From the Division of Gene Function in Animals, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0101, Japan
Received for publication, September 7, 2000
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ABSTRACT |
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The general transcription factor TFIID, which is
composed of the TATA box-binding protein (TBP) and a set of
TBP-associated factors (TAFs), is crucial for both basal and regulated
transcription by RNA polymerase II. The N-terminal small segment of
yeast TAF145 (yTAF145) binds to TBP and thereby inhibits TBP function.
To understand the physiological role of this inhibitory domain, which
is designated as TAND (TAF N-terminal
domain), we screened mutations, synthetically lethal with
the TAF145 gene lacking TAND
(taf145 In eukaryotes, transcriptional initiation of protein-coding genes
is precisely regulated by the concerted action of a large number of
proteins, e.g. general transcription factors (TFIIA, TFIIB,
TFIID, TFIIE, TFIIF, and TFIIH), negative and positive cofactors,
coactivators, and chromosome-modifying factors, in addition to RNA
polymerase II (reviewed in Refs. 1-5). The general transcription
factor TFIID, a multiprotein complex composed of the TATA-binding
protein (TBP)1 and more than
10 TBP-associated factors (TAFs), can recognize specifically a number
of core promoter elements such as the TATA box, initiator element, and
downstream promoter element (reviewed in Refs. 6-9). It nucleates the
assembly of the preinitiation complex around the transcriptional
initiation site by recruiting several other general transcription
factors and RNA polymerase II, either in a stepwise manner or as a
preassembled unit (i.e. RNA polymerase II holoenzyme)
(reviewed in Refs. 10-12). The importance of the binding of TFIID (or
TBP) to the core promoter in transcriptional regulation has been
extensively studied by various approaches (13-19). Biochemical studies
have demonstrated that suboptimal core promoter recognition by TFIID
might generate a substantial energetic barrier for initiating
transcription, however, gene-specific activators should overcome this
rate-limiting step by inducing conformational changes of TFIID
(16-19). Activator-bypass experiments, mostly performed in yeast, in
which TBP or TAFs were physically connected to the heterologous DNA
binding domain of gene-specific activators, showed that binding of
TFIID (or TBP) to the core promoter was indeed a rate-limiting step
in vivo that could be alleviated by artificial recruitment
of TFIID (reviewed in Refs. 12 and 20). The recently developed
DNA-cross-linking chromatin immunoprecipitation assay has made it
possible to test directly whether binding of TFIID (or TBP) to the core
promoter could be a bona fide regulatory step for activators
in living cells (14, 15, 21, 22). Results of assays for transcriptional
activity using more than 30 promoters correlates well with the degree
of TBP occupancy on the core promoter (14), arguing that TBP-TATA interactions should be the most critical step for gene regulation. These observations imply the presence of cofactors that modulate TBP-TATA interactions negatively and/or positively.
Several proteins have been reported to date that inhibit TBP-TATA
interactions (reviewed in Refs. 23-25). TAND (TAF
N-terminal domain), originally isolated from
the N terminus of Drosophila TAF230 (dTAF230), interacts
with TBP directly so that TAND prevents TBP from binding to the TATA
element (26-29). Functionally equivalent domains are conserved at the
N terminus of orthologous TAFs among various
species,2 (30, 31) suggesting
that TAND should be involved in certain principal functions of TFIID
(24, 32, 33). TAND consists of two subdomains, TAND1 and TAND2, each of
which binds to the concave and convex surface of TBP in a competitive
fashion with acidic activation domains and TFIIA, respectively (28, 30, 34). This structural configuration of the complex formed between TAND
and TBP was recently confirmed by NMR spectroscopic studies (29, 35).
Together with previous observations that activators and TFIIA are
essential factors for inducing conformational change of TFIID during
the course of activation (16, 17, 19), we assume that TAND-TBP
interactions should be one of the most important regulatory targets for
activators (32).
SAGA, a Gcn5-containing histone acetyltransferase coactivator complex,
plays a key role in the regulation of transcription of several genes,
e.g. HIS3 and TRP3 in Saccharomyces
cerevisiae (reviewed in Refs. 36 and 37). Whereas several yeast
TAFs (e.g. TAF90, TAF68, TAF60, TAF25, and TAF17) as well as
TBP are shared by TFIID and SAGA, histone acetyltransferase activities involved in coactivator function are encoded by distinct subunits, TAF145 and Gcn5, that are specific to TFIID and SAGA, respectively (36,
37). Interestingly, Spt3 and Spt8, which are SAGA-specific components,
bind to TBP and inhibit TBP-TATA interactions as observed for TAND in
TFIID (38). Although it remains to be determined whether Spt3- or
Spt8-TBP interactions are regulated by activators or other factors,
Spt8 appears to be dissociated from the SAGA complex under conditions
of activation so that such inhibitory interactions can be alleviated
(38).
Mot1, a member of the SWI2/SNF2 helicase family, is the third
intriguing factor that inhibits TBP-TATA interactions (39). It was
originally identified as the mot1-1 allele that leads to increased basal expression of many genes in S. cerevisiae
(40). Several lines of investigation uncovered it to be a 170-kDa
component of the TBP-TAF complex distinct from TFIID (41, 42) or as an
ADI (ATP-dependent inhibitor of TBP
binding to DNA) factor that dissociates TBP from the TATA element in an
ATP-dependent manner (39, 43). Recent studies provide
evidence that Mot1 can not only repress but also stimulate
transcription of certain genes presumably by regulating the
distribution of a limiting pool of TBP between promoter and nonpromoter
sites (44, 45). Interestingly, it was demonstrated that
MOT1, SPT3, and TOA1, which encodes
the larger subunit of TFIIA, interacted genetically with each other,
i.e. toa1 and spt3 are synthetically
lethal with mot1 and the Here, to identify factors that are functionally related to the TAF
N-terminal domain (TAND) of TAF145, we have screened for nsl
( Yeast Strains, Media, and Genetic Analyses--
Standard
techniques were used for yeast growth and transformation (46-48).
Yeast extract/peptone/dextrose (YEPD) and selective media have been
described (46). Transformation was done using the lithium acetate
procedure (49). Yeast strains used in this study are listed in Table
I.
The host strain, TMY4-2, used for the synthetic lethal screen was
constructed by targeted integration of the
taf145
TMY17-2c and TMY16-2c were made by mating-type interconversion from
TMY4-2 and CH1305, respectively. The YEp13-HO plasmid, expressing the
homothallic switching endonuclease endonuclease (53), was
transformed into TMY4-2 and CH1305, both of which are MATa,
to generate MATa/MAT
The YTK271 strain was generated from H2440 (kindly provided by Dr.
