Institut de Génétique et de
Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, Illkirch
Cédex, C.U. de Strasbourg, France,1
and Department of Molecular Physiology and Biophysics,
Vanderbilt University School of Medicine, Nashville, Tennessee
372322
Received 25 August 2000/Returned for modification 30 September
2000/Accepted 7 December 2000
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INTRODUCTION |
Transcription factor TFIID,
one of the general factors required for accurate and regulated
initiation by RNA polymerase II, comprises the TATA binding protein and
TATA binding protein-associated factors (TAFIIs)
(4, 15). The cDNAs encoding many human
TAFIIs (hTAFIIs) have been
isolated, revealing a striking sequence conservation with yeast
TAFIIs (yTAFIIs) and
Drosophila TAFIIs
(dTAFIIs). A subset of
TAFIIs are present not only in TFIID but also in
the SAGA, PCAF, STAGA, and TFTC complexes (7, 13, 23, 27, 36).
Genetic studies with yeast have shown that TAFIIs
play an important role in transcriptional regulation of many genes
(14). Temperature-sensitive mutations in
yTAFII145 and
yTAFII90 result in cell cycle arrest and
lethality, but the expression of only a small number of genes is
affected (3, 35). In contrast, tight temperature-sensitive
mutations in yTAFII17,
yTAFII25, yTAFII60, and
yTAFII61/68, which are present in the TFIID and
SAGA complexes, or in the TFIID-specific yTAFII40
have a more dramatic effect, the transcription of the majority of yeast
genes being affected (2, 21, 24-26, 29).
Initial sequence alignments indicated that
hTAFII80 (corresponding to
dTAFII60 and
yTAFII60), hTAFII31
(dTAFII40 and yTAFII17), and hTAFII20 (dTAFII30
and yTAFII61/68) presented obvious sequence similarity to histones H4, H3, and H2B, respectively (17,
20). Structural studies show that
dTAFII60 and dTAFII40
interact via a histone fold and form an H3-H4-like heterotetramer
(37). These findings, together with biochemical
experiments and genetic interaction data obtained with yeasts,
led to the proposal that TFIID and the other
TAFII-containing complexes contain a histone
octamer-like substructure composed of an
hTAFII80-hTAFII31
heterotetramer and two hTAFII20 homodimers
(8, 16).
Subsequent data show this model to be an oversimplification.
hTAFII28 and hTAFII18 are
also histone-like, since they interact via a histone fold domain (HFD)
to form a heterodimer (5). The SAGA, PCAF, TFTC,
and STAGA component SPT3 shows extensive sequence homology to
the HFDs of both hTAFII18 and
hTAFII28 in its N- and C-terminal regions,
respectively, and could potentially form a histone-like pair by
intramolecular interactions. Contrary to what was first suggested,
hTAFII20 does not homodimerize but rather
heterodimerizes with hTAFII135
(10). In yeasts, the hTAFII20 homologue yTAFII68 heterodimerizes with the SAGA
component yADA1, and it has been suggested that
yTAFII68 may also heterodimerize with
yTAFII48, a potential homologue of
hTAFII135, in yTFIID (28, 30). These
results indicate that there are many more histone-like pairs in TFIID
and SAGA than originally suspected. Recent electron microscopy studies
show that TFIID comprises three or four lobes arranged in a horseshoe
fashion around a central groove (1, 6). Within the present
limits of resolution it appears that no single lobe of TFIID would be
big enough to harbor all the known histone fold
TAFIIs, suggesting that they are shared among two
or more of the lobes.
We previously reported that hTAFII135 contained
an HFD with significant sequence homology to the SAGA component
yADA1 (10). Now we show that the
hTAFII135 HFD also shares significant
sequence homology with an HFD in yTAFII47
(34). In complementation experiments, the
yTAFII47 HFD is necessary and sufficient
for vegetative yeast growth. The temperature-sensitive phenotype of a
mutation in the yTAFII47 HFD can be rescued by
overexpression of yTAFII25 and, at less
restrictive temperatures, by yTAFII60,
yTAFII40, and yTAFII19. In
yeast two-hybrid and bacterial coexpression experiments, the yTAFII47 HFD mediates selective
heterodimerization with the conserved core domain of
yTAFII25. There are therefore both genetic and physical interactions between these two yTAFIIs,
suggesting that they form an additional histone-like pair in yTFIID.
Consistent with this idea, we show the conserved core domain of
yTAFII25 to be an HFD which shares homology with
the HFD of the hTAFII28 family.
