment containing the
RPS5 promoter, which was amplified by PCR using the primer
pair TK1291 (+SphI)-TK1292 (+XhoI) and genomic
DNA as a template. pM3228 was subsequently subjected to site-specific
mutagenesis to create pM3229 (TATAAA), pM3230 (TATAAAAA), pM3231
(TATATAAA), and pM3232 (TATATAAAAA), using the oligonucleotides TK1293,
TK1466, TK1467, and TK1468, respectively.
To enable the construction of CLN2-RPS5 hybrid promoters, a
SpeI site was created at the -68 bp position of pM1452 by
site-specific mutagenesis, using the TK1322 oligonucleotide. The 506-bp
SpeI/XhoI fragment containing the CLN2
initiator of the resulting plasmid pM3233 was subsequently replaced
with the 212-bp DNA fragment containing the RPS5 initiator,
which was amplified by PCR using the primer pair TK1321
(+SpeI)-TK1292 (+XhoI) and pM3228 (RPS5/-593) as
a template, to create pM3234 (shown as CLN2(-332)-RPS5 in
Fig. 2). For deletion analysis of the CLN2-RPS5 hybrid
promoter, pM3235 (CLN2(-126)-RPS5 in Fig.
2), pM3236 (CLN2(-96)-RPS5 in
Fig. 2), pM3237 (CLN2(-92)-RPS5 in
Fig. 2), pM3238 (CLN2(-88)-RPS5 in
Fig. 2), pM3239 (CLN2(-86)-RPS5 in
Fig. 2), pM3240 (CLN2(-84)-RPS5 in
Fig. 2), pM3241 (CLN2(-82)-RPS5 in
Fig. 2), and pM3242 (CLN2(-80)-RPS5 in Fig. 2) were constructed by replacing the 476-bp
SphI/XhoI fragment of pM3234 with DNA fragments
encoding the -126 (CLN2)~+139 (RPS5), -96
(CLN2)~+139 (RPS5), -92
(CLN2)~+139 (RPS5), -88
(CLN2)~+139 (RPS5), -86
(CLN2)~+139 (RPS5), -84
(CLN2)~+139 (RPS5), -82
(CLN2)~+139 (RPS5), and -80
(CLN2)~+139 (RPS5) fragments of the
CLN2-RPS5 hybrid promoter, respectively; these fragments
were amplified by PCR using pM3234 (CLN2(-332)-RPS5) as a template and
the following primer pairs: TK1436-TK1292, TK1435-TK1292,
TK1500-TK1292, TK1501-TK1292, TK1502-TK1292, TK1503-TK1292,
TK1504-TK1292, and TK1505- TK1292.
pM3244 (shown as RPS5/-87C in Fig. 3), pM3245
(RPS5TATAAA/-87C in Fig. 3), pM3248
(RPS5/-87A in Fig. 3), and pM3249 (RPS5TATAAA/-87A in Fig. 3) were
constructed by replacing the 765-bp SphI/XhoI
fragment of pM1452 with the DNA fragments containing the
RPS5 core promoter or its derivatives that were amplified by
PCR using the primer pairs TK1549-TK1292, TK1550-TK1292, TK1551-TK1292,
and TK1552-TK1292 and pM3228 (RPS5/-593) as a template. pM3246
(RPS5/-87C+SpeI in Fig. 3), pM3247
(RPS5TATAAA/-87C+SpeI in Fig. 3),
pM3250 (RPS5/-87A+SpeI), and pM3251
(RPS5TATAAA/-87A+SpeI in Fig. 3) were
constructed by replacing the 264-bp SphI/SpeI fragment of pM3234 (CLN2(-332)-RPS5
in Fig. 2) with the short DNA fragments generated by annealing two
oligonucleotide pairs, TK1541-TK1542, TK1545-TK1546, TK1543-TK1544, and
TK1547-TK1548, respectively.
For deletion analysis of the RPS5 promoter (Fig. 4), pM3252
(RPS5/-450 in Fig. 4), pM3253 (RPS5/-300 in
Fig. 4), and pM3254 (RPS5/-200 in Fig. 4) were constructed
by replacing the 765-bp SphI/XhoI fragment of
pM1452 with DNA fragments encoding the -450~+139-, -300~+139-,
and -200~+139-bp regions of the RPS5 promoter,
respectively; these fragments were amplified by PCR using the primer
pairs TK1624-TK1292, TK1625-TK1292, and TK1626-TK1292, respectively,
and pM3228 (RPS5/-593) as a template. pM3255
(RPS5/-593/
UASRAP1 in Fig. 4) was generated by removing the presumptive RAP1 binding site (-403~-415 bp) (44) of pM3228 (RPS5/-593) by site-specific mutagenesis using the
oligonucleotide TK1696.