A. G. Hinnebusch) (30) by targeted disruption of the SPT15 gene using a marker cassette that has a
URA3 gene between duplicated copies of a Salmonella
hisG gene segment (54). The cassette plasmid has the 5'-flanking
sequence (~500 bp upstream of the initiation codon) and 3'-flanking
sequence (~500 bp downstream of the termination codon) of the
SPT15 gene on each side of URA3 marker. These
flanking sequences were amplified by PCR with primers to create
EcoRI-BglII and SalI-BamHI
sites, respectively. The linear fragment digested with EcoRI
and SalI was used to transform H2440. The structure of the
disrupted gene was confirmed by Southern blotting. Since the
SPT15 gene is essential for viability, we dissected
heterozygously disrupted strains that had been transformed with the
TRP1-marked plasmid carrying the SPT15 gene.
Ura+ Trp+ haploid strains obtained from tetrad
analysis were grown on 5-FOA plates to excise the URA3
marker from the chromosome. The resulting Ura
YTK271 ( Synthetic Lethal Screen--
To perform an ade2 ade3
colony-sectoring assay for synthetic lethality (50), the TMY4-2 haploid
strain was transformed with pTM17. The pTM17 plasmid was constructed by
subcloning a 5.2-kb SalI-BamHI (partial) fragment
of pYN2 (30) carrying the TAF145 gene and a 3.7-kb
NheI-BamHI fragment of pDK255 (55) carrying the
ADE3 gene into the restriction enzyme sites between
SalI and XbaI of pRS316 (56). TMY4-2, bearing
pTM17, was grown in liquid SD medium lacking uracil (SD-Ura) to an
absorbance at 600 nm (A600) of around 0.7 and plated on YPD plates at a density of ~4000 cells/plate. The
plates were then UV-irradiated, resulting in ~5-7.5% survival (200-300 cells/plate), and incubated for 5 days at 25 °C. Red colonies were restreaked three times on YPD plates. Colonies that remained red during this cultivation process were subsequently counter-selected on 5-FOA-containing plates. To exclude the possibility that nonsectoring and 5-FOA-sensitive phenotypes might be due to
genomic integration of the pTM17 plasmid, mutant candidates were
transformed with the plasmids bearing the TAF145 or
taf145 Cloning of a Gene That Complements C40 and D7 nsl
Mutants--
C40 and D7 mutants were crossed to TMY17-2c bearing the
taf145
TMY4-2 is unable to grow at 35 °C due to the presence of the
taf145 Identification of Amino Acid Substitutions in SPT15 Gene of C40
and D7--
The mutations in the SPT15 gene of C40 and D7
strains responsible for synthetic lethality were identified by
sequencing. The 1.2-kb DNA fragment, including the entire
SPT15 gene, was amplified by PCR using the primer pairs
TK127 and TK128 from the isolated genomic DNA of these mutants. Direct
sequencing of amplified DNA fragments using TK222, TK223, TK224, and
TK225 as sequence primers revealed single C Construction of Plasmids Encoding Activation-defective TBP
Mutants--
pTM8 was constructed by ligating the 2.4-kb
EcoRI-BamHI fragment including the entire
SPT15 gene from the genomic inserts, obtained as described
above, into the EcoRI/BamHI sites of pRS314 (56).
pTM8 was subjected to site-specific mutagenesis (57) to create
spt15 alleles that were defective for activation by RNA
polymerase II or transcription by RNA polymerase III. Oligonucleotides TK688, TK16, TK215, TK1247, TK1248, TK1270, TK1269, TK1268, TK1267, TK1416, TK1417, and TK1524 were used to generate the plasmids pM1228
(P65S), pM1581 (K138T/Y139A), pM496 (N159D), pM1861 (R220H), pM1862(Y231A), pM1863 (F148H), pM1864 (T153I), pM1865 (E236P), pM1866
(F237D), pM1867 (N159L), pM1868 (V161A), and pM2008 (S118L), respectively.
To prepare histidine-tagged TBP mutant proteins, pM1578 was constructed
by ligating the 0.7-kb PCR-amplified NdeI-BamHI
fragment encoding TBP into the expression vector, pET28a (Novagen).
Site-specific mutagenesis was performed on pM1578 using the same sets
of oligonucleotides as described above to generate pM1229 (P65S), pM16
(K138T/Y139A), pM494 (N159D), pM1871 (R220H), pM1872 (Y231A), pM2004
(F148H), pM1873 (T153I), pM1874 (E236P), pM1875 (F237D), pM1876
(N159L), pM1877 (V161A), and pM2005 (S118L).
Phenotypic Analyses--
To confirm the presence of the
synthetic lethal phenotype in different general genetic backgrounds,
YAK303 and YAK307 were transformed with pRS314-based plasmids encoding
TBP derivatives as described above and then incubated on 5-FOA plates
at 30 °C for 5 days. To test the complementing activities of various
plasmids by a red/white sectoring assay, colonies transformed with
these plasmids were streaked onto YPD plates and then incubated at
25 °C for 8-10 days. Recovery of the TS phenotype was assayed by comparing the growth rates at 25 and at 35 °C of yeast transformants incubated on YPD plates for 3-4 days.
Plasmids Encoding Activation Domains or TBP Mutants Fused with
the GAL4 DNA Binding Domain--
pM471 was constructed by replacing
the 1240-bp SphI fragment of pGAD424
(CLONTECH) that contains the GAL4 activation
domain, whose expression is regulated by an ADH1 promoter and
terminator, with the corresponding 1094-bp SphI fragment
from pGBT9 (CLONTECH) that contains the GAL4 DNA
binding domain under the control of the same regulatory sequences. For
expression of various activators in yeast cells, pM1569, pM967, pM1440,
pM1570, pM524, and pM468 were constructed by ligating DNA fragments
encoding GAL4 (aa 842-874), GCN4 (aa 107-144), ADR1 TADIV (aa
642-704), EBNA2 (aa 426-462), VP16 (aa 457-490), and yTANDI (aa
10-42) activation domains, respectively, into pM471 as described
previously (58). To express GAL4-TBP derivatives in yeast cells, pM1572
(wild type), pM1573 (P65S), pM1878 (K138T/Y139A), pM1879 (N159D),
pM1884 (R220H), pM1885 (Y231A), pM2006 (F148H), pM1881 (T153I), pM1882
(E236P), pM1883 (F237D), pM1886 (N159L), pM1887 (V161A), and pM2007
(S118L) were constructed similarly by ligating
EcoRI-BamHI, PCR-amplified fragments into pM471.
Primer pairs used were TK21/TK22 (wild type, P65S, K138T/Y139A, N159D,
R220H, Y231A, F148H, T153I, N159L, V161A, and S118L), TK21/TK1326 (F237D), and TK21/TK1327 (E236P).