Recently, yTAFII65 was identified as a novel
component of yTFIID (30). Now we demonstrate that
yTAFII65 contains an HFD with homology to that of
yTAFII17, dTAFII40, and
hTAFII31. In contrast to the HFD of
yTAFII47, the yTAFII65 HFD
is not sufficient for growth. Nevertheless, deletion or mutation of
this domain results in temperature sensitivity, showing that it is
important for yTAFII65 function. Surprisingly,
the yTAFII65 HFD also selectively heterodimerizes
with yTAFII25, which thus has two
heterodimerization partners in TFIID.
Finally, as yTAFII47 and
yTAFII65 are not present in SAGA, we sought a
heterodimerization partner for yTAFII25 in this
complex. Our results indicate that ySPT7 contains an HFD with homology to that of yTAFII47. This domain mediates
selective heterodimerization with yTAFII25.
Together our results reveal the existence of novel histone-like pairs
in the TFIID and SAGA complexes. They highlight the important
functional and structural role played by this motif in these complexes
and provide evidence that the histone-like yTAFIIs assemble into at least two distinct
substructures within TFIID.
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MATERIALS AND METHODS |
Yeast strains.
The yeast strains used in this study
are YSLS67 (Mata ura3-1
leu2-3,112
trp1-1
his3-11,15
ade2-11 can1-100
taf47
hisg-hisg [pRS416-TAF47]), YSLS67/47
(MATa ura3-1
leu2-3,112
trp1-1 his3-11,15
ade2-11 can1-100
taf47hisg-hisg [pAS3-TAF47)],
YSLS67/47HFD (MATa ura3-1
leu2-3,112
trp1-1
his3-11,15
ade2-11 can1-100 taf47hisg-hisg
[pAS3-TAF47(1-81)]), YSLS67/47m1 (MATa ura3-1
leu2-3,112
trp1-1
his3-11,15
ade2-11 can1-100
taf47hisg-hisg [pAS3-TAF47(R13D,
I14E)]), YSLS67/VP16AD47HFD (MATa
ura3-1 leu2-3,112
trp1-1
his3-11,15
ade2-11 can1-100
taf47hisg-hisg [pASV3-TAF47(1-81)]), YSLS58 (MATa
ura30 leu20
his31 lys20
MET15 KAN taf65 [pRS416-TAF65]), YSLS58/65
(MATa ura30
leu20 his31
lys20 MET15 KAN taf65
[pAS3-TAF65]), YSLS58/65HFD (MATa
ura30 leu20
his31 lys20 MET15 KAN
taf65 [pAS3-TAF65(103-510)]), YSLS58/65m1
(MATa ura30
leu20 his31
lys20 MET15 KAN taf65
[pAS3-TAF65(L64P, L67P)]), YSLS58/VP16AD65
(MATa ura30
leu20 his31
lys20 MET15 KAN taf65
[pASV3-TAF65]), and L40 [MATa
trp1-901
leu2-3,112
his3-200 ade2
LYS2::(LexAop)4-HIS3 URA3::(LexAop)8-Lac].
Construction of recombinant plasmids.
All yeast and
bacterial expression vectors were constructed by PCR using primers with
the appropriate restriction sites, and constructs were verified by
automated DNA sequencing. Details of constructions are available on
request. LexA fusions were constructed in the multicopy vector pBTM116
containing the TRP1 marker, and the VP16 fusions were
constructed in the multicopy vector pASV3 containing the
LEU2 marker (10). For complementation,
wild-type or mutated yTAFIIs were cloned in the
multicopy pAS3 plasmid with a LEU
marker.
Two-hybrid, complementation, and high-copy-number
temperature-sensitive suppression assays.
All yeast strains were
transformed by the lithium acetate technique. For two-hybrid
assays, transformants were selected on Trp
Leu plates. Quantitative 
-galactosidase
assays on individual L40 transformants were determined as previously
described (10). Reproducible results were obtained in
several independent experiments, and the results of a typical
experiment are shown in the figures. Yeast strains YSLS67 and YSLS58,
used for plasmid shuffling of TAF47 and TAF65,
were derived from YJR10 (34) and YSLS41 (30) by sporulation and tetrad dissection. For complementation assays, the
rescue plasmids indicated in the relevant figures were transformed and
the wild-type TAF/URA3 plasmid was shuffled out by two
passes on media containing 5-fluoroorotic acid. For suppression of the yTAFII47(R13D, I14E) mutant strain, cells were
transformed with high-copy-number plasmids with a URA3
marker expressing the indicated yTAFIIs and
serial dilutions of the transformants were spotted at 30, 34, and
36°C. Plates at the restrictive temperatures were photographed after
3 days of growth. In all experiments cultures were grown in yeast
extract-peptone-dextrose unless selection was necessary, in
which case all cultures were grown in the appropriate selective
synthetic dextrose (SD) medium.