pM3256 (shown as UAS90bp+RPS5/-87 in
Fig. 5) was created by ligating the 90-bp DNA fragment encoding
-450~-361-bp of RPS5-UAS, which was amplified by PCR using the
primer pair TK1624-TK1697 and pM3228 (RPS5/-593) as a
template, into the SphI site of pM3244
(RPS5/-87C). Similarly, pM3257
(UAS150bp+RPS5/-200 in Fig. 5) and
pM3258 (UAS150bp+RPS5/-87 in Fig. 5)
were created by ligating the 150-bp (-450~-300 bp of RPS5-UAS) DNA fragment amplified by the primer pair TK1624-TK1733 into the
SphI sites of pM3254 (RPS5/-200) and pM3244 (RPS5/-87C),
respectively. To construct pM3259 (UAS150bp+CYC1/-174 in
Fig. 5), pM1588 was created first by replacing the 260-bp
SpeI/XhoI fragment of pM1585 (20) with the 344-bp
PCR fragment containing the CYC1 promoter amplified by the
primer pair TK1136-TK1137. pM3259 was subsequently created by ligating
the 150-bp (-450~-300 bp of RPS5-UAS) DNA fragment amplified by the
primer pair TK1734-TK1735 into the SpeI site of pM1588.
pM3260 was created by ligating the DNA fragment containing four repeats
of the GAL4 binding site, which was amplified by PCR using the primer
pair TK1627-TK1628 and pM2190 as a template, into the SphI
site of pM3244 (RPS5/-87C). The PvuII fragment of pM3266
containing the entire reporter gene was moved into pRS316 (45) to
change the auxotrophic marker from LEU2 to URA3.
The resulting plasmid pM3279 is shown as
UASGAL+RPS5/-87C in Fig. 5.
Plasmids Encoding Activation Domains Fused with the GAL4 DNA
Binding Domain--
The plasmids expressing activators in yeast cells,
pM524 (GAL4DBD-VP16C (amino acids 457-490)), pM1570 (GAL4DBD-EBNA2
(amino acids 426-462)), pM967 (GAL4DBD-GCN4 (amino acids 107-144)),
and pM471 (GAL4DBD) have been described previously (20). pM3261 (GAL4DBD-RAP1 (amino acids 630-690)) was similarly constructed by
ligating the DNA fragment encoding the RAP1 activation domain (amino
acids 630-690) (46) that was amplified using the primer pair
TK1694-TK1695 into the EcoRI/PstI sites of pM471.
Northern Blot Analyses--
Northern blot analyses were
performed as described previously (20). All mRNAs derived from
mini-CLN2 hybrid gene reporter constructs were detected by
the 32P-labeled 411-bp XhoI/HindIII
fragment isolated from pM1452.
 |
RESULTS |
The TATA Element Is Important for Transcription from the CLN2 and
CYC1 Promoters in taf145 Mutants--
We previously demonstrated that
transcription of a subset of genes at 37 °C is drastically impaired
in the taf145-N568
and -T657K mutants but
only slightly impaired in the taf145-Y570N mutant, despite
the much slower growth phenotypes of all of these mutants at 37 °C
(20). Interestingly, the impaired transcription from the
TUB2 promoter in the former two mutants can be rescued by
creating a consensus TATA element, indicating that the TATA element
compensates for the loss of TAF145 function (20). Therefore, we assume
that the TATA element will become more crucial to the transcription of
a subset of genes once TAF145 is mutated. To examine this possibility
further by reciprocal experiments, we tested whether the TATA element
is required for transcription from the CLN2 and
CYC1 core promoters, both of which were not affected in our
taf145 mutants (20). To monitor the transcriptional activities of these promoters, we employed the mini-CLN2
hybrid gene reporter system that we had previously developed, in which the loading amount of RNA can be easily normalized (20). In this
system, the expression of the endogenous CLN2 gene, which is
not affected in our mutants, can be used as an internal standard because a single probe (shown at the top in Fig.
1A) detects both transcripts
simultaneously, i.e. the transcript from the reporter construct (marked with a black arrow in Northern blot
analyses, as shown in Fig. 1B) and from the endogenous
CLN2 gene (Fig. 1B, white arrow). The transcript
derived from the former is ~1 kilobase pair shorter than that
derived from the latter because the SpeI-NcoI fragment encoding an essential carboxyl-terminal region of
CLN2 was excised to generate the mini-CLN2 gene
(Fig. 1A).

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Fig. 1.
Promoter-specific TATA dependence in
taf145 mutants. A, schematic
representation of the reporter plasmids used in this experiment. The
positions of the UAS, TATA element, transcriptional initiation site,
open reading frame of the CLN2 gene, and probe for Northern
analysis are shown in the top row. The
CLN2TATA reporter construct was generated by removing an
internal SpeI/NcoI fragment from the intact
CLN2 gene, as shown in the second row.