In Vivo Activation Measured by Preparation of Recombinant Proteins and GST Pulldown
Assay--
Hexahistidine-tagged TBP derivatives were expressed in
E. coli BL21(DE3) (Novagen) by induction with 0.4 mM isopropyl-1-thio-
Yeast TAND (6-96 aa) and VP16 activation domain (457-490 aa) were
expressed in E. coli (DH5
To study interactions between GST-TAND and TBP derivatives, E. coli extracts containing GST-TAND (30 pmol) and TBP derivatives (30 pmol) were mixed in 100 µl of 0.2 M KCl/buffer D at
4 °C for 30 min, incubated with 10 µl of glutathione-Sepharose 4B
(Amersham Pharmacia Biotech) for another 30 min, and washed three times with 500 µl of 0.2 M KCl/buffer D. The complexes on the
beads were eluted by boiling in SDS sample buffer and analyzed by
Western blotting with polyclonal antibodies to TBP. Interactions
between GST-VP16 and TBP derivatives were examined similarly except
that two different concentrations of KCl (0.1 M and 0.2 M) were used in the binding and washing buffers.
Coimmunoprecipitation Analysis--
Immunoblot and
coimmunoprecipitation analyses were performed as described previously
(28, 58).
Gel Retardation Assays--
The gel retardation assay was
performed as described previously (26, 59) except that 2 µg/µl BSA
was added to the reaction mixture. For TBP-TATA element interactions,
20 ng of TBP was used, and the complex was analyzed in a 4%
polyacrylamide gel (59:1) containing TGMg buffer (25 mM
Tris, 192 mM glycine, 2 mM MgCl2) and 5% (v/v) glycerol using TGMg as a running buffer. When TFIIA binding to the TBP-TATA complex was tested, 10 ng of TBP and12 ng of
TFIIA were added to the reaction mixture, and the complex was analyzed
in a 4% polyacrylamide gel (59:1) containing 0.5× TBE buffer (89 mM Tris, 89 mM boric acid, 2 mM
EDTA, pH 8.0) and 5% (v/v) glycerol using 0.5× TBE as a running buffer.
Screen for nsl Mutants That Are Synthetically Lethal with the Loss
of TAND Function--
Recently we proposed that TAND, located at the N
terminus of TAF145, might be involved in transcriptional regulation by
acidic activators (32). However, as target gene(s), whose expression depends on the TAND-TBP interactions, have not been identified despite
our extensive efforts, it seems likely that some other regulatory
systems may exist in yeast cells that compensate for the loss of
TAND-TBP interactions. To identify genes involved in such a parallel
pathway, we screened nsl genes displaying synthetic lethal
interactions with the taf145 gene that lacks TAND
(taf145
The host strain, TMY4-2, used for our screen was constructed to harbor
a taf145 Two nsl1 Mutants Carry Different Amino Acid Substitutions in the
SPT15 Gene Encoding TBP--
C40 and D7 mutants segregated into the
plasmid-dependent red/white sectoring phenotype 2:2 when
backcrossed to an isogenic parent strain harboring the chromosomal
taf145
To identify possible mutation(s) in the SPT15 gene of both
mutants, we sequenced PCR-amplified genomic fragments encompassing the
entire open reading frame plus 5'- and 3'-adjacent DNA regions (~500
bp each) of the SPT15 gene. We found the single amino acid substitutions, S118L and P65S, in the coding region of the
SPT15 gene of C40 and D7 mutants, respectively. Thus, we
next asked whether these TBP mutations were sufficient to reproduce the
nsl phenotype and whether such a phenotype depended on a
particular genetic background. To address these questions, we
constructed yeast strains containing either the wild type
TAF145 gene (YAK303) or taf145 Transcriptional Activation Is Impaired in spt15/nsl1
Mutants--
The spt15-S118L allele was previously isolated
as one of the TBP mutants that was specifically impaired in its
response to acidic activators (63). Yeast strains containing the S118L
mutant as the sole source of TBP are deficient for activation by Gcn4, Gal4, and Ace1, whereas transcription from pol I (rDNA), pol III (tRNA-I), TATA-less pol II (TRP3), and constitutive pol II (DED1 and
RPS4) promoters is not impaired (63). On the other hand, the P65S
mutant was reported to be defective for transcription in
vitro from pol II (CYC1) as well as pol III (5 S rDNA and tRNA-L) promoters (64). These TBP mutants, S118L and P65S, showed poor growth
at higher temperatures (63, 64), and the TS phenotype of the latter was
exploited to isolate the BRF1 gene (65) (also called as
TDS4 (66) and PCF4 (67)) encoding a component of the pol III-specific general transcription factor, TFIIIB. As the
overexpression of BRF1 suppressed the TS phenotype of the P65S mutant,
transcription by pol III should be more severely affected than that by
pol II in this mutant (65).
We proposed that TAND may play an important role in transcriptional
activation by acidic activators (32). In this regard, it is intriguing
that the activation-defective TBP mutant, S118L, was isolated in our
screen as one of the nsl alleles. We reasoned that the P65S
mutant also might be deficient in the response to acidic activators.
Thus, we examined activation efficiencies in the P65S mutant that had
been backcrossed to an isogenic wild type strain more than three times
so as to avoid the effect of other unrelated mutations. The
Weakened interactions of TBP, either with the TATA element, TFIIA, or
activation domains, were reported to lower the activation efficiencies
by acidic activators (63, 69-72). Thus, to identify which defects were
most relevant to the nsl phenotype, we examined the
abilities of these TBP mutants to bind to the TATA element and
interactions with TFIIA, TAND, and VP16. TBP-P65S bound to the TATA
element at normal levels, whereas binding of TBP-S118L was reduced to
50% or less of the wild type (Fig. 2B, upper panel). Although previous studies demonstrated that these TBP mutants lacked
TATA binding activity (63, 64), we found that they could bind to the
TATA element when they were produced in bacterial cells incubated at
16 °C. In contrast to TATA binding, TBP-S118L interacted with TFIIA
much less weakly than the wild type form (a very faint signal was
detected in Fig. 2B, lower panel, lane 4), whereas TBP-P65S
did not form any detectable amount of the TFIIA-TBP-TATA complex under
the same conditions (Fig. 2B, lower panel, lane 6). Such
affected interactions with TFIIA were previously observed for the other
TBP mutant, N2-1 (K138T/Y139A), that also is defective in response to
acidic activators (70). The interaction of TBP-S118L with TAND was
weaker than that of TBP-P65S or the wild type form (Fig. 2C,
upper panel). Thus, serine 118 is important for the interaction
with TFIIA and TAND (Fig. 2, B and C), whereas proline 65 appears to be specifically required for the interaction with
TFIIA (Fig. 2, B and C). Consistently,
biochemical and structural studies argue that TFIIA and TAND share, at
least in part, the interaction surface of TBP (30, 35). Interestingly,
both TBP mutants bound to the acidic activation domain of VP16 more
avidly than the wild type form (Fig. 2C, lower panel).
Strong interactions became more evident when the complex was washed
with higher concentrations of salt in the buffer. Contrary to this, the
other type of activation-defective TBP mutant, L114K, was reported to
be impaired in the interaction with the activation domain of VP16 (72).
These two opposite traits of TBP mutants could lead to the same outcome
(i.e. activation deficiency) according to our "two-step
hand off model" where interactions, either too weak or too strong,
between the activation domain and the concave surface of TBP may
prevent the activation process (32).