Coexpression in Escherichia coli.
Coexpression in E. coli was performed as previously
described (9a, 10). All plasmids were constructed by PCR,
and details are available on request. The
yTAFII47, yTAFII65, and
SPT7 histone fold regions were expressed as glutathione
S-transferase (GST) fusion proteins in pGEX2T. Native
untagged, yTAFII25,
hTAFII30, and
yTAFII68 HFDs were expressed from a
modified version of the vector pACYC184 (New England
Biolabs). Plasmids pairs were introduced into E. coli strain
BL21(DE3), and double transformants were selected on plates containing
ampicillin and chloramphenicol. Bacteria were amplified to an optical
density at 600 nm of 0.45 and induced for 4 h at 25°C with 1 mM
isopropyl--D-thiogalactopyranoside (IPTG).
Cell were lysed by sonication in buffer (25 mM Tris-HCl [pH 6.0] and
0.4 M NaCl), and the soluble fraction was collected after
centrifugation at 14,000 rpm for 20 min at 4°C in an Eppendorf centrifuge. Aliquots of the soluble fraction from a 10-ml bacterial culture were then incubated with glutathione-Sepharose (Pharmacia). Binding and washing were done essentially as described previously (10). Bound proteins were analyzed by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis and staining with Coomassie
brilliant blue.
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RESULTS |
yTAFII47 contains an HFD which suffices for vegetative
growth.
Previously we reported that
hTAFII135 amino acids 870 to 944 showed
significant sequence homology to H2A, NC2 and yADA1
(10) (Fig. 1). The
hTAFII135 HFD also shows significant homology to yTAFII48, the hTAFII135
homologue in yTFIID (28, 30) (Fig. 1). Database searches
also revealed a significant similarity between the HFD of the
TAFII135 family and the N-terminal region of
yTAFII47 (Fig. 1). In the
yTAFII47 HFD, the conserved RI(V, M) residues are
found in the 1 helix, followed by an amphipathic 2 helix with
numerous conserved hydrophobic residues. The 3 helix is characterized by the presence of D(V, I, L) residues. This pattern of
sequence conservation is analogous to that seen amongst other histone
fold proteins (5, 10, 32). Several metazoan sequences encoding potential proteins with homology to the
yTAFII47 HFD were also detected in these searches
(Fig. 1) (32). This observation indicates that
yTAFII47 is a histone-like protein belonging to an evolutionarily conserved family. In addition to the 1, 2, and
3 helices, which comprise the minimal HFD, there is a potential additional C helix in this family.
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FIG. 1.
Alignment of the HFD sequences of the
hTAFII135 family with those of ADA1 and the
yTAFII47 family. h, human; y, Saccharomyces
cerevisiae; d, Drosophila melanogaster; S.pombe,
Schizosaccharomyces pombe; Anopheles, Anopheles
gambiae; mouse, Mus musculus; Candida,
Candida albicans; zebra fish, Danio
rerio. The positions of the predicted helices and loops are
indicated above the sequence based on homology with H2A (10,
22). Positions with conserved, mainly hydrophobic, amino acids
are in white on a black background. Other residues conserved within the
yTAFII47 family are boxed in gray. Amino acids were
classified as follows: small residues, P, A, G, S, and T; hydrophobic
residues, L, I, V, A, F, M, C, Y, and W; polar and/or acidic
residues, D, E, Q, and N; basic residues, R, K, and H. Threonine
residues are occasionally present in otherwise hydrophobic positions.
The amino acids sequences shown without numbers are predicted from
genomic, expressed sequence tag, or sequence tagged site sequences. The
accession numbers for the indicated sequences are as follows: S.
pombe, SPT:CAB90151; Candida, 396380B03;
Anopheles, GB CN501GI9 AL143170; zebra fish, GB
AW343321fi76b06.y1; mouse, GB AA692266ur52c07.
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To determine whether the yTAFII47 HFD is
important for function, we performed complementation experiments by
plasmid shuffle in a yTAFII47 null strain.