Arrows indicate the initiation site and direction of
transcription. Sequences derived from the CYC1 and
RPS5 genes are indicated by striped and
shaded boxes, respectively. Synthetic binding sites
for GAL4 fusion activators were linked upstream of the
CYC1 promoter to generate the
UASGAL+CYC1TATA/-174 reporter plasmid,
although GAL4 fusion activators were not coexpressed in this
experiment. TATA elements (open squares) of the
CLN2TATA and
UASGAL+CYC1TATA/-174 reporter plasmids were
replaced with GAGA elements (closed squares) by
site-specific mutagenesis to generate CLN2GAGA and
UASGAL+CYC1GAGA/-174 reporter plasmids,
respectively. Conversely, the TAAAAT sequence of the RPS5 promoter
(RPS5/-593) was replaced with various TATA elements as indicated in
the bottom row. B, Northern blot analysis of
mRNA with a CLN2-specific probe to test the requirement
for the TATA element for transcription from the CLN2,
CYC1, and RPS5 promoters. Total RNA was isolated
from wild type or mutant strains (Y570N, N568 , and T657K) harboring
the indicated reporter plasmids 2 h after a temperature shift to
37 °C (lanes 5-8) or after being continuously incubated
at 25 °C over the same time period (lanes 1-4) and were
blotted for hybridization with a radioactive CLN2 probe. The
upper band, marked with a white arrow, corresponds to
mRNA derived from the endogenous CLN2 gene, whereas the
lower band, marked with a black arrow, corresponds to
mRNA derived from the mini-CLN2 gene on the reporter
plasmid. The middle band, marked with an asterisk in the
third (UASGAL+CYC1TATA/-174) and
fourth (UASGAL+CYC1GAGA/-174)
rows presumably corresponds to transcripts initiated from an
unknown promoter on the reporter plasmid under these particular culture
conditions.
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We constructed two parental reporter plasmids:
CLN2TATA,
which contains upstream sequences up to 332 bp from the transcriptional start site of the CLN2 promoter, and
UASGAL+CYC1TATA/-174, which contains upstream
sequences 174 bp from the transcriptional start site of the
CYC1 promoter (Fig. 1A). In our mutants, these
reporter plasmids were expressed normally at 37 °C as previously
demonstrated (Fig. 1B, top section). The consensus
TATA elements of both reporter plasmids were changed to GAGA elements
by two-nucleotide substitutions to generate two novel TATA-less
reporter plasmids,
CLN2GAGA and UASGAL+CYC1GAGA/-174 (Fig. 1A).
When these two plasmids were introduced into yeast cells, they produced
much lower amounts of transcript at 37 °C in the N568
and T657K
mutants (Fig. 1B, top section). Together with the previous
reciprocal results showing the stimulatory effect of the TATA element
in the TUB2 promoter, this shows that the TBP-TATA element
interaction is necessary in order to sustain normal levels of
transcription in a broad set of genes when a particular function of
TAF145 is lost.
Impaired Transcription from the RPS5 Promoter Is Partly Restored by
Creating a Canonical TATA Element--
Green and co-workers (19, 47,
48) initially isolated taf145 mutants (ts1 and ts2)
in which a set of genes is affected that overlaps with, but is distinct
from, the set of genes affected in our mutants (20). For instance, the
CLN2 gene ceases its expression promptly after being shifted
to the restrictive temperature in the ts1 and ts2 mutants (48), whereas
it is continuously expressed in our mutants (20). On the other hand,
ribosomal protein genes such as RPS5 are shut off in both
groups of mutants (19, 20). Intriguingly, the creation of a consensus
TATA element was able to restore the transcription from the
TUB2 promoter in our mutants (20) but was not able to
restore transcription from the RPS5 promoter in the ts2
mutant (19), showing that the TATA requirement is different in these
two mutants. Thus, we next wished to test whether a consensus TATA
element has a stimulatory effect on transcription from the
RPS5 promoter in our mutants. First, we introduced the same
two-nucleotide substitution, i.e.
TAAAAT to TATAAA, in an
RPS5/-593 reporter plasmid (Fig. 1A), as previously tested
by Green and Shen (19). Unexpectedly, this substitution increased
RPS5 promoter activity in our mutants as well, although only
slightly (Fig. 1B, bottom section). These observations
suggest that the difference in the TATA requirement between these two mutants depends on the sort of promoter tested but not on allelic differences. Interestingly, in the ts2 mutant, the impaired
transcription of the RPS5 promoter was restored by
introducing both the TATA-surrounding and upstream sequences of the
ADH1 promoter, but not by introducing only the upstream
sequences of the ADH1 promoter (19). These observations
strongly suggest that the TATA-surrounding sequences (but not the TATA
element itself) are important in determining TAF145 dependence. We
wondered whether the RPS5 promoter might lack this unknown
DNA element(s) surrounding the TATAAA sequence that is required for
TAF145 independence and therefore would be impaired in our mutants as
well. Inspection of the TATA-surrounding sequences of the
CLN2 and CYC1 promoters showing TAF145
independence in our mutants, as described above (Fig. 1B, top
section), revealed that they were TATATAAAAA and
TATATAAAAC, respectively. We next tested five consecutive A
residues, such as CLN2 -TATA (TATAAAAA), or the reiterated
TA sequences found in both CLN2-TATA and
CYC1-TATA (TATATAAA), or the combination effect of these two
elements (TATATAAAAA), for the ability to restore transcription from
the RPS5 promoter in our mutants (Fig. 1B, bottom
section). We found that these constructs could not rescue the
transcription significantly more than when they were changed to the
simplest TATAAA element. The failure of the CLN2- or
CYC1-type consensus TATA elements to confer TAF145
independence to the RPS5 promoter suggests that an unknown determinant of TAF145 dependence surrounds the TATA element but may not
overlap the TATA element itself. More simply, another possibility is
that the consensus TATA element may function well when combined with
the TUB2, CLN2, and CYC1 initiators
but not with the RPS5 initiator, at least in our mutants.