Although the molecular defect found in TBP-S118L and TBP-P65S that
contributes most significantly to the nsl phenotype has remained elusive, the results described above prompted us to test whether other activation-deficient TBP mutants also show synthetic lethal interactions with the taf145 Activation-defective TBP Mutants Show Synthetic Lethality with the
taf145 Gene Lacking TAND--
A large number of TBP mutants have been
isolated so far, and most have been characterized at the molecular
level (reviewed in Refs. 1, 73, and 74). Special attention has been
paid to a class of TBP mutants displaying activation-specific defects. Earlier genetic screens seeking such yeast TBP mutants identified several amino acid substitutions such as V71A, P109A, F116Y, S118L, F148L, N159D, N159L, and V161A (63, 69). Most of these residues are
located on the DNA-binding surface of TBP. These mutants are defective
in TATA binding, suggesting that activators may facilitate the TBP-TATA
complex formation or stabilize it. Other studies report the isolation
of K138T/Y139A, F148H, T153I, E236P, and F237D substitutions as other
types of activation-defective TBP mutants (70, 71). As described above,
the K138T/Y139A mutant associated with the TATA element normally, but
it had specifically lost the ability to bind TFIIA (70). The F237D
mutant associated with the TATA element with a similar affinity as the
wild type form but with an altered conformation so that it interacted
quite poorly with TFIIA and TFIIB (71). On the other hand, the F148H, T153I, and E236P mutants interacted normally with the TATA element, TFIIA, TFIIB as well as the acidic activation domains, implying that it
should be impaired in binding to unknown factors that are important for
activation (71).
To see the correlation between the activation defects and the
nsl phenotype, we selected different types of TBP mutants
like N159D, N159L, and V161A (defective for TATA binding) (63, 69), K138/139A and F237D (defective for TFIIA binding) (70, 71), F148H,
T153I, and E236P (impaired interaction with unknown factors) (71), and
R220H and Y231A (specifically impaired in pol III-driven transcription)
(75), and we determined their nsl phenotypes (Fig.
3A). As described for S118L
and P65S mutants, we transformed the yeast strains, YAK303 and YAK307,
with a centromeric TRP1 plasmid expressing each TBP mutant,
and we examined their growth on 5-FOA plates at 30 °C. Like the
S118L and P65S mutants, other activation-defective TBP mutants also
exhibited synthetic growth defects with the
taf145
We next investigated how these TBP mutants interacted with the TATA
element, TFIIA, TAND, and VP16 (Fig. 3, B and C),
although similar analyses had been conducted for some of these mutants previously. Consistent with these studies (63, 69), N159D, N159L, and
V161A mutants were affected in their binding to the TATA element (Fig.
3B, upper panel; lanes 7, 14 and 15), and such defects were not rescued by the addition of TFIIA (Fig. 3B, lower panel, lanes 3, 10, and 11). K138/139A and F237D
mutants were impaired in TFIIA binding (lower panel, lanes 2 and 8), but they could bind to the TATA element (upper
panel, lanes 9 and 12). In this regard, they resemble
S118L and P65S mutants. Note that the TATA binding activity of the
K138T/Y139A mutant was sensitive to the presence of BSA in the reaction
buffer. Much stronger TATA binding could be seen in the absence of BSA
(upper panel, compare lane 6 and 9).
F148H, T153I, E236P, R220H, and Y231A mutants interacted with both the
TATA element and TFIIA almost normally. On the other hand, as shown in
Fig. 3C, decreased interactions with TAND were observed for
F237D, F148H, R220H, and Y231A mutants. Considering that the latter two
mutants, i.e. R220H and Y231A, were viable when combined
with the taf145 TBP Mutants Defective for the Post-recruitment Step Exhibit a
Stronger nsl Phenotype--
To see the correlation between the degree
of the nsl phenotype and that of activation defects, we
compared activation efficiencies of these TBP mutants, described above,
under the same conditions where VP16, GAL4, and GCN4 activation domains
fused with GAL4DNA binding domain were used as activators (Fig.
4A). Consistent with previous
studies (see also Fig. 2), all TBP mutants, except R220H and Y231A that
are supposed to be specifically impaired in pol III transcription, were
found to be more or less affected in activation by these activators
(Fig. 4A). Nonetheless, we did not see any distinct
correlations among the degree of the nsl phenotypes and the
activation defects or the sort of activation domains present and the
molecular defects observed. It was noteworthy that F148H and T153I
mutants were affected in activation to a similar level as other TBP
mutants (Fig. 4A) although they showed a weaker
nsl phenotype than the others (Fig. 3A).
Previous studies demonstrated that transcription could be activated in
the absence of activators by artificial recruitment of TBP that is
physically connected to a heterologous DNA binding domain (76-78).
This simple in vivo recruitment assay could predict roughly
which step(s) is impaired in each activation-defective TBP mutant (71).
The rationale of how to interpret the result was originally provided by
Stargell and Struhl (71), namely if an activation-defective TBP can
activate transcription when it is recruited to the promoter, its defect
must be involved in the step(s) before recruitment to the TATA element.
Conversely, if the TBP mutant fails to activate transcription under the
same conditions, it should lack the ability to proceed to
post-recruitment steps. We transformed wild type strains with the
reporter plasmid harboring the GAL1 promoter-driven lacZ
gene as well as the effector plasmid expressing each TBP mutant fused
to the GAL4 DNA binding domain (Fig. 4B). Consistent with
previous studies, K138T/Y139A, E236P, and F237D mutants activated
transcription less efficiently than the wild type gene (71, 79).
Besides these mutants, P65S, S118L, N159D, N159L, and V161A mutants
also showed lower activities than the wild type gene in this assay. In
contrast, F148H, T153I, R220H, and Y231A mutants activated
transcription at similar levels to the wild type gene. Thus the defects
in the post-recruitment step tend to represent a stronger
nsl phenotype.
Integrity of the TFIID Complex Containing TAF145 Lacking TAND and
the Activation-defective TBP Mutant--
We previously demonstrated
that the same amount of TAF145 protein was coprecipitated with TBP from
cell lysates prepared either from wild type or In this study, we screened nsl genes that have genetic
interaction with TAND of TAF145. The NSL1 gene isolated in
our screen was found to be allelic to the SPT15 gene
encoding TBP. We also identified the amino acid substitutions, S118L
and P65S, for the two spt15/nsl1 alleles. They are recessive
in both the nsl and TS phenotypes in different general
genetic backgrounds. Previous studies demonstrated that the S118L
mutant was deficient in activation by acidic activators (63); however,
it is unknown whether the P65S mutant has similar defects or not. On
the other hand, the P65S mutant was reported to be defective in pol III
transcription (64) although the S118L mutant seems to suffer only pol
II-specific defects. Since TAF145 is a pol II-specific general
transcription factor, it is likely that the P65S mutant is also damaged
in activation by acidic activators like the S118L mutant; we found that
this is the case. Intriguingly, all of the other activation-defective TBP mutants that we tested also showed synthetic lethal interactions with the taf145TAND), in Saccharomyces cerevisiae by exploiting a red/white colony-sectoring assay. Our screen yielded several recessive nsl (
TAND
synthetic lethal) mutations, two of which,
nsl1-1 and nsl1-2, define the same
complementation group. The NSL1 gene was found to be
identical to the SPT15 gene encoding TBP. Interestingly,
both temperature-sensitive nsl1/spt15 alleles, which harbor
the single amino acid substitutions, S118L and P65S, respectively, were
defective in transcriptional activation in vivo. Several
other previously characterized activation-deficient spt15
alleles also displayed synthetic lethal interactions with taf145
TAND, indicating that TAND and TBP
carry an overlapping but as yet unidentified function that is
specifically required for transcriptional regulation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
spt3
phenotype is partially suppressed by overexpression of Toa1 (45). These
observations strongly indicate that TBP-TATA interactions are
intricately regulated in vivo by a wide variety of factors
such as TFIID, SAGA, Mot1, and TFIIA.