Expression vectors for wild-type or mutated
yTAFII47 proteins were constructed (Fig.
2A), and their ability to rescue growth
of the null strain was evaluated. As previously described,
yTAFII47 is essential for vegetative yeast growth (34). Expression of full-length
yTAFII47 efficiently restored the
growth of the null strain at 30°C, whereas no growth was seen
with the expression vector alone (1 and 3 in Fig. 2B). Growth was also
rescued by yTAFII47(1-81) containing only the minimal HFD (construct 4 in Fig. 2B). These two strains showed comparable growth rates in liquid culture (data not shown). This 1-81
domain rescued growth both when expressed as a native protein and when
expressed as a fusion with the VP16 activating domain from a two-hybrid
expression vector (construct 5 in Fig. 2B). Mutation of the highly
conserved amino acids R13 and I14 in the 1 helix (construct m1 in
Fig. 2A) abolished the ability of yTAFII47(1-81) to rescue growth (construct 6 in Fig. 2B). In contrast, this mutation did not abolish yTAFII47 function in the context
of the full-length protein (construct 2 in Fig. 2B). The (1-353)m1
mutant did, however, show a temperature-sensitive phenotype, as it did
not rescue growth at 37°C while both the wild-type protein and the
1-81 deletion rescued growth at this temperature (compare constructs 1 and 3 in Fig. 2C). The VP16-TAFII47(1-81) fusion
also showed a temperature-sensitive phenotype (construct 4 in Fig. 2C).
These results indicate that the yTAFII47 HFD is
an essential functional domain necessary and sufficient for vegetative
yeast growth.
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FIG. 2.
The yTAFII47 HFD is sufficient for growth.
(A) The sequence of the yTAFII47 HFD is shown along with
that of mutant m1. The locations of the potential helices and loops
are indicated above the sequence. The ability of each mutant to
complement the null strain is indicated on the right. TS, temperature
sensitive. (B and C) Growth of yeasts plated at the indicated
temperatures. 5-FOA, 5-fluoroorotic acid.
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Genetic interaction between yTAFII47 and other
histone-like yTAFIIs.
Possible genetic interactions
between yTAFII47 and other
yTAFIIs were examined. To look for such
interactions, we tested the ability of other
yTAFIIs to rescue the temperature-sensitive
phenotype of the yTAFII47(1-353)m1 allele when
overexpressed at the nonpermissive temperature.
The yTAFII47(1-353)m1 strain did not grow
at 34°C (Fig. 3). At 34°C, the
temperature-sensitive phenotype was efficiently suppressed by
overexpression of yTAFII25 and partially
suppressed by overexpression of yTAFII60,
yTAFII40, and
yTAFII19 (Fig. 3). At 36°C, however, only
yTAFII25 could suppress the temperature-sensitive
phenotype (Fig. 3). No significant growth was seen when any of the
other yTAFIIs were overexpressed at either
temperature (Fig. 3), while all strains showed equivalent growth at
30°C (data not shown). Analogous results were obtained with the
yTAFII47(1-81)-VP16 allele (data not shown).
These results show a genetic interaction between yTAFII47 and three other known histone-like
yTAFIIs, yet the strongest interaction was
observed with yTAFII25, which has not
previously been described as histone-like.
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FIG. 3.
Genetic interactions among histone-like
yTAFIIs. The growth of serial dilutions of strains with the
yTAFII47(1-353)m1 allele at 34 and 36°C is shown. The
overexpressed yTAFIIs used to rescue the growth at the
nonpermissive temperatures are shown on the left.
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The HFD of yTAFII47 mediates heterodimerization with
yTAFII25.
The strong genetic interaction between
yTAFII47 and yTAFII25
prompted us to determine whether they also interact physically. The
yTAFII47 HFD was fused to the VP16 activation
domain or the LexA DNA binding domain and tested for interactions with
LexA or VP16 fusions of the core domain of
yTAFII25 and the HFDs of other
yTAFIIs in a series of yeast two-hybrid
experiments. Interactions were assessed by measuring -galactosidase
activity in strain L40, which harbors a LexA-responsive LacZ gene
(10, 33).
In two-hybrid assays, a strong interaction between
yTAFII47 and
yTAFII25(74-206) was observed (Fig.