The Initiator Region of the RPS5 Promoter Supports a Normal Level
of Transcription When Combined with the TATA Region of the CLN2
Promoter--
In the ts2 mutant, as described above, a hybrid promoter
connecting ~600 bp upstream of the ADH1 promoter,
including the TATA element, to the initiator region of the
RPS5 promoter, was shown to be expressed independently of
TAF145 function (19). This indicates that the RPS5 initiator
does not require TAF145 function, at least in the ts2 mutant, when
combined with an appropriate TATA element and UAS derived from a
heterologous promoter. Therefore, we tested analogously whether
upstream sequences including the TATA element of the CLN2
promoter could provide a similar TAF145-independent function to the
initiator region of the RPS5 promoter in our mutants. The
-332- to -68-bp region of the CLN2 promoter amplified by
PCR as a SphI-SpeI fragment was fused to the -73
bp position of the RPS5 promoter, resulting in the hybrid
reporter plasmid CLN2(-332)-RPS5 (Fig.
2A). This construct was
expressed normally in our mutants (Fig. 2B, top
panel), attesting to the fact that the RPS5 initiator is fully functional when combined with the TATA element and UAS of the
CLN2 promoter, even in our mutants.

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Fig. 2.
Deletion analysis of CLN2-RPS5
hybrid promoters to delineate the region that confers TAF145
independence on the CLN2 promoter. A,
schematic representation of the reporter plasmids used in this
experiment. The -68 to +438-bp region of the CLN2 promoter
of CLN2TATA (as shown in Fig. 1A) was
replaced with the region from -73 to +139 bp of the RPS5
promoter (shaded) to generate the CLN2(-332)-RPS5 reporter
plasmid (top row). The region containing the CLN2
promoter was deleted successively from the 5'-end as described.
SphI was originally located at the -332 bp position of the
CLN2 promoter, whereas SpeI was created by
site-specific mutagenesis to enable the fusion of the CLN2
and RPS5 promoters. Both restriction sites were utilized
here for the constructions. B, Northern blot analysis of
mRNA with a CLN2-specific probe. Wild type or mutant
strains carrying the indicated reporter plasmids were cultured at
25 °C or 37 °C. Total RNA isolated from these cultures was
analyzed as described in Fig. 1.
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To delineate the region of the CLN2 promoter that is
responsible for conferring TAF145 independence to the RPS5
initiator, we successively deleted portions of the CLN2(-332)-RPS5
hybrid promoter from the 5'-terminus (Fig. 2A).
Unexpectedly, even CLN2(-82)-RPS5, which retains only 1 bp upstream of
the TATA element, was normally expressed in our mutants (Fig.
2B). The consensus TATA element was essential for supporting
normal levels of transcription from this hybrid promoter, because
CLN2(-80)-RPS5 lacking the TATA element significantly decreased its
expression specifically at 37 °C in our mutants (Fig. 2B,
bottom panel). Consistent with this, the substitution of the TATA
element with a GAGA element in CLN2(-126)-RPS5 decreased transcription
by a similar amount (data not shown). It is also noteworthy that the
reiterated TA sequence overlapped with TATA element is not a
determinant of TAF145 independence (compare CLN2(-84)-RPS5
and CLN2(-82)-RPS5 in Fig. 2B), which is
consistent with the results described above (Fig. 1B, bottom
section). These observations are in agreement with the importance
of a consensus TATA element for transcription from the CLN2
and CYC1 promoters (Fig. 1B, top section) but
appear to contradict the slight stimulatory effect of consensus TATA sequences on transcription from the RPS5 promoter (Fig.
1B, bottom section).
The RPS5 Core Promoter Activity Is Slightly Affected in taf145
Mutants, but Can Be Restored by Creating a Consensus TATA
Element--
It is surprising that a very short sequence derived from
the CLN2 promoter, i.e.
ATATAAAAAAATAG flanked by SphI and
SpeI sites, was enough to confer TAF145 independence to the
RPS5 initiator (Fig. 2B). Given that creation of
several TATA elements could not restore the impaired transcription from
the RPS5 promoter (Fig. 1), only a few candidates can be
listed as possible determinants of TAF145 independence. First, the
CLN2(-82)-RPS5 hybrid promoter has an A residue at just 1 bp upstream
of the TATA element, but the original (i.e.
