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Molecular and Cellular Biology, April 2000, p. 2385-2399, Vol. 20, No. 7
Division of Gene Function in Animals, Nara
Institute of Science and Technology, Ikoma, Nara 630-0101, Japan
Received 28 September 1999/Returned for modification 29 November
1999/Accepted 10 January 2000
The general transcription factor TFIID, which is composed of
TATA-binding protein (TBP) and an array of TBP-associated factors (TAFs), has been shown to play a crucial role in recognition of the
core promoters of eukaryotic genes. We isolated Saccharomyces cerevisiae yeast TAF145 (yTAF145)
temperature-sensitive mutants in which transcription of a specific
subset of genes was impaired at restrictive temperatures. The set of
genes affected in these mutants overlapped with but was not identical
to the set of genes affected by a previously reported
yTAF145 mutant (W.-C. Shen and M. R. Green, Cell
90:615-624, 1997). To identify sequences which rendered transcription
yTAF145 dependent, we conducted deletion analysis of the
TUB2 promoter using a novel mini-CLN2 hybrid
gene reporter system. The results showed that the yTAF145
mutations we isolated impaired core promoter recognition but did not
affect activation by any of the transcriptional activators we tested. These observations are consistent with the reported yTAF145 dependence of the CLN2 core promoter in the mutant isolated by Shen
and Green, although the CLN2 core promoter functioned
normally in the mutants we report here. These results suggest that
different promoters require different yTAF145 functions for efficient
transcription. Interestingly, insertion of a canonical TATA element
into the TATA-less TUB2 promoter rescued impaired
transcription in the yTAF145 mutants we studied. It
therefore appears that strong binding of TBP to the core promoter can
alleviate the requirement for at least one yTAF145 function.
In eukaryotes, transcriptional
initiation by RNA polymerase II requires a set of general
transcriptional factors (TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH)
(reviewed in references 16, 72, and
78) and the SRB-MED complex associated with the
carboxy-terminal domain of RNA polymerase II (reviewed in references
65 and 66). These factors
nucleate on the core promoter of eukaryotic class II genes to form a
preinitiation complex in an ordered stepwise fashion (reviewed in
references 9, 23, and 78) or are
recruited in a simpler sequence involving a small number of
preassembled units (reviewed in references 43 and
73). In either case, the first step, which is the
sequence-specific binding of TFIID (76), is thought to be a
major rate-limiting step during transcription and a focal point for the
activity of transcriptional activators (14, 40, 54).
TFIID is a multiprotein complex composed of TATA-binding protein (TBP)
and an array of TBP-associated factors (TAFs); in total, the complex
includes 8 to 12 molecules ranging in size from 15 to 250 kDa (reviewed
in references 11, 51, 91, and
93). Almost all of these TAFs are conserved among
evolutionarily divergent organisms (humans, Drosophila
melanogaster, and Saccharomyces cerevisiae), albeit
with a few exceptions (for instance, no orthologue of
Drosophila TAF110 or human TAF130 (dTAF110/hTAF130) is found in yeast). This level of conservation suggests that TAFs play a
fundamental role in eukaryotic transcription (reviewed in references 5, 11, 81, and 91). Earlier
biochemical studies in vitro demonstrated that TAFs are obligatory
cofactors for activation, since TBP alone can mediate basal
transcription but, unlike TFIID, cannot support activated transcription
(reviewed in references 11, 20, and
42). However, the concept of an absolute requirement for TAFs in activation has been challenged by a number of recent studies. First, several groups reported that activation can be successfully reconstituted in an in vitro transcription system using
yeast or human components that include TBP but no detectable amounts of
TAFs (24, 39, 44, 69, 103). Second, in vivo depletion of
functional TAFs in yeast cells demonstrated that the absence of certain
TAFs had little effect on the activation mediated by a variety of
activators, including Gcn4, Ace1, Gal4, and Hsf (63, 98).
However, TAFs do play important roles in TFIID recognition of core
promoter elements. TFIID requires TAFs for core promoter binding,
especially when the canonical TATA element is absent (reviewed in
references 11, 21, and 97).
TAF-DNA interactions appear to compensate for the lack of direct
TBP-TATA interactions on TATA-less promoters (58, 74).
Indeed, a number of TAFs have been identified which recognize sequence
elements near or downstream of the initiation site (10, 13, 38,
68, 96). Moreover, TAFs were also shown to mediate
transcriptional synergism between TATA and initiator elements along
with TFIIA and TAFII- and initiator-dependent cofactors
(TICs) (21, 56). The requirement for TAFs in core promoter
recognition has been further demonstrated by genetic studies. In vivo
TAF depletion experiments demonstrated that Saccharomyces
cerevisiae yeast TAF145 (yTAF145) is not generally required for
transcriptional activation (63, 98) but is essential for a subset of genes (84, 99). Importantly,
promoter-swapping experiments provided evidence that yTAF145 dependence
is conferred by sequences within the core promoter region rather than
upstream activating sequences (UAS) (84). These observations
argue that the principal role of TAFs is to recognize core promoter elements.