4A, column 1). This
interaction required only the HFD of
yTAFII47(1-81) and was abolished by mutation m1
in the 1 helix (Fig. 4A, columns 2 and 3). In the context of the
full-length yTAFII47, the m1 mutation reduced but
did not abolish the interaction (Fig. 4A, column 4). In contrast, we
detected no interactions between the HFD of
yTAFII47 and the HFDs of
yTAFII40 or yTAFII19 (which
themselves strongly interact in two-hybrid assays [data not shown]),
yTAFII60, yTAFII68,
yTAFII48, and yTAFII65 (summarized in Table 1). Moreover, we did
not observe homodimerization of yTAFII47.
Similarly, with the exception of yTAFII65 (see
below), yTAFII25 did not interact with the HFDs
of the other yTAFIIs tested (Fig. 4A, columns 5 to 7, and Table 1), although a possible homodimerization was seen
(Table 1; also, see Discussion). These results indicate that
yTAFII47 selectively heterodimerizes with
yTAFII25.
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FIG. 4.
Selective heterodimerization between
yTAFII47 and yTAFII25 in two-hybrid assays. (A)
Quantification of -galactosidase activity in two-hybrid assays. The
VP16-yTAFII47, VP16-yTAFII40,
VP16-yTAFII19, and yTAFII48 fusions shown below
each column were assayed in a LexA-yTAFII25(74-206)
background as indicated above the graph. -gal, -galactosidase.
(B) Alignment of yTAFII25 and hTAFII30 with
members of the hTAFII28 family from yeast and D.
melanogaster. Conserved positions are in white against a black
background. The positions of the helices and loops of
hTAFII28 are indicated. Alignment of the 3 helix of
yTAFII25 and hTAFII30 with that of the other
indicated histone fold proteins is also shown. (C) The sequences of the
conserved region of yTAFII25 and hTAFII30 are
shown. Conserved amino acids are white on a black background. The end
points of the deletions tested in two-hybrid assays are indicated by
the arrows below the sequence. , internal deletion. (D) Mapping of
the yTAFII25 region required for interaction with
yTAFII47. The LexA-yTAFII25
(LexA-hTAFII30) deletions indicated below the graph were
assayed in the VP16-yTAFII47(1-81) strain.
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Sequence alignments have shown that hTAFII30,
yTAFII25, and their homologues from other species
have a bipartite structure with a highly conserved C-terminal domain
and an unconserved N-terminal region (12) (Fig. 4C). In
yTAFII25, it is the conserved C-terminal region
(amino acids 74 to 206) which mediates interaction with the
yTAFII47 HFD (Fig. 4A and D, column 2). We
therefore compared this region of yTAFII25 to the
other known histone-like TAFIIs. In doing this,
we noted a significant similarity between the sequences of
yTAFII25/hTAFII30 and the
HFD of the hTAFII28 family of proteins (Fig. 4B).
This similarity predicted the existence of potential N, 1, and
2 helices within yTAFII25 and
hTAFII30, the nonconserved sequence corresponding
to an insertion in the L2 loop of yTAFII25 (Fig.
4B). In contrast, the proposed 3 helix of
yTAFII25 contains the conserved D(V, I, L) pair
and shows better homology to several other known histone-like proteins
than to hTAFII28.
We next tested the effect of deleting various regions of the
yTAFII25 HFD on heterodimerization with
yTAFII47 (Fig. 4C). Deletion of the N and a
large part of the extended L2 loop
[yTAFII25(84-206)] had no effect on
interaction (Fig. 4D, column 3). However, deletion of the LN led to a
loss of interaction despite the fact that the proposed 1 remained in
this construct [yTAFII25(90-206)] (Fig. 4D,
column 4). At the C terminus, interaction with
yTAFII47 was not affected by deletion up to amino
acid 199, leaving intact the proposed 3 helix
[yTAFII25(74-199)] (Fig. 4D, column 5), whereas interaction was abolished when the 3 was truncated (74-186) (column 6). The conserved C-terminal domain of
hTAFII30 interacted with
yTAFII47 as efficiently as
yTAFII25
[hTAFII30(116-218) and yTAFII25(74-206)] (Fig. 4D, columns 1 and 2).
Together, these results indicate that the conserved C-terminal domain
of the yTAFII25 family contains an HFD which
heterodimerizes with yTAFII47.