TAF145-dependent) RPS5 promoter and its variants have a C residue at the corresponding position. Note that the residue 1 bp upstream of the TATATA sequences in CLN2(-84)-RPS5S is A
(i.e. ATATATA), whereas the corresponding position of
RPS5/-593 variants is C (i.e. CTATATA). Second, the
distance between the TATA element and the transcriptional start site of
CLN2(-82)-RPS5 is different from that of TAF145-dependent
RPS5 promoters. Third, the region just downstream from the
TATA element of CLN2(-82)-RPS5 promoter is A-rich (Fig.
2A), whereas the corresponding region of RPS5 promoters is
T-rich (Fig. 3A). To determine
whether these elements are responsible for providing TAF145
independence, we first constructed a parental plasmid, RPS5/-87C, by
removing the upstream sequences of the RPS5 promoter but
retaining only one base (C) flanked between the SphI site
and the TAAAAT sequence (Fig. 3A). We then tested for TAF145
dependence in our mutants (Fig. 3B). Unexpectedly, however,
transcription from the RPS5/-87C promoter decreased only partially at
37 °C (Fig. 3B), in stark contrast to the dramatic loss
of transcription from the longer RPS5 promoter under the
same conditions (Fig. 1B, bottom section). The expression
profile of the RPS5/-87C promoter is much like that of the
CLN2(-80)-RPS5 promoter (Fig. 2B, bottom panel). More surprisingly, the conversion of the TAAAAT sequence in RPS5/-87C to a
TATAAA sequence completely restored the partial loss of transcription from the RPS5 core promoter (see
RPS5TATAAA/-87C in Fig. 3B).
This stimulatory effect on transcription by creating a consensus TATA
element parallels the case of the TUB2 core promoter (20), as well as that of the CLN2-RPS5 hybrid promoters (compare
CLN2(-82)-RPS5 and CLN2(-80)-RPS5 in Fig.
2B). It is again puzzling that the same substitution
creating a consensus TATA sequence was not as effective in the longer
RPS5 promoters (Fig. 1B, bottom section).

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Fig. 3.
TAF145 dependence of the RPS5
core promoter. A, schematic representation of the
reporter plasmids used in this experiment. The -87 to +139-bp region
of the RPS5 promoter was fused with the mini-CLN2
gene to generate an RPS5/-87C reporter plasmid (top row).
The TAAAAT sequence of RPS5/-87C was substituted with a TATAAA
sequence (RPS5TATAAA/-87C), or a
SpeI site was introduced to adjust the distance between the
TAAAAT sequence and the transcriptional initiation site
(RPS5/-87C+SpeI), or both modifications were added
simultaneously (RPS5TATAAA/-87C+SpeI).
The C residue 1 bp upstream of the TAAAAT sequence on RPS5/-87C was
substituted with an A residue (RPS5/-87A), and the same set of
modifications were added to RPS5/-87A to generate
RPS5TATAAA/-87A, RPS5/-87A+SpeI, and
RPS5TATAAA/-87A+SpeI as indicated.
B, Northern blot analysis of mRNA with a
CLN2-specific probe. Wild type or mutant strains carrying
the indicated reporter plasmids were cultured at 25 or 37 °C. Total
RNA isolated from these cultures was analyzed as described in Fig.
1.
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The slightly impaired transcription in our mutants could not be
restored by the adjustment of the distance between the TAAAAT sequence
and the transcriptional start site through the introduction of a
SpeI site (RPS5/-87C+SpeI), nor by the
conversion of a C residue to an A residue at 1 bp upstream of the
TAAAAT sequence (RPS5/-87A), nor by the combination of these two
changes (RPS5/-87A+SpeI) (Fig. 3B), indicating
that these elements are not determinants of TAF145 independence. In
contrast, the creation of a consensus TATA element
(RPS5TATAAA/-87C,
RPS5TATAAA/-87C+SpeI, RPS5TATAAA/ -87A, and
RPS5TATAAA/-87A+SpeI in Fig.
3B) always restored the transcription levels, indicating that the TATA element is a major determinant of TAF145 independence as
observed for the TUB2, CLN2, and CYC1
promoters. Such an apparent contradiction between the RPS5
core promoter (Fig. 3) and its longer version (Fig. 1) prompted us to
reinspect and interpret these results more carefully. We immediately
became aware of a difference between the two versions of the promoter,
in that the ratios of the lower band (black arrows) to the
upper band (white arrows) are much higher in Fig. 1 than in
Fig. 3. The upper band, which represents the expression of the
endogenous CLN2 gene, should be constant in any of the
strains we tested. Therefore, if the intensity of the lower bands was
normalized by that of the upper bands, it is evident that the
expression of the longer promoters is much higher than that of the core
promoters (e.g. compare RPS5/-593 in Fig.