TAND synthetic lethal)
mutations that cause lethality in combination with the
TAF145 gene that lacks TAND
(taf145
TAND) in S. cerevisiae. Our
screen identified two distinct temperature-sensitive (TS) alleles of
the NSL1 gene, as alleles of the SPT15 gene which encodes TBP. Further characterization suggests that
activation-defective TBP mutants tend to display synthetic lethal
interactions with the taf145
TAND gene. This is
in accordance with a hypothetical role of TAND in transcriptional
regulation that we have recently proposed. Taken together with previous
observations that the TS phenotype of TAND-lacking strains can be
suppressed by overexpressing TBP or TFIIA, TAND is likely to assist TBP
function rather than simply inhibit it, at least in
vivo.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
S. cerevisiae strains used
TAND allele into the original
TAF145 locus of the CH1305 strain (kindly provided by Dr.
Connie Holm) (50) as follows. pTM6 plasmid was constructed by
subcloning a 1.2-kb HindIII fragment of pGT5 (51) carrying the URA3 gene and a 2.4-kb NotI-BglII
(partial) fragment of pM10 (28) carrying a 3'-truncated
taf145
TAND allele into the HindIII and NotI-BamHI sites, respectively, of
pBluescript II (Stratagene). TMY4-2 was generated by a
recombination-mediated two-step gene replacement procedure (52), first
by transforming CH1305 to Ura+ with pTM6 that had been
linearized at the unique BglII site, and then by screening
for a Ura
Ts+ segregant bearing the
taf145
TAND allele on 5-FOA plates. The gene
replacement was confirmed by PCR.
diploid cells. After segregating the
YEp13-HO plasmid away, cells were sporulated and dissected to isolate
MAT
haploid cells, i.e. TMY17-2c and
TMY16-2c.
Trp+ strains were subsequently transformed with the
URA3-marked plasmid carrying the SPT15 gene
(pYN11). The YTK271 strain (Ura+ Trp
) was
selected by plasmid segregation. YAK289, 293, 493, 495, 582, 584, 586, 588, 620, 622, 633, 636, 938 strains were then generated from YTK271 by
a plasmid shuffling technique.
spt15 strain) was crossed with YKII1
(28) (
taf145 strain) and then dissected to
obtain the haploid strain, YAK284, carrying double deletions of
TAF145 and SPT15 genes. As TAF145 and
SPT15 are both essential genes, the growth of YAK284 is
supported by pYN11/SPT15 (URA3 marker) and
pM11/TAF145 (TRP1 marker). YAK303 andYAK307 were
constructed by replacing pM11 of YAK284 with pM3217 and pTM26, respectively.
TAND genes. True synthetic lethal
mutants should show restored sectoring and growth on 5-FOA plates only
upon transformation with the plasmid bearing the wild type
TAF145 gene. From a total of ~40000 colonies screened, 14 strains (A1, A6, A22, A38, B9, B16, C2, C40, C60, C72, D7, D16, E4, and
E10) had the synthetic lethal phenotype with the
taf145
TAND gene (i.e.
nsl phenotype). C40 and D7 strains were further
characterized in this study.
TAND allele, and in both cases red to
white sectoring was observed, indicating that these mutations were
recessive. Diploids were sporulated, and 18 tetrads were dissected and
analyzed phenotypically. When four spores were recovered, the sectoring
phenotype and the 5-FOA lethality segregated 2:2, indicating that the
synthetic lethality was presumably caused by mutation at a single
locus. Crosses between mutant segregants from C40 and D7 assigned them to a complementation group.
TAND allele but forms normal sized
colonies at 25 °C. C40 and D7 strains also show the TS phenotype
even though they contain the pTM17 plasmid expressing the wild type
TAF145 gene. Multiple backcrosses with TMY17-2c allowed
determination of whether the TS phenotype was linked to synthetic
lethality. As both mutations are linked to the TS phenotype, mutant
segregants derived from C40 and D7 were transformed with a low copy
number plasmid library (ATCC77162) yielding ~100,000 transformants on
SD-Leu plates when grown at 25 °C for 12-18 h and then shifted to
35 °C and incubated for 7 days. Plasmids containing complementing
genomic DNA fragments were recovered from the positive colonies and
were amplified in Escherichia coli DH5
. These plasmids
were retransformed into C40 and D7 strains to confirm the
complementation of the red/white sectoring, 5-FOA lethality, and the TS
phenotype. Insert DNA boundaries were sequenced and compared with the
yeast genome data base. Overlapping regions from chromosome V were
obtained in all cases, and subcloning indicated that the presence of
the SPT15 ORF was sufficient to complement all mutant
phenotypes shown by C40 and D7.
T point
mutations at 353 bp in C40 and at 193 bp in D7, which result in the
amino acid substitutions S118L and P65S, respectively. Proof that these
mutations conferred synthetic lethality was obtained by testing the
nsl phenotype of taf145 alleles bearing these
mutations produced by site-specific mutagenesis (57) as described
below. Oligonucleotides used in this study are listed in Table
II.
Oligonucleotides used in this study
-Galactosidase
Activity--
For the artificial recruitment experiments, plasmids
encoding GAL4-TBP derivatives were introduced into the CH1305 strain containing pB20, a multicopy URA3 plasmid with the
GAL1 promoter upstream of the lacZ structural
gene (kindly provided by Dr. A. G. Hinnebusch). The resulting
strains were grown to an A600 of 0.7 in YPD
medium and then treated with repeated freeze/thaw cycles to measure the
-galactosidase activity as described previously (32). To measure
activation by classical activation domains, yeast strains bearing
mutant TBP derivatives (Table I) were transformed with pB20 and
plasmids expressing various activators.