Recently, several TAFs were reported to be integral components not only
of TFIID but also of large histone acetyltransferase (HAT) complexes
(reviewed in reference 88), including yeast SAGA
(26) and mammalian PCAF complex (71), TFTC
(8, 102), and STAGA (57). Earlier genetic studies
in yeast demonstrated that several components of these complexes (such
as ADAs and SPTs) are involved in transcriptional regulation (reviewed
in reference 27), and more recent in vitro studies
clearly show that SAGA stimulates activator-induced transcription on
chromatin templates in an acetyl coenzyme A-dependent manner (36,
95). Additionally, mammalian TFTC can substitute for TFIID in
transcriptional initiation from TATA-containing and TATA-less promoters
as well as in activation by GAL4-VP16 in vitro (102). These
observations raise the intriguing possibility that the HAT complexes
described above and TFIID may function redundantly in vivo. In this
respect, it is notable that TFIID also has HAT activity
(62). In yeast, five TAFs (yTAF90, -60, -17/20, -25, and
-68/61) are components of both TFIID and SAGA (26). Of
these, histone-like TAFs, such as yTAF68/61 (histone H2B-like), yTAF60
(histone H4-like), and yTAF17/20 (histone H3-like), and the
non-histone-like yTAF25 were reported to be required for transcription
of a broader range of genes than other TAFs (1, 29, 34, 61, 64,
67, 80). More importantly, unlike yTAF145, these histone-like
TAFs are apparently involved in transcriptional activation as well
(1). The observation that shared TAFs appear to be more
crucial for in vivo transcription and activation than TFIID-specific
TAFs supports the notion that TFIID and SAGA are functionally redundant.
In mammalian cells, a point mutation in TAF250 (an orthologue of
yTAF145) was shown to cause late G1 cell cycle arrest in temperature-sensitive ts13 cells (31, 33, 79, 83).
Interestingly, only a subset of genes, including cyclin D1 and cyclin
A, were affected in these cells (55, 82, 90, 100). This
parallels the phenotype of yTAF145 mutant cells, which are
also arrested in G1 phase (98) and do not show
general transcriptional defects at restrictive temperatures
(99). However, the region that renders the cyclin A gene
promoter TAF250 dependent in ts13 cells was mapped to UAS in addition
to core promoter sequences (101). This suggests that
specific activator function is impaired in ts13 cells, in contrast to
the yTAF145 mutant, in which core promoter recognition does
not operate normally (84). Recently, however, yTAF145 has
also been shown to be required for specific activator function
(45). The transactivation domain IV (TADIV) of ADR1, a yeast
activator that regulates transcription of the ADH2 gene (17), functions much less efficiently in a
yTAF145 mutant (45). Meanwhile, it has been
demonstrated that Drosophila TAF110 and TAF60 are important
for activation by dorsal, a transcription factor that
regulates twist and snail in embryos
(104). Despite these observations, the full extent to which
TAFs are required for activation in vivo remains unclear.
To further investigate TAF function in vivo, we isolated a number of
novel yTAF145 temperature-sensitive (TS) mutants.
Interestingly, the expression profiles of some genes in these mutants
were not identical to those in a previously reported yTAF145
mutant (84, 99). Deletion analysis of the TUB2
promoter demonstrated that core promoter elements rather than UAS are
responsible for yTAF145 dependency in our mutants. Various activation
domains activated a reporter construct normally when directed by the
CYC1 core promoter but not when controlled by the
TUB2 core promoter. Thus, transcriptional activators appear
to be unable to compensate for the loss of yTAF145 function in core
promoter recognition. Interestingly, in contrast to the earlier
yTAF145 mutant (84), insertion of a canonical TATA element restored transcription directed by the TUB2
promoter in our mutants.
Construction of a mutated plasmid library by error-prone
PCR.
To generate randomly mutated yTAF145 libraries, we
divided the entire open reading frame (ORF) of the yTAF145
gene into four regions, denoted I (amino acids [aa] 5 to 230), II (aa
231 to 535), III (aa 536 to 817), and IV (aa 818 to 1066), flanked by a
set of unique restriction enzyme recognition sequences (Fig. 1A). An MluI site at the
junction between regions I and II and an NheI site at the
end of region IV were inserted into template pYN2 (41) by
site-directed mutagenesis (49) using primers TK65 and TK3.
Oligonucleotides used in this study are listed in Table
1. We designated the resulting plasmid
pM34. The sequence of region III, flanked by BglII and
BstEII sites, was amplified by error-prone PCR
(12) using primers TK66 and TK67. A random mutant library
was generated by replacing the BglII/BstEII
fragment of pM34 with the resulting error-prone PCR products. The
plasmid library was transformed into yeast to isolate conditional
alleles of the yTAF145 gene as described below.
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Impaired Core Promoter Recognition Caused by Novel
Yeast TAF145 Mutations Can Be Restored by Creating a Canonical TATA
Element within the Promoter Region of the TUB2
Gene
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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FIG. 1.
Structure of and growth properties conferred by
conditional alleles of the yTAF145 gene obtained by
error-prone PCR. (A) Schematic diagram of wild-type and three TS
alleles of yTAF145. The entire yTAF145 ORF was
divided into four regions flanked by a set of unique restriction enzyme
sites. Region III was randomly mutagenized by error-prone PCR, and
screening of the resulting plasmid library yielded the TS alleles
Y570N, N568 
, and T657K. The TAND
and the HAT domain of yTAF145 are hatched at the top of panel A. (B)
Comparison of TS phenotypes. Strains carrying wild-type or mutant
alleles were grown on YPD (yeast extract-peptone-dextrose) plates at 25 and 37°C for 3 days.
TABLE 1.
Oligonucleotides used in this study
Yeast strains, genetic analyses, and isolation of conditional
alleles.