1B with RPS5/-87C in Fig. 3B). These
observations strongly suggest that the longer promoter represents the
sum of the effects of the basal transcription supported by the core
promoter and the activated transcription mediated by UASs located
somewhere between the -593 and -87 bp positions of the
RPS5 promoter. Only the basal transcription is represented
in the core promoter RPS5/-87C. Hence, we are inclined to think that
transcriptional activation of the longer promoter is significantly
impaired in our mutants, whereas basal transcription of either the
longer or the core promoter is only partially affected, leading to much
larger transcriptional defects for the longer promoter than for the
core promoter. Importantly, only the defect in core promoter
recognition appears to be restored by creating a consensus TATA
sequence (Fig. 3B). Careful inspection revealed that the
creation of various TATA sequences in the longer promoter also
appeared to restore basal transcription as they increased the ratio of
the lower bands (reporter transcripts) to the upper bands (endogenous
CLN2 transcripts) slightly but reproducibly (Fig. 1B, bottom
section, lanes 7 and 8).
UAS Function of the RPS5 Promoter Is Significantly Impaired in
taf145 Mutants--
To verify the assumption described above, we
deleted upstream promoter sequences of RPS5/-593 successively to
generate a series of truncated promoter constructs, RPS5/-450,
RPS5/-300, and RPS5/-200 (Fig.
4A), and compared the promoter
activities of these constructs in wild type and taf145
mutants (Fig. 4B). In our mutants, a significant decrease in
transcription was observed at 37 °C for RPS5/-593 and RPS5/-450,
whereas only a slight decrease was observed for RPS5/-300 and
RPS5/-200 (Fig. 4B). These observations indicate that
transcriptional activation by the UAS located between -450 and -300
bp is significantly impaired in our mutants. Previous computational
analysis predicted that the region from -415 to -403 bp might be the
binding site for RAP1 (44), a transcription factor that appears to
regulate the expression of many ribosomal protein genes (49). To
examine whether RAP1 is responsible for such UAS activity, we
constructed RPS5/-593/
UASRAP1 by removing the region
from -415 to -403 bp from RPS5/-593. The expression profile of
RPS5/-593/
UASRAP1 was found to be quite similar to that
of RPS5/-593 (Fig. 4). Taken together, these observations suggest that
the RPS5 promoter may be activated by an unknown transcription factor(s) other than RAP1, which binds to the region between -450 and -300 bp and requires TAF145 function to activate the
transcription.

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Fig. 4.
Deletion analysis of the RPS5
promoter to delineate the region that confers TAF145
dependence. A, schematic representation of the reporter
plasmids used in this experiment. The region from -593 to +139 bp of
the RPS5 promoter was fused with the mini-CLN2
gene to generate the RPS5/-593 reporter plasmid as shown in Fig. 1
(top row). The region containing the RPS5
promoter was deleted successively from the 5'-end to generate
RPS5/-450, RPS5/-300, and RPS5/-200 as described. The potential ABF1
and RAP1 binding sites postulated in silico (44) are
indicated by vertical bars. The RAP1 binding site of
RPS5/-593 was deleted by site-specific mutagenesis to generate
RPS5/-593/ UASRAP1. B, Northern blot analysis of mRNA
with a CLN2-specific probe. Wild type or mutant strains
carrying the indicated reporter plasmids were cultured at 25 or
37 °C. Total RNA isolated from these cultures was analyzed as
described in Fig. 1.
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Functional Interactions between UAS and Core Promoters Are
Selectively Impaired in taf145 Mutants--
Our observations described
above indicate that TAF145 function is required not only for core
promoter recognition but also for transcriptional activation. However,
our previous study concluded that the CYC1 core promoter
could mediate the normal level of activated transcription by various
activators, such as GAL4, GCN4, EBNA2, and VP16, even in the same
taf145 mutants (20). Therefore, we next wished to ask
whether the defect in transcriptional activation observed for the
RPS5 promoter is confined to a specific UAS or core promoter
structure or both. The fragments containing the RPS5-UAS,
i.e. -450~-361 bp (90 bp) and -450~-300 bp (150 bp), were fused with either the RPS5/-87 or the RPS5/-200 core promoter to
generate three reporter plasmids, UAS90bp+RPS5/-87,
UAS150bp+RPS5/-200, and UAS150bp+RPS5/-87
(Fig. 5A). They showed quite
similar expression profiles to that of RPS5/-593 (Fig. 5B),
indicating that the sequences intervening between the UAS and the core
promoter are not required to reconstitute TAF145-dependent
transcriptional activation and that the UAS and the core promoter of
the RPS5 promoter can be manipulated independently. We therefore fused
UAS150bp to a heterologous CYC1 core promoter to
generate the hybrid reporter plasmid UAS150bp+CYC1/-174 (Fig. 5A) and tested for TAF145 dependence (Fig.