-D-galactopyranoside for
6 h at 16 °C in M9 medium, and the cells were then resuspended in 10 ml of 0.5 M KCl, buffer C (25 mM Hepes,
pH 7.6, 0.1 mM EDTA, pH 8.0, 12.5 mM
MgCl2, 10% (v/v) glycerol, 0.1% Nonidet P-40, 1 mM dithiothreitol) per liter of culture volume. After
sonication, cell debris was removed by centrifugation, and the
supernatant was stored at
30 °C. For electrophoretic mobility
shift assays, cell lysates were subjected to
Ni2+-nitrilotriacetic acid resin (Qiagen) to purify TBP
derivatives. TBP, in the cleared lysate or purified through the
Ni2+-resin, was quantitated by SDS-PAGE and Coomassie
Brilliant Blue staining.
) as GST fusion proteins.
Isopropyl-1-thio-
-D-galactopyranoside induction was
conducted at 37 °C for 2 h in LB media, and the cell pellet was
resuspended in 10 ml of 0.2 M KCl, buffer D (20 mM Tris-HCl, pH 7.9, 0.2 mM EDTA, pH 8.0, 12.5 mM MgCl2, 10% (v/v) glycerol, 1 mM
dithiothreitol, 1 mM phenylmethylsulfonyl fluoride), per
liter of culture volume. After sonication, GST fusion proteins were
quantitated as described above. Hexahistidine-tagged TFIIA was prepared
as described previously (30).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
TAND), by exploiting an ade2/ade3-based
red/white colony sectoring assay (50, 55). The principle of a synthetic
lethal screen is that, although a single mutation is tolerable for the
cell, a combination of mutations in functionally related pathways
results in severe growth inhibition or cell death. This type of screen
was often used for the identification of unknown component(s)
regulating a common process in a wide variety of biological phenomena
(60-62).
TAND allele at the original
chromosomal locus by homologous recombination and was then transformed
with pTM17, a centromere-based plasmid that contains the URA3 and ADE3
nutritional markers and the wild type TAF145 gene. This host
strain, TMY4-2, is red on YPD media but forms white sectors when the
plasmid pTM17 is lost under nonselective conditions. Thus, we can
expect that nsl mutant strains should show no white sectors
in colonies since the TAND function on pTM17 becomes essential for
growth. TMY4-2 was mutagenized by UV irradiation, and of approximately
~40000 surviving colonies screened, 30 failed to show red/white
sectoring at 25 °C. Of these, 14 regained a sectoring phenotype when
transformed with a plasmid carrying the wild type TAF145
gene, but not with the taf145
TAND gene, and
failed to grow on 5-FOA media (i.e. pTM17 plasmid carrying
the URA3 and TAF145 genes is essential for cell
growth). These observations strongly suggest that they contain
mutations that are synthetically lethal with the
taf145
TAND gene. Two of these nsl
mutants, C40 and D7, were further characterized in this study.
TAND gene, indicating that their
nsl phenotypes resulted from single gene mutations. A
complementation test revealed that they were recessive and allelic, thus hereafter this gene is designated as NSL1
(
TAND synthetic lethal
1). We also refer to mutant alleles of C40 and D7 as
nsl1-1 and nsl1-2. Both nsl1 alleles
seemed to exhibit TS growth phenotypes since all nsl1 spores
obtained through backcross experiments with a parental strain harboring
the chromosomal TAF145 or
taf145
TAND gene were TS even in the presence
of the plasmid, pTM17, expressing the wild type TAF145 gene
(data not shown). Thus we tried to isolate the wild type
NSL1 gene by complementing the TS phenotypes of these
nsl1 mutants. nsl1-1 and nsl1-2
mutants were transformed with a partial Sau3A yeast genomic library,
and several complementing colonies were isolated from both mutants.
Retransformation analysis established that growth recovery at 35 °C
was dependent on the presence of the plasmids that were isolated from
the original colonies (data not shown). Consistent with the allelismic
test, restriction enzyme digestion and sequencing analysis revealed that genomic inserts included in plasmids, derived from either mutant
(i.e. nsl1-1 or nsl1-2), overlapped
with each other (data not shown). Importantly, only the
SPT15 gene encoding TBP was found in all inserts. Thus we
subcloned an ~2.4-kb EcoRI-BamHI fragment, from
which only the SPT15 gene could be expressed, into a
centromeric vector and tested it using the complementation assay. The
plasmid carrying the subcloned fragment rescued the TS growth defects
of both mutant strains (Fig. 1),
suggesting that NSL1 might be allelic to
SPT15.
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Fig. 1.
Growth comparison of nsl1-1
and nsl1-2 mutants transformed with the single
copy plasmids containing SPT15 (+TBP)
or no insert (+vector). These strains were
streaked on SD plates and incubated at 25 or 35 °C for 3 days.
TAND
gene (YAK307) on a centromeric LEU2 plasmid as well as the
wild type SPT15 gene on a centromeric URA3
plasmid in combination with double deletions of chromosomal
TAF145 and SPT15 genes. These strains have
different general genetic backgrounds from the one used in the original
genetic screen for the nsl mutants. The
spt15-S118L and spt15-P65S alleles were
reconstructed on a centromeric TRP1 plasmid by site-directed
mutagenesis to exclude any other possible mutations. These plasmids
were transformed into YAK303 and YAK307 strains described above and
tested for their growth on 5-FOA plates. We reasoned that if
spt15-S118L and spt15-P65S are responsible for
the nsl phenotype, only strains carrying the wild type
TAF145 gene (i.e. derived from YAK303) would be
viable on 5-FOA plates that select for cells that had lost the
URA3 and SPT15 containing plasmid. Consistent
with this expectation, yeast strains carrying the
taf145
TAND gene (i.e. derived from
YAK307) grew well on 5-FOA plates only when they expressed the wild
type SPT15 gene but not when they expressed the
spt15-P65S or spt15-S118L alleles (Fig.
3A). We also confirmed that these mutant alleles were
recessive and TS in the genetic background of YAK303 strains as
observed for the original mutant strains. These observations support
the notion that these TBP mutations, S118L and P65S, are synthetically
lethal with the taf145
TAND gene even under the
different general genetic background. Similar conclusions were
confirmed by genetic experiments in which spores harboring the
combination of taf145
TAND and
spt15-S118L or taf145
TAND and
spt15-P65S alleles were never recovered, as more than 20 asci were dissected for each diploid (data not shown).
-galactosidase activity from the Gal4 upstream activating
sequence-dependent reporter plasmid was measured when
various activation domains fused to Gal4 DNA binding domain were
coexpressed in the cell (Fig.
2A). As we expected, activation efficiencies were constantly lower in the P65S mutant than
that in the wild type strains. TAND1, which binds the concave surface
of TBP, can function as a strong activation domain when recruited onto
the promoter by the Gal4 DNA binding domain (32). Interestingly,
activation by TAND1 was most severely affected in the P65S mutant
(4.2% of the wild type), whereas activation by TADIV of ADR1, which
requires the presence of intact TFIID for its function (68), was least
affected (50.7% of the wild type).
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Fig. 2.