Standard techniques were used for yeast growth and
transformation (2, 28). Yeast strains were derived from
Y22.1, which carries a deletion of the chromosomal yTAF145
coding region and the wild-type yTAF145 gene on a
URA3-based low-copy-number vector (41). To
isolate conditional alleles of yTAF145 from the randomly mutated plasmid library, a plasmid shuffling technique was used (7). 5-Fluoroorotic acid-resistant colonies harboring mutant yTAF145 genes on plasmids derived from pM34 were incubated
on synthetic dextrose (SD) plates for 3 days at 30 or 37°C to compare their growth properties. Plasmid DNA was isolated from candidate TS
clones and retransformed to confirm the plasmid linkage of the TS
phenotype. The BglII/EcoRI (464-bp) and
EcoRI/BstEII (380-bp) fragments of pM34 (wild
type), which between them contain the entire sequence of region III
(Fig. 1A), were individually replaced with the corresponding regions of
candidate plasmids to determine which fragment conferred the TS
phenotype. The amino acid residue(s) responsible for the TS phenotype
was finally determined by sequencing and site-directed mutagenesis
(49). Yeast strains used in this study are listed in Table
2.
|
Plasmids encoding conditional alleles of yTAF145
gene.
pM34 was subjected to site-specific mutagenesis
(49) to recreate the conditional yTAF145 alleles.
Oligonucleotides TK175, TK176, and TK178 were used to generate the
plasmids pTM25 (Y570N), pTM32 (N568), and pTM43 (T657K), respectively.
Construction of mini-CLN2 hybrid reporter
gene.
For construction of mini-CLN2 reporter plasmids,
the parental plasmid encoding CLN2 was obtained by screening
yeast genomic libraries. pM1450 was constructed by ligating the 2.8-kb
SphI/XbaI fragment including the entire
CLN2 gene from the parental plasmid into the
SphI/XbaI sites of YEplac181 (25).
pM1451 was constructed by exchanging the PvuII fragment of
pRS315 (86) with the PvuII fragment from pM1450,
which included the CLN2 gene, to enable site-directed
mutagenesis (49). pM1452 was created by digesting pM1451
with SpeI and NcoI to remove a 1,047-bp internal
fragment from the CLN2 ORF, blunt ending the linearized
vector, and religating (pM1452 is shown as CLN2 in Fig.
4A). pM1453 was constructed by ligating a 216-bp SphI
fragment containing the UAS of CLN2 into the SphI
site of pM1452 (pM1453 is shown as
UASCLN2+CLN2 in Fig. 4A); the
UAS-containing fragment was amplified by PCR using primers TK962 and TK860.
80~+172
(fragment from 80 to +172), 129~+172, and 829~+172 fragments
of the TUB2 promoter, respectively; these fragments were
amplified by PCR using the primer pairs, TK1105 and TK1053, TK1078 and
TK1053, and TK1052 and TK1053, respectively. pM1584 was constructed by
ligating the 216-bp SphI fragment containing the UAS of the
CLN2 gene from pM1453 into the SphI site of pM1583.
pM1586 was created by ligating four repeats of the GAL4
binding site, constructed by annealing two oligodeoxynucleotides, TK521
and TK522, into the SpeI site of pM1585 (which was
constructed from pM1584 by linker insertion at the SphI
site). The PvuII fragment of pM1586 containing the entire
reporter gene was moved into pRS316 (86) to change the
auxotrophic marker from LEU2 to URA3. The resulting plasmid pM1587 is shown as
UASGAL+TUB2/80 in Fig. 5A. pM1591 was created
by replacing the 770-bp SpeI/XhoI fragment of
pM1587 encompassing the TUB2 promoter with a DNA fragment
encompassing the CYC1 promoter; the latter fragment was
amplified as an SpeI/XhoI fragment by PCR with
the primers TK1136 and TK1137 (pM1591 is shown as
UASGAL+CYC1/174 in Fig. 5A).
Plasmids encoding activation domains fused with the GAL4 DNA binding domain. pM471 was constructed by replacing the 1,240-bp SphI fragment of pGAD424 (Clontech) that contains the GAL4 activation domain, expression of which is regulated by an ADH1 promoter and terminator, with the corresponding 1,094-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, pM1594, pM1569, pM967, pM1440, pM1570, pM524, and pM468 were constructed by ligating DNA fragments encoding ABF1 (aa 600 to 731), GAL4 (aa 842 to 874), GCN4 (aa 107 to 144), ADR1 (aa 642 to 704), EBNA2 (aa 426 to 462), VP16 (aa 457 to 490), and yTANDI (aa 10 to 42) activation domains, respectively, into pM471. The activation domains were amplified by PCR using the primer pairs TK1130 and TK1131, TK212 and TK213, TK208 and TK209, TK937 and TK938, TK184 and TK185, TK189 and TK187, and T844 and TK202, respectively.