5B). Interestingly, UAS150bp can activate the
CYC1 core promoter at almost the same levels as the original
RPS5 core promoter (Fig. 5B, bottom panel). More
importantly, this activation is not affected in our mutants (Fig.
5B), suggesting that the RPS5 core promoter
requires TAF145 function for activated transcription by
UAS150bp, whereas any activator bound to
UAS150bp does not require the same TAF145 function, at
least when it stimulates the CYC1 core promoter. Next, we
tested reciprocally whether the RPS5 core promoter could be
activated by other activators in our mutants. To compare the effects of various activation domains in the same system, we fused GAL4 binding sites to the RPS5 core promoter to generate a reporter
plasmid, UASGAL+RPS5/-87C (Fig. 5A). The
effector plasmids, expressing various activation domains connected to
the GAL4 DNA binding domain (i.e. GAL4-RAP1, -VP16, -EBNA2,
and -GCN4), were introduced into yeast cells together with
UASGAL+RPS5/-87C to test for TAF145 dependence in
transcription (Fig. 5C, bottom section). Surprisingly, all
of these activation domains were able to stimulate transcription, even
for the RPS5 core promoter. Although
RPS5-UAS150bp (Fig. 5B, bottom section) and
GAL4-RAP1 (Fig. 5C, top section) could activate the
CYC1 core promoter at comparable levels, only the latter
could activate the RPS5 core promoter in our mutants at the
nonpermissive temperature (Fig. 5, B and C).
These observations indicate that certain combinations of UAS and core
promoters are selectively impaired in their functional interactions in
our mutants, although each component appears to be normal if combined
with a different partner. Thus, we conclude that there are several activation pathways, even for a single activator or a single core promoter, which vary in their requirement for TAF145 function.

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Fig. 5.
TAF145 dependence was observed only when the
RPS5 UAS was combined with the RPS5
core promoter in taf145 mutants.
A, schematic representation of the reporter plasmids used in
this experiment. The regions derived from the CLN2,
RPS5, and CYC1 genes are shown by open,
shaded, and striped boxes, respectively.
RPS5-UAS was fused with the RPS5 and
CYC1 core promoters in its two forms, i.e.
UAS90bp (-450~-361 bp) or UAS150bp
(-450~-300 bp), to generate UAS90bp+RPS5/-87,
UAS150bp+RPS5/-200, UAS150bp+RPS5/-87, and
UAS150bp+CYC1/-174. Synthetic binding sites for GAL4
fusion activators were linked upstream of the CYC1 and
RPS5 core promoters to generate
UASGAL+CYC1/-174 and
UASGAL+RPS5/ -87C, respectively. B and
C, Northern blot analysis of mRNA with a
CLN2-specific probe. Wild type or mutant strains carrying
the indicated reporter plasmids were cultured at 25 or 37 °C. Total
RNA isolated from these cultures was analyzed as described in Fig. 1.
In C, activator expression plasmids (GAL4DBD-RAP1, -VP16C,
-EBNA2, and -GCN4, or GAL4DBD alone as a negative control) were
introduced into yeast cells with the reporter plasmid as indicated to
measure the activation efficiencies.
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DISCUSSION |
Here, we report that TAF145 function is critically important for
activated transcription from the RPS5 promoter. However, earlier studies done by Shen and Green (19) concluded differently, that
TAF145 function was required only for the core promoter recognition of
the RPS5 gene. They examined in the ts2 mutant whether
ADH1-UAS or (dG:dC)42 could activate the
RPS5 core promoter (-135~+39), which lacks binding sites
for any known yeast activators and therefore cannot mediate efficient
transcription by itself. It was suggested that a homopolymeric sequence
such as (dG:dC)42 disrupts nucleosome structures so that it
can activate a core promoter even in the absence of an activator. Both
chimeric promoters, ADH1UAS-RPS5core and
(dG:dC)42-RPS5core, produced much lower amounts of mRNA
in the ts2 mutant when the temperature was raised to 37 °C. In
addition, reciprocal experiments demonstrated that RPS5-UAS
could activate the ADH1 core promoter. These observations
allowed them to conclude that TAF145 function is not involved in
activating the RPS5 gene. However, we think that the same
observations could be interpreted differently. ADH1-UAS and
(dG:dC)42 may have failed to activate the RPS5
core promoter even though the basal activity of the core promoter was
only slightly impaired. If this is the case, then the impaired
transcription from the RPS5 promoter in the ts2 mutant might
be due to the core promoter-specific activation defect, i.e.
RPS5-UAS could activate the ADH1 core promoter
but could not activate its own core promoter, as we found in our
experiments using different taf145 mutants. To distinguish
these two possibilities, it will be necessary to examine the
transcriptional activity at 37 °C of an RPS5 core
promoter in the ts2 mutant that is not connected to any UAS. If the
original interpretation of Shen and Green (19) is correct, it
would be intriguing to know the reason why these two classes of
taf145 mutants (ts2 and ours) show distinct phenotypes in
transcription from the RPS5 promoter and whether it might be related to the differential effect of these taf145 alleles
on CLN2 transcription as described previously (20, 48).