In vivo activation and biochemical
properties of TBP-P65S and TBP-S118L
mutants. A, GAL4-dependent
transcriptional activation in the wild type (solid bars) and
nsl1-2 (spt15-P65S) mutant strains (open
bars). Expression vectors encoding various activation domains
fused to the GAL4 DNA binding domain were transformed into yeast, and
transcription activity was determined by measuring the lacZ
reporter activity expressed from a reporter plasmid. B, gel
mobility shift analyses of the TBP-TATA element interactions
(upper panel) and TFIIA-TBP-promoter DNA interactions
(lower panel). Adenovirus major-late promoter ( 119 to +61)
was used as a probe. The amounts of protein used for assays were 20 ng
of TBP (upper panel) and 10 ng of TBP and 12 ng of TFIIA
(lower panel). The positions of the TBP-DNA complex and
TFIIA-TBP-DNA complex are indicated by an asterisk on the
right. C, interaction of TBP derivatives with
GST-TAND and GST-VP16. GST fusion proteins were incubated with an
equimolar amount of TBP. Complexes were trapped by
glutathione-Sepharose beads, washed extensively with the buffer
containing 0.2 M KCl (TAND-TBP interactions) and 0.1 or 0.2 M KCl (VP16-TBP interactions), and then analyzed by
SDS-PAGE followed by Western blotting.
TAND gene.
TAND gene but not with the wild type
TAF145 gene. In contrast, the mutants, R220H and Y231A,
which are specifically defective for transcription by pol III, did not
cause any deleterious effect on growth with either alleles of the
TAF145 gene. Despite all of the spt15/nsl1
alleles tested here that exhibit the TS phenotype on their own (data
not shown), only the activation-defective TBP mutants had the
nsl phenotype. Therefore, we assumed that the P65S mutant
was isolated as a nsl1-2 mutant in our original screen due
to its inefficient activation by pol II rather than to its impairment
in pol III transcription.
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Fig. 3.
A, activation-defective TBP mutants show
the nsl phenotype. The TRP1-marked plasmids encoding TBP
derivatives, as indicated on the left, were individually
introduced into the strains with double deletions of TAF145
and SPT15 genes but which instead contained either the
LEU2-marked plasmid encoding the wild type gene or the
taf145 TAND gene, as indicated at the
top, in addition to the URA3-marked plasmid encoding wild
type TBP. The resulting transformants were grown on 5-FOA plates at
30 °C for 5 days. B, TBP-TATA and TFIIA-TBP-DNA
interactions were analyzed as described in Fig. 2. Note that TBP-K138T,
Y139A binds weakly to the TATA element in the presence of 2 µg/µl
BSA (lane 6), whereas it binds as well as the wild type in
the absence of BSA (lanes 8 and 9). C,
TBP-TAND and TBP-VP16 interactions were analyzed as described in Fig.
2.
TAND gene, we believe that such deficiency may not be directly related to the nsl phenotype.
With regard to the interactions with VP16, all TBP mutants retained the
binding activity although some of these mutants, i.e. N159D, N159L, V161A, F237D, and E236P, bound to VP16 more strongly than the
wild type in the buffer containing 0.2 M KCl (Fig.
3C, lower panel) which was similar to the result obtained
with the S118L and P65S mutants (Fig. 2C, lower panel).
Interestingly, F148H and T153I mutants, which appeared to bind to VP16
with normal affinities, showed a weaker nsl phenotype (Fig.
3A). Collectively, the nsl phenotype can be
achieved by various types of activation-defective TBP mutants but not
by pol III transcription-defective TBP mutants. It is notable that
stronger interactions with VP16 seem to be most closely correlated to
this phenotype.
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Fig. 4.
A, activation by acidic activators in
TBP mutants. -Galactosidase activities of a
GAL4-dependent reporter system were measured in strains
containing the indicated TBP derivatives and either of three
activators, i.e. GAL4DBD-VP16AD, GAL4DBD-GAL4AD, or
GAL4DBD-GCN4AD. The values are represented as a percentage of the value
obtained in the wild type strain containing the corresponding
activators. Note that the host strain used here is different from that
in Fig. 2A. B, artificial recruitment assay of
GAL4DBD-TBP derivatives. The relative
-galactosidase activities of a
GAL4-dependent reporter plasmid were measured in the wild
type strain expressing the indicated GAL4DBD-TBP derivatives. Each
value represents the average of three different isolates of each strain
(A and B).
TAND strains (28).
The reason differences between these two strains were not observed was
probably because other regions of TAF145 or other TAF components
contributed to the stability of the TAF145-TBP interactions and/or
supported the integrity of TFIID. Therefore, it is likely that the
nsl phenotype may occur due to the instability of TFIID
enhanced by combining two different mutations within the same complex.
To explore this possibility, we constructed yeast strains in which the
chromosomal SPT15 gene was deleted and instead carried two
plasmids, one expressing the HA-tagged
taf145
TAND gene (LEU2 marked plasmid) and the
other expressing either wild type or activation-defective TBP (TRP1 marked plasmid). All strains were viable due to the presence of the
wild type TAF145 gene on the chromosome. We tried to examine the integrity of TFIID in these strains by measuring the amounts of
TAF145
TAND and TAF61 proteins that were coprecipitated with TBP
(Fig. 5). Precipitates were blotted onto
a membrane and probed with anti-HA, TAF145, TAF61, and TBP antibodies,
respectively. The signal detected by the anti-HA antibody represents
the amount of TAF145
TAND proteins, whereas the signal detected by
anti-TAF145 antibody is derived from the sum of wild type TAF145 and
TAF145
TAND proteins. Note that nontagged wild type TAF145 proteins
and HA-tagged TAF145
TAND proteins migrated at the same position on
this gel. When the signals were normalized with the amount of
precipitated TBP (lowest panel), it appeared that similar
amounts of TAF145 and TAF61 proteins were included in the TFIID complex
in all yeast strains. In contrast, smaller amounts of TAF145
TAND
proteins were coprecipitated with TBP in the N159D mutant. Such
differences were not observed for other TBP mutants. However, it is
still possible that even the N159D mutant can make a stable TFIID
complex with the TAF145
TAND protein if the wild type TAF145 protein
is not present in the cell, although we cannot test this possibility directly due to its synthetic lethality. Collectively, the instability of the TFIID complex does not simply explain why these TBP mutants display the nsl phenotypes.
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Fig. 5.
Coimmunoprecipitation analysis to test the
integrity of the TFIID complex containing both
TAF145 TAND protein and TBP derivatives.
Whole cell extracts were prepared, using glass beads, from yeast
strains containing the wild type gene or the indicated TBP derivatives
and the HA-tagged TAF145
TAND proteins. Note that these strains have
the nontagged wild type TAF145 gene on the chromosome to support
viability. Aliquots of whole cell extract proteins were
immunoprecipitated with anti-TBP polyclonal antibodies
(even-numbered lanes) or preimmune antibodies
(odd-numbered lanes). Proteins coprecipitating with TBP were
fractionated on SDS-PAGE, transferred to nitrocellulose membranes, and
probed with the indicated antibodies.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
TAND gene. In contrast, similar
effects were not observed for TBP mutants that were specifically
impaired in pol III transcription. Further characterization of the
biochemical properties of these activation-defective TBP mutants
revealed that enhanced interaction with VP16 appeared to be closely
related to the nsl phenotype (Table
III). In addition, an artificial
recruitment assay produced further evidence for the good correlation
between the defect in the post-recruitment step and the nsl
phenotype (Table III).