Northern and slot blot analyses. Cells were grown to log phase at 25°C, a portion of each culture was shifted to 37°C, and incubation was continued for 2 h. Cell density was determined, and equal numbers of cells were harvested from 25 and 37°C cultures. Total RNA was isolated as described previously (37). Briefly, cells were washed once in water, resuspended in 400 µl of lysis buffer (10 mM Tris-HCl [pH 7.5], 10 mM EDTA, 0.5% sodium dodecyl sulfate [SDS]), mixed with 400 µl of unbuffered phenol by vortexing, and then incubated at 65°C for 1 h. The tubes were placed on ice for 10 min and centrifuged at 4°C for 10 min. The aqueous phase was reextracted with phenol-chloroform and then precipitated with ethanol. RNA pellets were washed with 70% ethanol and resuspended in water. DNA was removed by treatment with RNase-free DNase (Boehringer Mannheim) at 37°C for 1 h. For Northern analysis, total RNA (20 µg) was resolved on 1% denaturing agarose gels, transferred to GeneScreen Plus (NEN Research Products) membranes according to the manufacturer's instructions, and fixed to the membranes by UV cross-linking using a Stratalinker (Stratagene). Blots were hybridized overnight at 42°C with the appropriate radioactive probes in a buffer containing 5× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 50% formamide, 5× Denhardt's solution, 0.5% SDS, and 0.2 mg of salmon sperm DNA per ml. The hybridized blots were washed three times in 1× SSC buffer at 65°C for 15 min and then autoradiographed.
Slot blot analysis was performed as described previously (48). Total RNA (2 µg in 1 µl) was incubated with 30 µl of denaturing solution (17.5 µl of formamide, 2 µl of formaldehyde, 1.75 µl of 10× morpholinepropanesulfonic acid [MOPS] buffer [0.2 M MOPS {pH 7.0}, 50 mM sodium acetate, 10 mM EDTA] 8.75 µl of deionized water) at 60°C for 15 min and then placed on ice. Cold Tris-EDTA (TE) buffer (35 µl) was added to each sample, and the samples were applied to GeneScreen Plus (NEN Research Products) nylon membranes using a Bio-Dot microfiltration apparatus (Bio-Rad). The samples were washed twice with 2× SSC buffer, and RNA was fixed to the membranes by UV cross-linking. The blots were hybridized overnight at 37°C with a radioactive poly(dT) probe in a buffer containing 4× SSC, 10× Denhardt's solution, 0.5% SDS, and 0.1 mg of salmon sperm DNA per ml. The hybridized blots were washed several times with 1× SSC at 37°C for 10 min and then autoradiographed.Probes for RNA analyses. For Northern blot analysis of endogenous genes, DNA fragments surrounding the initiating methionine were amplified by PCR from yeast genomic DNA, purified, and 32P labeled using a random priming method. The PCR primers used were as follows: TK249 and TK250 for ACT1, TK1186 and TK1187 for ADH1, TK1224 and TK1225 for PGK1, TK1169 and TK1170 for DED1, TK489 and TK490 for CLN1, TK491 and TK492 for CLN2, TK1194 and TK1195 for CLN3, TK1165 and TK1166 for CLB1, TK1167 and TK1168 for CLB2, TK1188 and TK1189 for CLB5, TK531 and TK532 for TUB2, TK477 and TK478 for RPL32, TK1231 and TK1232 for RPS30, and TK493 and TK494 for RPS5.
For detection of mRNA derived from mini-CLN2 hybrid gene reporter constructs, the 411-bp XhoI/HindIII fragment was isolated from pM1452 and 32P labeled by random priming. The poly(dT) probe was 32P labeled by incubating 50 ng of poly(rA)/poly(dT)12-18 (Amersham Pharmacia Biotech), 4 µl of 5× first-strand synthesis buffer (GIBCO BRL), 1 µl of 100 mM dithiothreitol, 1 µl of [
-32P]dTTP (400 Ci/mmol) and
1 µl (200 U) of Moloney murine leukemia virus reverse transcriptase
(GIBCO BRL), in a final volume of 20 µl at 37°C for 1 h. The
reaction mixture was incubated at 70°C for 15 min, and then 80 µl
of TE buffer was added. Unincorporated nucleotides were removed with a
Sephadex G50 spin column.
Antibodies, immunoblot, and coimmunoprecipitation analyses. Polyclonal antibodies directed against yTAF61 were raised in rabbits using a recombinant His-tagged yTAF61 polypeptide (aa 1 to 360), expressed in bacteria, and gel purified as an antigen. Polyclonal antibodies against yTAF145 and TBP were described previously (47).
Immunoblot and coimmunoprecipitation analyses were performed as described previously (47).| |
RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Isolation of yTAF145 conditional alleles. It has been demonstrated that yTAF145 is not required for transcription of most genes (63, 98), but is essential for a subset of genes such as G1 or B-type cyclins and ribosomal proteins (84, 99). Interestingly, the sequences that confer yTAF145 dependency were mapped to the core promoter rather than UAS in these genes (84). More recently, genome-wide expression analysis using DNA microchip technology showed that the levels of mRNAs representing only about a third of the 5,441 genes analyzed are reduced by twofold or greater within 45 min after temperature shift in a TS yTAF145 mutant (34). Importantly, however, these observations were obtained using a limited number of conditional mutants that were originally isolated by S. S. Walker and colleagues (ts1 and ts2) (98). On the other hand, yTAF145 has been shown to possess multiple functions, including TAND (TAF N-terminal domain) activity, which negatively regulates TBP function (3, 41, 47), and HAT activity (62), both of which are thought to be involved in certain aspects of transcription. Thus, we believe that there remains a good chance of isolating additional yTA145 conditional alleles which affect transcription in different ways from ts1 and ts2 (98), by conducting more extensive screening.