Our results indicate that transcription factors bound to the
RPS5-UAS require a particular TAF145 function to activate
the RPS5 core promoter but not to activate the
CYC1 core promoter. Furthermore, other activators that we
tested here do not require the same TAF145 function to activate the
RPS5 core promoter. Thus, the functional interaction between
the RPS5-UAS and the RPS5 core promoter is
impaired selectively in our mutants, implying that a single activator
may exploit several alternative activation pathways in response to core
promoter structures. Consistent with this supposition, there have been
several observations regarding core promoter-specific activation
domains. For instance, GAL4-VP16 activates the TATA and
initiator-containing core promoter more strongly than the TATA-less but
initiator-containing core promoter, whereas Sp1 activates these two
promoters almost comparably (50). USF-specific region, another
activation domain that is found in mouse USF2, activates the adenovirus
major late minimal promoter, but not the E1b minimal promoter (51).
Insertion of initiator rescues USF-specific region function on the E1b
minimal promoter, suggesting that the USF-specific region is an
initiator-specific activation domain (51). Transcription factors
derived from mammalian viruses (e.g. ICP4 and Zta) are also
reported to carry core promoter-specific activation domains (52, 53).
Although it is still unclear how such specificity between the activator
and the core promoter is established, the difference in the requirement
of activators for general transcription factors, coactivators, and/or
cofactors might be related to this phenomenon. In this respect, it is
notable that different classes of mammalian transcription factors, such as retinoid X receptor-retinoic acid receptor, cAMP response
element-binding protein, and signal transducer and activator of
transcription 1, require different configurations and different
enzymatic components of the large histone acetyltransferase coactivator
complex, which includes CBP/p300, NCoAs, and PCAF, in order to activate
their target promoters properly (54). In yeast, similar activation domain-specific cofactor requirements have been observed. For instance,
the VP16 and GAL4 activation domains require Srb4 function for
activation, but the Ace1 and Hsf1 activation domains do not (55).
Similarly, the GCN4 activation domain requires TAF17 function for
activation, but the Ace1 and Hsf1 activation domains do not (55).
Interestingly, the Ace1 and Hsf1 activation domains do show an
intricate requirement for TFIIE in CUP1 gene activation (56). Activation of the CUP1 promoter by either Ace1 or Hsf1 requires TFIIE function, but a CUP1-UAS-containing binding
site for both activators circumvents the requirement for TFIIE (56). Thus, there might be a certain combinatory effect of different activators for general transcriptional factor and/or cofactor requirements. Given that the core promoter structures are intimately linked to the activator function in prokaryotes (reviewed in Refs. 57-59), it should be more commonly observed in eukaryotes as well that
a single activator takes multiple activation pathways depending on the
structure of the core promoter. We believe, as a next step, it will be
important to identify an activator or activators that show a core
promoter-specific TAF145 requirement in our mutants and to characterize
the impaired functional interactions between such an activator and
TFIID at the molecular level.
Our taf145 mutants showed defects in both core promoter
recognition and transcriptional activation. Similarly, in the ts2 mutant, not only core promoter recognition (19) but also activation (21) and derepression (22) were affected on apparently different target
genes. It is not easy to determine whether such multiple defects occur
on all of the target genes, or whether each target gene has a specific
defect. For instance, given that the integrity of core promoter
recognition is a prerequisite for efficient activation, it is
impossible to examine the effect of taf145 mutation on these two molecular events separately. Our observation that the creation of a
TATA element restored the basal transcription from the RPS5 core promoter but not the activated transcription from the
RPS5-UAS implies that activated transcription is not simply
an enhanced form of basal transcription. In this respect, we propose
that these two molecular events, i.e. core promoter
recognition and activation, are functionally separate and independent
processes, at least in terms of their TAF145 requirements. Consistent
with this, the impaired transcription from the core promoter of an major histocompatibility complex class I gene was shown to be partially rescued by the presence of its own UAS or a heterologous SV40
enhancer in mammalian taf250/ccg1 mutant cells (60).
This suggests that the same taf250/ccg1 mutation
affects these two molecular events differently, in that the core
promoter recognition is more severely damaged than the activation step
in this particular case. In addition, it should be noted that the
activator and core promoter specificities were also observed for
mammalian cells; the cyclin D1 core promoter cannot be activated by its
own UAS, although it can be activated well by GAL-p65, -p53, and -VP16, as shown by an in vitro transcription assay using the
nuclear extract prepared from a taf250/ccg1 mutant
(61). Therefore, the function of this particular TAF (TAF145/TAF250),
which is involved in both core promoter recognition and activation of
at least a subset of genes, must be conserved from yeast to human. It
would be of special interest to know the molecular mechanisms of how a
specific combination of core promoter and activation domains determines
the degree of TAF145/TAF250 requirement in these two organisms.