To observe direct heterodimerization of yTAFII47
and yTAFII25, these proteins were coexpressed in
E. coli. A native version of
yTAFII25(74-206) or
hTAFII30(116-218) was coexpressed with a GST
fusion of the yTAFII47 HFD. When expressed alone,
the GST-yTAFII47 fusion is largely insoluble and
little soluble protein is recovered on the glutathione-Sepharose beads
(Fig. 5A, lane 1). In contrast, coexpression with the yTAFII25 or
hTAFII30 HFD solubilizes
GST-yTAFII47, which is retained in the form of a
complex with each of these proteins on the beads (Fig. 5A, lanes 2 and
3). At first, the GST-yTAFII47 chimera appears to
be more abundant than the untagged yTAFII25 or
hTAFII30 protein. However, the GST moiety of the
chimera stains strongly with Coomassie brilliant blue. When this
disproportionate staining is taken into account, the
yTAFII47-yTAFII25 ratio
would be closer to the 1:1 ratio expected for a heterodimeric complex. This is a selective heterodimerization, since neither solubilization nor complex formation was seen when
GST-yTAFII47(1-81) was coexpressed with the HFD
of yTAFII68 (lane 4), previously shown to
heterodimerize with yADA1 in this assay (10),
yTAFII40, or yTAFII19 (data
not shown). In control experiments, the native
yTAFII25(74-206) and hTAFII30(116-218) proteins were insoluble when
expressed alone and were not retained on glutathione beads (Fig. 5D,
lanes 4 and 5). These results confirm the direct heterodimerization of
the TFIID components yTAFII47 and
yTAFII25, indicating that they form a novel
histone-like pair.
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FIG. 5.
Coexpression of yTAFII47 and
yTAFII65 with yTAFII25 in E.
coli. (A) Bacteria were transformed to express the proteins
shown above each lane. Following extract preparation, the soluble
protein retained on glutathione-Sepharose beads was analyzed by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis and staining with
Coomassie brilliant blue. The locations of the GST-yTAFII
fusions and the retained yTAFII25 and
hTAFII30 proteins are indicated. (B)
yTAFII25-ySPT7 heterodimerization in E.
coli. The locations of the GST-ySPT7 fusion and
yTAFII25 are shown. (C) Heterodimerization with
yTAFII25 deletion mutants. The GST-yTAFII47,
GST-yTAFII65, and GST-SPT7 proteins were coexpressed with
the untagged yTAFII25 deletion mutants as indicated. The
soluble proteins retained on glutathione-Sepharose beads are shown. (D)
Lack of evidence for yTAFII25 and hTAFII30
homodimerization. The GST fusions of yTAFII25 and
hTAFII30 were expressed alone or in combination with the
native HFDs indicated above each lane as in panel A.
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Solubilization and complex formation were also observed when
GST-yTAFII47(1-81) was expressed with
yTAFII25(74-206) in which the extended L2
loop has been deleted (Fig. 5C, lane 1). A similar result was seen with
yTAFII25(84-199), whereas almost no
soluble complex was observed with
yTAFII25(90-206) (Fig. 5C, lanes 2 and 3). Thus, as observed in the two-hybrid experiments,
yTAFII47 and yTAFII25
heterodimerization does not require the extended L2 loop but does
require the LN region.
yTAFII65 contains a functional HFD which mediates
selective heterodimerization with yTAFII25.
The above
results reveal the presence of considerably more histone fold proteins
in TFIID than originally suspected. This prompted us to examined the
sequences of other yTAFIIs for the presence of
potential HFDs. Analysis of the novel yTFIID subunit yTAFII65 (30) indicates the presence
of a potential HFD with similarity to
yTAFII17/dTAFII40/hTAFII31
between amino acids 37 and 103 at the N terminus of the protein (Fig.
6A). To determine whether this is a
functional domain of the protein, we tested the ability of
yTAFII65 mutants to complement the growth of the yTAFII65 null strain.
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FIG. 6.
yTAFII65 contains a histone fold motif. The
sequence of yTAFII65 is aligned with the sequences of
members of the yTAFII17 family. The conserved positions are
white against a black background, and the positions of the helices
and loops are indicated. The sequences of the m1 and m2 mutants are
indicated below the wild-type sequence. (B) The structures of the
yTAFII65 mutants used in complementation experiments are
schematized. The HFD is depicted as a black box. TS, temperature
sensitive. (C and D) Growth of yeasts plated at the indicated
temperatures. 5-FOA, 5-fluoroorotic acid.