To isolate conditional alleles showing a wide variety of phenotypes, we divided the entire yTAF145 ORF into four regions separated by unique restriction sites, each of which could be replaced with a corresponding PCR-amplified DNA fragment by digesting with the appropriate restriction enzymes and religating (Fig. 1A). In this study, we used error-prone PCR (12) to introduce random mutations into the most highly conserved section of yTAF145, region III, which overlaps the HAT domain. Three conditional alleles (plasmids 2, 50, and 51) were isolated from the mutated library by plasmid shuffling (7) and growth rate screening. Sequence analysis showed that plasmids 2 and 51 each contained two mutations in the yTAF145 ORF, encoding the amino acid changes N568
(deletion of N568) and R580G in plasmid 2 and K559I and Y570N in
plasmid 51, while plasmid 50 had just one mutation site (encoding the amino acid change T657K). Single residue substitution or truncation by
site-directed mutagenesis (49) demonstrated that the
N568, Y570N, and T657K mutations are chiefly responsible for the TS phenotypes of mutants 2, 51 and 50, respectively. The growth phenotypes of cells carrying wild-type or singly mutated alleles
(Y570N, N568, and T657K) at
permissive (30°C) and nonpermissive (37°C) temperatures are shown
in Fig. 1B. Note that the Y570N allele displayed a weaker TS
phenotype than the others.
Transcription of a subset of genes is specifically reduced at
restrictive temperatures.
To test the specificity of the effects
of these TS alleles on gene expression in vivo, total RNA was isolated
from wild-type and mutant strains harvested 2 h after a
temperature shift to 37°C and analyzed by slot blotting (Fig.
2A) and Northern blotting (Fig. 2B). Slot
blot analysis using an oligo[d(T)] probe measured levels of
poly(A)+ RNA in these mutants. The results were consistent
with those of previous studies (99) and showed that there
was no substantial reduction in the synthesis of poly(A)+
RNA in these mutants. However, careful inspection revealed that the
N568 and T657K mutations caused slight decreases in total poly(A)+ RNA levels, although the reduction was much
smaller than that induced by rpb1-1, a mutation of the
largest subunit of RNA polymerase II (92).
|
and
T657K mutants and slightly reduced in the Y570N mutant. These
observations, together with the slot blot analysis results discussed
above, suggest that N568 and T657K produce more severe defects than
Y570N. Interestingly, CLN2 mRNA was expressed at the
wild-type level in our mutants, in stark contrast to the ts2 mutant in
which it is almost undetectable at 37°C (84, 99). In
addition, the N568 and T657K mutations significantly affected
transcription of mRNAs encoding CLB1, CLB2, TUB2, and RPL32 and partially reduced
transcription of ACT1, DED1, CLN1, and
CLN3 but had no effect on ADH1 or
PGK1. Therefore, these novel conditional mutants exhibit
gene expression profiles which are distinct from that of the ts2 mutant
(84, 99), suggesting that different yTAF145 functions might
be affected in our mutants.
Expression of yTAF145 mutant proteins and stability of the TFIID
complex.
Expression of mutant alleles under nonpermissive
conditions was monitored by immunoblotting (Fig.
3A). The yTAF145 mutant proteins (Y570N,
N568, and T657K) were stably expressed for at least 4 h after
the temperature shift to 37°C, after which the levels of these
proteins gradually declined. This is another distinctive feature of our
mutant alleles, since ts1 and ts2 mutant proteins were reported to be
rapidly degraded and almost undetectable 2 h after the temperature
shift to 37°C (98).
|
A novel reporter system using the mini-CLN2 hybrid
gene.
The observation that our yTAF145 mutations
affected transcription of TUB2 but not CLN2
prompted us to develop a novel reporter system to delineate the regions
of the TUB2 promoter that are responsible for yTAF145
dependency. We used a portion of the CLN2 gene as a
reporter, instead of the commonly used lacZ gene
(84), so that a single probe could monitor the expression of
reporter constructs and endogenous CLN2 simultaneously.
Since the latter was not affected by our yTAF145 mutations,
it could be used as an internal standard. To distinguish reporter gene
mRNAs from those derived from the endogenous CLN2 gene, we
shortened the CLN2 gene in the reporter plasmid by removing
an internal SpeI/NcoI fragment (Fig.
4A).
Since the deleted fragment encodes one
of the essential -helixes of the cyclin box (helix 5), the
mini-CLN2 reporter constructs could be expected to produce
nonfunctional (i.e., nontoxic) CLN2 proteins (35). Indeed,
the mini-CLN2 gene driven by various promoters at different
expression levels did not affect the growth rates of host yeast cells
in any of our experiments (data not shown).
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CLN2) or
without (CLN2) UAS (84) was introduced into
yeast on a low-copy-number plasmid. Transcription of the mini-CLN2 gene on these reporter plasmids was monitored by
Northern blot analysis using a CLN2-specific probe (Fig.
4B). As expected, two specific bands were detected, corresponding to
mRNAs derived from endogenous CLN2 and from the
mini-CLN2 reporter gene. Expression of the lower
mini-CLN2 band was not detected when cells were transformed with the empty vector plasmid and was enhanced by
UASCLN2 function.
We then tested whether this reporter system could be used to analyze
the function of heterologous promoters. The core promoter of the
TUB2 gene, including 100 bp of the coding region, was fused to the mini-CLN2 gene (TUB2/80) and this
construct was assayed for transcription in yeast cells (Fig. 4C). The
band derived from TUB2/80 migrated at the expected size
relative to the UASCLN2+CLN2 signal. Like the CLN2 core promoter, the TUB2
core promoter was also augmented by UASCLN2
(UASCLN2+TUB2/80). These results
indicate that the mini-CLN2 hybrid gene reporter system is a
useful and convenient tool for analysis of heterologous promoter
function, especially in our yTAF145 mutants.