Summary of various properties of TBP mutants
We recently proposed that TAND might be involved in the initial stage
of activation by acidic activators (32). Some functional similarities
have been found between TAND1 (that is the N-terminal subdomain of TAND
that binds to the concave surface of TBP) and acidic activation domains
like VP16, GAL4, and EBNA2 (32). Such unexpected functional
similarities prompted us to build a two-step hand off model in which
TAND1, bound to the concave surface of TBP, could first be displaced by
an acidic activation domain (AD) and the AD could be successively
displaced by the TATA element (32). We believe that such transfer of
TBP from TAND to the TATA element may trigger the isomerization process
of TFIID that leads to increased stimulation of transcription.
According to this model, TAND must play two different roles,
i.e. when activators are absent near the promoter, TAND
inhibits TBP-TATA interactions to prevent leaky transcription, whereas
it must be able to release TBP once activators come close to the
promoter. Taken that TFIID can recognize the core promoter, not only by
TBP-TATA interactions but also by TAF-DNA interactions (reviewed in
Refs. 8 and 7), TBP might be converted into an active form at a much
closer position to the TATA element when it is liberated from TAND by
the action of activators. If this is the case, TAND performs dual
functions as a negative and positive regulator. Previous studies
demonstrated that the TS phenotype of
taf145TAND strains could be suppressed by
overexpression of TBP and TFIIA (30, 31). The assumption described
above may explain why TFIID that lacks TAND needs higher concentrations
of TBP or TFIIA to perform its normal function.
Our studies demonstrate that activation-defective TBP mutants confer
severe damage to yeast strains carrying the
taf145TAND gene. It is especially intriguing
that TBP mutants defective in the post-recruitment step produce a
stronger nsl phenotype (Table III). As described above, it
is believed that TAND is involved in the initial step during
activation, i.e. stable binding of TFIID to the promoter
(pre-recruitment step). The combined defects of the pre- and
post-recruitment steps can be expected to yield the lowest activation
and thereby inhibit cell growth. In this regard, the results obtained
here support our previous model that TAND is involved in
pre-recruitment steps.
Another issue to be discussed is how these TBP mutants are impaired in the post-recruitment step. TBP may function together with several other transcription machineries besides TFIID, e.g. SAGA and RNA pol II holoenzyme (reviewed in Ref. 23). Thus, it is possible that these TBP mutants affect the function of other machineries involved in post-recruitment steps. Although we have not yet examined the integrity of those machineries, most of these TBP mutants appear to be normal at least in the integrity of TFIID. However, it is still likely that TFIID may be impaired in its interactions between TBP-TAF or TBP-TFIIA that are required specifically for the post-recruitment steps. To determine which complex is most severely damaged in these TBP mutants, we need to separate each complex and test its activity individually using in vitro transcription experiments.
Recently, TBP has been shown to exist, at least in part, as an
independent form of TAFs (21, 22). TAFs are recruited in much smaller
amounts to TAF-independent (TAFind) promoters when compared with
TAF-dependent (TAFdep) promoters. Consistently, TBP is
recruited, in a manner apparently independent of TAF function, to the
TAFind promoters. Conversely, TAFs are recruited independently of TBP
function to TAFdep promoters. These observations strongly suggest that
there are at least two transcriptionally active forms of TBP,
i.e. associated form and nonassociated form with TAFs (21,
22). Thus it might be possible that activation-defective TBP mutations
affect the TAF-nonassociated form of TBP predominantly, whereas
TAF145TAND affects only the TAF-associated form of TBP, so that
double mutants can decrease the expression of a much broader range of
genes. It has yet to be determined whether these alternative forms of
TBP, other than TFIID, correspond to the free TBP molecule or any other
known or unknown TBP-containing complexes.
It is still unclear whether TAND is involved in the activation of most
genes or just a particular set of genes in vivo. Earlier studies demonstrated that activation may occur even in the absence of
TAFs in vitro as well as in vivo (80, 81). More
recent genome-wide expression analyses have shown that the requirements for various TAFs are not the same among different genes probably because some TAFs are shared by TFIID and SAGA, which are functionally redundant (reviewed in Refs. 82-84). Consistently shared TAFs such as
TAF17, TAF25, and TAF60 appear to be more generally required for gene
expression than TFIID-specific TAFs such as TAF145 and TAF150 (84).
However, there are exceptions like TAF40, which is a TFIID-specific TAF
but nonetheless required by most promoters (85). Since our preliminary
genome-wide expression analysis showed that TAF145TAND produced much
smaller effects,3 we believe
that the possible detrimental effect of TAF145
TAND on gene
expression might be concealed in vivo by compensatory interactions between TFIID and other machineries like SAGA, as just
recently observed for TAF145 and Gcn5 (84). TAF145 and Gcn5, both of
which encode catalytic subunits of histone acetyltransferase in TFIID
and SAGA, appear to regulate the expression of a large fraction of
genes through the function of either complex. In this regard, it would
be intriguing to examine the redundancy between TAND and Spt3/Spt8 that
are analogous subunits, which negatively regulate TBP function in each
complex. Isolation of other NSL genes could solve the
functional redundancies in vivo that are accomplished by
multiple TBP-interacting factors.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. K. Kasahara for helpful discussions. We also thank Drs. A. G. Hinnebusch, Y. Nakatani, and C. Holm for yeast strains and plasmids and Drs. Y. Ohya and K. Matsumoto for yeast genomic libraries.
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FOOTNOTES |
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* This study was supported by grants from the Ministry of Education, Science, and Culture of Japan, by the CREST Japan Science and Technology Corp., the Uehara Memorial Foundation, the Asahi Glass Foundation, the NAITO Foundation, the Sumitomo Foundation, and NOVARTIS Foundation for the Promotion of Science.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Dept. of Biochemistry and Molecular Genetics,
Health Sciences Center, University of Virginia, Charlottesville, VA 22908.
§ To whom correspondence should be addressed: Division of Gene Function in Animals, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0101, Japan. Tel.: 81-743-72-5531; Fax: 81-743-72-5539; E-mail: kokubo@bs.aist-nara.ac.jp.
Published, JBC Papers in Press, October 16, 2000, DOI 10.1074/jbc.M008208200
2 T. Ichimiya, M. Yuhki, K. Kasahara, M. Kawaichi, and T. Kokubo, unpublished observations.
3 Y. Tsukihashi and T. Kokubo, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: TBP, TATA-binding protein; pol, polymerase; TAFs, TBP-associated factors; 5-FOA, 5-fluoroorotic acid; bp, base pair; aa, amino acid; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; kb, kilobase pair; HA, hemagglutinin; BSA, bovine serum albumin; TS, temperature-sensitive; AD, activation domain.
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