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Deletions or mutations in the yTAFII65 HFD were
generated (Fig. 6A and B) and used to complement the
yTAFII65 null strain. At 30°C, growth was seen
with the full-length protein, whereas no growth was seen when the null
strain was complemented with yTAFII65(1-140)
containing only the HFD (Fig. 6C, constructs 2 and 3). Surprisingly,
growth at 30°C was also seen using the deletion 103-510, in which
the HFD is deleted, and with mutant (1-510)m1, which contains a double
amino acid substitution in the 2 helix (Fig. 6C, constructs 4 and
5). These two strains were, however, temperature sensitive, since they
did not grow at 37°C, while growth was seen with the wild-type
protein (Fig. 6D, constructs 1 to 4). Therefore, in contrast to
yTAFII47, the yTAFII65 HFD is not sufficient for vegetative growth. The HFD is nevertheless an important functional domain at 37°C, since its deletion or mutation generates a temperature-sensitive phenotype.
We next tested the ability of yTAFII65(1-140) to
heterodimerize with the HFDs of other yTAFIIs
in the two-hybrid assay. Surprisingly, a selective interaction was seen
only with yTAFII25 (Table 1 and Fig.
7B, column 2). Interaction with
yTAFII25 was seen with full-length
yTAFII65 (Fig. 7B, column 1), and this
interaction was abolished by mutation m1, which also generated a
temperature-sensitive phenotype (Fig. 7A, column 4). Interaction with
the yTAFII65 HFD alone was
reduced compared to yTAFII65(1-510)
or (1-140) [yTAFII65(37-107) (Fig. 7A,
column 3). Nevertheless, interaction with
yTAFII25 was totally abolished when two
amino acid changes were introduced into the 1 and 2 helices of
the yTAFII65 HFD
[yTAFII65(37-103)m2 in Fig. 6A and 7B, column
5]. Interaction with yTAFII65 required the
conserved C-terminal domain of yTAFII25 that was
required for interaction with yTAFII47 (data not
shown). However, in contrast to yTAFII47,
yTAFII65 does not interact with
hTAFII30 (Fig. 7B, column 6).
View larger version (21K):
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[in a new window]
|
FIG. 7.
The histone fold of yTAFII65 is required for
heterodimerization with yTAFII25. (A) Structures of the
yTAFII65 deletion mutants used in the two-hybrid assays.
(B) The VP16-yTAFII65 deletions indicated below the graph
were assayed in the LexA-yTAFII25(74-206) strain. For
column 6, yTAFII65 was assayed in the
LexA-hTAFII30(116-218) strain. -gal,
-galactosidase.
|
|
As described above for yTAFII47, direct
yTAFII65-yTAFII25
heterodimerization was verified by coexpression in E. coli.
The yTAFII65(36-107) HFD was fused to GST and
expressed either alone or in combination with the HFD of
yTAFII25, hTAFII30, or
yTAFII68. As observed for
yTAFII47, the GST-yTAFII65
protein was essentially insoluble when expressed alone, while
solubilization and complex formation were observed when
GST-yTAFII65 was coexpressed with yTAFII25(74-206) (Fig. 5A, lanes 5 and
6). In agreement with the two-hybrid assay data,
heterodimerization was also observed with yTAFII25(74-206) and
yTAFII25(84-199) but was strongly reduced with (90-206) (Fig. 5C, lanes 4 to 6). Similarly, no complex was
formed with the hTAFII30 core domain (Fig. 5A,
lane 7), and in an additional control no heterodimerization was
seen with the yTAFII68 HFD (Fig. 5A, lane 8).
Therefore, while yTAFII47 can heterodimerize
with yTAFII25 or hTAFII30,
heterodimerization with yTAFII65 is selective for
yTAFII25 and is not seen with the closely related
hTAFII30. These results indicate that the TFIID
component yTAFII65 selectively and directly
heterodimerizes with yTAFII25 to form an
additional histone-like pair.
yTAFII25 heterodimerizes with the SAGA component
ySPT7.
The above results indicate that
yTAFII25 heterodimerizes with
yTAFII47 and yTAFII65.
yTAFII25 is present in TFIID and SAGA, yet both
yTAFII47 and yTAFII65 are
TFIID specific and are not present in SAGA (29, 30). We
therefore sought a potential heterodimerization partner for
yTAFII25 in the SAGA complex. Database searches
with the yTAFII47 HFD showed that ySPT7 contains
a potential HFD between amino acids 975 and 1051 with similarity to
that of yTAFII47 (Fig. 1) (32). This
observation prompted us to test the ability of the ySPT7 HFD to
interact with that of yTAFII25. In two-hybrid
assays, a strong interaction between ySPT7(964-1051) and
yTAFII25(74-206) was observed (Fig.