Impaired transcription of reporter constructs driven by
TUB2 promoter.
Next, we wished to test whether
gene-specific transcriptional defects could be reproduced in a plasmid
background. We compared the functions of CLN2 and
TUB2 promoters in wild-type and our mutant strains by using
the mini-CLN2 hybrid gene reporter system. As expected, the
CLN2 promoter
(UASCLN2+CLN) mediated efficient
transcription under all conditions (Fig. 4D). In contrast, the
TUB2 promoter (TUB2/829) containing 829 bp
upstream of the initiation site (Fig. 4A) (30) did not
function normally in N568 or T657K mutants under nonpermissive
conditions (Fig. 4E). Transcription of the endogenous CLN2
gene was normal in both of these mutants. These results demonstrate
that the mini-CLN2 reporter system can produce gene-specific
transcriptional defects originally observed in the chromosomal context.
We therefore employed this system to delineate the sequences that
confer yTAF145 dependency on the TUB2 promoter in N568
and T657K mutants.
129 and
TUB2/80, that contained different portions of the
TUB2 promoter (Fig. 4A). The TUB2/129 insert
contains the binding site for ABF1, which is important for constitutive
expression of TUB2 (30), whereas the
TUB2/80 insert lacks this site. Transcription of both of
these plasmids was similar to that of TUB2/829 (Fig. 4G
and 5B). It therefore appears that the core promoter, rather than UAS,
of the TUB2 gene renders its expression yTAF145 dependent in
our mutants, as was reported for the CLN2 promoter in the
ts2 mutant (84).
Activators do not overcome transcriptional defects of
TUB2 promoter in yTAF145 mutants.
The
results presented above indicate that the TUB2 core promoter
is not recognized in the normal fashion by TFIID containing N568 or
T657K mutant subunits. Furthermore, ABF1, a transcription factor that
regulates TUB2 gene expression in vivo (30), does not overcome such transcriptional defects, since transcription of
TUB2/829, TUB2/129, and TUB2/80
reporter plasmids were comparably damaged (Fig. 4; also data not
shown). However, it has been demonstrated that activator function
depends on core promoter structures; for example, GAL4-VP16 requires
the TATA element much more strongly than GAL4-Sp1 (22). In
addition, there are several classes of activator which target different
basal factors so as to stimulate different steps in transcription
(6). We therefore tested a number of different activators
for their ability to overcome the transcriptional defects in our
mutants (Fig.
5). To
measure the activator effects in the same background, we fused each
activation domain (ABF1 [52], GAL4
[60], GCN4 [19], ADR1
[45], EBNA2 [15], VP16
[77], and yTAND1 [47]) to the GAL4
DNA binding domain, which binds to the multiple recognition sites
linked to the TUB2 or CYC1 promoter in the
mini-CLN2 reporter plasmids
(UASGAL+TUB2/80 and
UASGAL+CYC1/174, respectively, in
Fig. 5A). In this experiment,
UASGAL+CYC1/174 functions as a
control, since transcription driven by the CYC1 promoter was
not affected in our mutants (Fig. 5C). We introduced effector and
reporter plasmids together into wild-type or mutant strains and
measured transcription of the mini-CLN2 gene under
permissive and nonpermissive conditions (Fig. 5B and C). GAL4 and VP16
activated both promoters strongly, while the other activators displayed
some preference for one promoter over the other. For instance, ABF1
activated the TUB2 promoter more strongly than the
CYC1 promoter, but yTAND1 activated the CYC1
promoter more strongly than the TUB2 promoter. As expected,
transcription driven by the CYC1 promoter was not affected
by yTAF145 mutations under any conditions. However, all of
the activators we tested failed to support normal levels of activated
transcription from the TUB2 promoter at 37°C in N568 and T657K mutants (Fig. 5B). These results indicate that
transcriptional activators cannot overcome the transcriptional defects
in our mutants. Notably, TADIV of ADR1 activated transcription from the CYC1 promoter even under nonpermissive conditions in our
mutants (Fig. 5C). In contrast, ADR1 TADIV displayed more than fourfold reduction in activation of transcription driven by the GAL1
promoter, even under permissive conditions, in the ts1 mutant
(45). It has yet to be determined whether this discrepancy
is due to the different core promoters (i.e., CYC1 versus
GAL1) or directly caused by the yTAF145
mutations.
|
TATA element restores impaired transcription driven by TUB2 core promoter. Previous double-shutoff experiments showed that, following in vivo depletion of yTAF145 protein, the TRP3 and HIS3 (+1) promoters ceased transcription much faster than the DED1 and HIS3 (+13) promoters (63). Since canonical TATA elements were found in the latter but not in the former promoters, it was suggested that a nonconsensus TATA element may be the determinant of yTAF145 dependency (63). However, more recent experiments delineating the region that confers yTAF145 dependency on the RPS5 promoter revealed that it overlaps with but does not consist entirely of the nonconsensus TATA element (84).
We examined the transcription levels of several endogenous genes in our mutants (Fig. 2B). While not all of these promoters are well characterized, we noticed a tendency for the affected genes to be driven by promoters lacking a canonical TATA element. For instance, the ADH1 (94), PGK1 (70), DED1 (87), and CLN2 (89) promoters, all of which contain canonical TATA elements, were much less affected by the yTAF145 mutations than the TUB2 (30), RPS30 (4), and RPS5 (85) promoters, which lack TATA elements. The ACT1 promoter was an exception, being somewhat affected even though it lacks the consensus TATA element (59); this is probably because ACT1 mRNA is quite stable, having a half-life greater than 25 min (32). To verify directly whether the nonconsensus TATA element was a major determinant of yTAF145 dependency in our mutants, we created a canonical TATA element (TATAAA) at a position55 bp from
the initiation site of the TUB2 promoter in the
mini-CLN2 reporter plasmid
UASGAL+TUB2/80. The modified
reporter plasmid was introduced into wild-type and mutant yeast strains and tested for basal and activated transcription under permissive and
nonpermissive conditions (Fig. 6A). The
canonical TATA element partially but reproducibly restored
transcription driven by the TUB2 promoter in N568 and
T657K mutants at 37°C. Importantly, a nonspecific GAGA sequence
similarly created at the same position did not restore the impaired
transcription (Fig. 6B), indicating that the rescue effect was sequence
specific. It appears that strong interaction between TBP and a
canonical TATA element can partially compensate for the impaired
function of yTAF145 protein in our mutants. This is yet another respect
in which our mutants differ from the ts2 mutant (84).
|
| |
DISCUSSION |
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|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Novel conditional alleles, N568 and
T657K, confer similar phenotypes.
In this study, we
isolated three novel conditional alleles (Y570N,
N568, and T657K) of the yTAF145
gene. Several lines of evidence from analysis of transcription levels
and growth rates suggested that N568 and T657K were more defective
than Y570N (Fig. 1, 2, 4, and 5). The N568 and T657K mutants showed
reduced growth rates even at 30°C and almost ceased to grow at
33°C, while the Y570N mutant grew well under the same conditions.
More detailed analysis revealed that T657K causes a slightly stronger
TS phenotype than N568 (data not shown).
and T657K mutants displayed nearly the same transcriptional
defects in our analyses (Fig. 2, 4, 5, and 6), suggesting that both
mutations affect the same function of yTAF145. It has been clearly
demonstrated that yTAF145 and its orthologs (e.g., dTAF230/250,
hTAF250) carry out multiple functions, including TAND activity which
negatively regulates TBP function (3, 41, 47), HAT activity
(62), and serine/threonine kinase activity which
autophosphorylates and transphosphorylates the large subunit of TFIIF
(18). Only the HAT domain (62) overlaps with the mutation sites (Fig. 1), but it has yet to be determined whether our
mutations affect HAT activity or other, as-yet-unknown functions. A
dramatic loss of transcription caused by the yTAF17 or
yTAF60 mutation was observed only when the conditional
alleles were "tight" (61). Weaker or leaky alleles had
much weaker effects on transcription even if they exhibited tight TS
phenotypes on agar plates (61). Thus, Y570N might
simply be a weaker allele that is slightly impaired in the same
function as the two tighter alleles N568 and
T657K. Alternatively, Y570N and N568/T657K may be
impaired in different yTAF145 functions. More extensive studies such as
examination of genome-wide gene expression profiles will be required to
clarify this point.
The novel conditional alleles reported here differ from ts1 and
ts2.
Our conditional alleles, especially N568 and
T657K, differ from previously reported yTAF145
alleles (98) in several respects. First, Y570N, N568, and
T657K mutant proteins are stably expressed for at least several hours
after the shift to the restrictive temperature, and the structure of
TFIID appears not to be strongly affected by our yTAF145 mutations
(Fig. 2). In contrast, ts1 and ts2 mutant proteins were rapidly
degraded within 1 h after the temperature shift and appeared to
induce loss of some other TAFs (98), so that mutant TFIID
containing ts1 or ts2 subunits would be expected to be present at very
low levels, if at all, at the restrictive temperature.
T657K
and ts2 mutations impair the transcription of an overlappingly
(ADH1, RPS5, RPS30, and
CLB5) but not identical (CLN2) set of genes.
Third, another remarkable difference between the two sets of mutants is
in the TATA dependency of transcription (Fig. 6). A previous study
demonstrated that yTAF145 dependency was conferred on the
CLN2, RPS5, and RPS30 genes by the
core promoter sequence (84). Interestingly, the region
downstream of the initiation site was not required for
yTAF145-dependent transcription of the CLN2 gene
(84). Furthermore, the determinant of yTAF145 dependency in
the RPS5 gene was mapped to the region surrounding the
nonconsensus TATA element (TAAAAT) but not the TAAAAT
sequence itself. In our mutants, like ts2, the region of the
TUB2 promoter responsible for yTAF145 dependency mapped to
the core promoter rather than UAS (Fig. 4). Remarkably, however, the
TUB2 core promoter could be converted into a
yTAF145-independent (or less-dependent) promoter by creating a
consensus TATA element upstream of the initiation site. It therefore
appears that the absence of a canonical TATA sequence is one of the
most important determinants of yTAF145 dependence in our mutants.
However, further analysis of a range of core promoters would be
essential to generalize such assumptions.
Function of yTAF145 protein in vivo. There is still some controversy over the in vivo function of TAFs (reviewed in reference 29). At present, two classes of TAFs are recognized: TFIID-specific and -nonspecific TAFs, the latter being common to TFIID and SAGA (or homologous complexes, such as mammalian TFTC, PCAF complex, and STAGA) (reviewed in reference 88). Interestingly, it seems that the latter class of TAFs are more generally required for transcription than the former class. For instance, genome-wide transcription analysis showed that 67% of yeast genes displayed a significant dependence on yTAF17, whereas only 16% were dependent on yTAF145, as judged by comparing kinetics of total mRNA reduction for 45 min after temperature shift with that of the rpb1-1 mutant (34). There are several possible explanations for such biases in TAF requirements (reviewed in reference 29), of which the most likely is that TFIID and SAGA function redundantly in transcription. Specific malfunctions of TFIID (e.g., yTAF145 or yTAF67 mutations) (34, 61) or SAGA (e.g., GCN5 or SPT20 mutations) (29, 34) are much less detrimental to general transcription than disorders simultaneously affecting both TFIID and SAGA (e.g., yTAF61, yTAF60, yTAF17, or yTAF25 mutations) (1, 34, 61, 64, 67, 80). Indeed, such functional redundancy has been demonstrated between mammalian TFIID and TFTC, at least in in vitro transcription experiments (102). If this were the case, mutations of the TFIID-specific factor yTAF145 would be expected to abrogate TFIID-specific functions. Alternatively, the difference in TAF requirements for general transcription might be simply due to the difference of taf alleles that were tested, since TFIID-specific yTAF40 inactivation results in TFIID depletion (SAGA remains largely unaffected) and a rapid loss of PolII-driven transcription (46). However, it is still possible that yTAF40 might be shared by TFIID and other unknown and redundant transcription factor complex besides SAGA. In any case, it should be emphasized that without knowing the real kinetics of loss of TAF function in TFIID following the temperature shift, we cannot make any accurate interpretations regarding the requirement for TFIID function for transcription of genes where no effect was seen.
Although the intrinsic function of TFIID is not yet entirely revealed, accumulating in vitro evidence indicates that TFIID is involved in transcriptional activation and the recognition of core promoter elements, such as the TATA box, initiator sequences, and downstream promoter elements (reviewed in references 10 and 11). A limited number of in vivo experiments using conditional TAF knockout strains suggest that common TAFs are involved in activation (1, 67), whereas TFIID-specific TAFs are involved in both activation (45) and core promoter recognition (Fig. 4) (84). More extensive in vivo studies may clarify whether common TAFs are also involved in both aspects of TFIID function. In any case, it is clear that TFIID is widely but not universally required for activation as well as core promoter recognition in vivo. Different yTAF145 conditional alleles have been shown to have different effects on transcription. ts1 mutants (98) were shown to be impaired in activation by TADIV of ADR1 (45), whereas ts2 mutants (98) showed a defect in recognition of the core promoter of the CLN2 gene (84). N568 and T657K mutants responded to TADIV of ADR1
and recognized the CLN2 promoter normally but failed to
transcribe genes under the control of the TUB2 promoter unless a canonical TATA element was provided near the initiation site.
It has yet to be determined whether these apparent differences are due
to the mutation site per se or to the specific core promoter and
activation domains tested.
TATA-dependent transcription in our mutants.
Recent
experiments using DNA cross-linking-immunoprecipitation assays have
shown that TBP binding to the promoter is stringently controlled in
vivo and stimulated by concerted action of activators and RNA
polymerase II holoenzyme (50, 53). Interestingly, TBP
binding to the RPS5 promoter was specifically compromised in
the ts1 mutant, suggesting that yTAF145 facilitates TBP binding in a
promoter-specific manner (53). Given that the canonical TATA
element failed to restore transcription driven by the RPS5 promoter in the ts2 mutant, it is likely that TBP alone cannot bind to
the TATA element in vivo without the aid of yTAF145 and/or other TAFs
that were codegraded under nonpermissive conditions (98).
Consistent with this idea is the observation that mutations of TBP
which removed most of the TAFs from TFIID also produced promoter-specific transcriptional defects (75). In N568
and T657K mutants, transcriptional defects were restored by creating a
canonical TATA element (Fig. 6). It therefore appears that TBP can be
positioned properly on a canonical TATA element even by yTAF145 mutant
proteins. In other words, the molecular defects in our mutants are
apparently confined to the yTAF145 function that supports TBP function
on TATA-less promoters. In vivo DNA cross-linking-immunoprecipitation
analysis will help to clarify this point.
| |
ACKNOWLEDGMENTS |
|---|
We thank A. G. Hinnebusch, K. Kasahara, and Y. Nakatani for helpful discussions and critical reading of the manuscript. We also thank A. Kobayashi for yTAF61 antibodies and plasmids, T. Kotani and K. Kasahara for plasmids, and Richard A. Young for RPB1 and rpb1-1 yeast strains.
This study was supported by grants from the Ministry of Education, Science, and Culture of Japan, the CREST Japan Science and Technology Corporation, the Uehara Memorial Foundation, the Asahi Glass Foundation, and the NOVARTIS Foundation (Japan) for the Promotion of Science.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Division of Gene Function in Animals, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0101, Japan. Phone: 81-743-72-5531. Fax: 81-743-72-5539. E-mail: kokubo{at}bs.aist-nara.ac.jp.
Present address: Department of Biochemistry and Molecular Genetics,
Health Sciences Center, University of Virginia, Charlottesville, VA 22908.
| |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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