8A, column 6, and B, column 1). The
yTAFII25-ySPT7 two-hybrid interaction was
comparable to that seen with both yTAFII47 and
yTAFII65 (Fig. 8A, columns 1, 4, 5, and 6).
View larger version (30K):
[in this window]
[in a new window]
|
FIG. 8.
Heterodimerization of the SAGA components
yTAFII25 and ySPT7. (A) The VP16 fusions shown below each
column were transformed into the LexA-yTAFII25(74-206)
strain. (B) The LexA fusions shown below each column were transformed
into the VP16-SPT7(964-1051) strain. -gal, -galactosidase.
|
|
Interestingly, the ySPT7 HFD interacted not only with
yTAFII25(74-206) but also with
yTAFII25(90-206), which did not interact with
either yTAFII47 or yTAFII65
(Fig. 8B, columns 1 and 2). This indicates that the determinants of
yTAFII25 required for interaction with ySPT7 are
not exactly the same as those required for interaction with
yTAFII47 and yTAFII65. In
contrast, ySPT7 did not heterodimerize with
hTAFII30(116-218) or with the other SAGA
components yTAFII68 and ADA1, showing that
heterodimerization with yTAFII25 is highly specific (Fig. 8B, columns 3 to 5).
Heterodimerization between yTAFII25 and ySPT7 was
verified directly by coexpression in E. coli. The
GST-ySPT7(964-1051) fusion protein was insoluble when expressed alone,
whereas in the presence of yTAFII25(74-206) a
soluble heterodimeric complex was formed (Fig. 5B, lanes 1 and 2).
Therefore, the SAGA components yTAFII25 and
ySPT7 directly heterodimerize to form a histone-like pair. Consistent with the two-hybrid assay results, no complex was
formed with hTAFII30(116-218) or with
yTAFII68 (lanes 3 and 4). In contrast to what was
observed with yTAFII47 and
yTAFII65, complex formation with
yTAFII25(90-206) was as efficient as that
with yTAFII25(74-206) and
yTAFII25(84-199) (Fig. 5C, lanes 7 to 9).
This is in good agreement with the two-hybrid assay data and confirms
that there is a differential requirement for the LN loop in
heterodimerization with ySPT7 compared with
yTAFII47 and yTAFII65.
Potential homodimerization of yTAFII25.
It has
previously been reported that both yTAFII25 and
hTAFII30 can interact with themselves (18,
19). Indeed, in our two-hybrid experiments, we observed an
interaction between LexA and VP16 fusions of the
yTAFII25 HFD (Fig. 8A, column 7, and Table 1). We
therefore addressed the potential yTAFII25
homodimerization in the coexpression assay. In contrast to
GST-yTAFII47, GST-yTAFII65, and GST-ySPT7, both full-length yTAFII25 and the
yTAFII25 (hTAFII30) HFD GST
fusions were soluble when expressed alone (Fig. 5D, lanes 1 to 3).
However, when these GST fusions are coexpressed with the corresponding
native HFDs, the untagged HFDs are not retained on the GST-Sepharose
beads (Fig. 5D, lanes 6 to 8) as they are when expressed with
GST-yTAFII47, GST-yTAFII65,
or GST-ySPT7. Thus, while we readily observe heterodimerization using
this assay, we fail to detect homodimerization of
yTAFII25 and hTAFII30 via their HFDs.
| |
DISCUSSION |
Novel histone-like components in TFIID and SAGA.
We previously
reported that hTAFII135 and yADA1 contained HFDs
(10). Here we show that yTAFII47,
yTAFII25, and yTAFII65 have
significant sequence similarities with other
TAFIIs shown experimentally to contain histone
folds. These similarities are comparable to those described for other
histone-like proteins (9). Although definitive proof that
these are bone fide HFDs will require that their structures be
determined, the sequence similarities and the results of our
coexpression studies suggest that yTAFII47,
yTAFII65, yTAFII25, and
ySPT7 are potential histone-like proteins which heterodimerize to form
novel pairs in the TFIID and SAGA complexes. Thus, in total there are
nine histone-like yTAFIIs, and the known genetic
and physical interactions suggest that they are organized in at least
two substructures within TFIID.
Our database searches using the hTAFII135 HFD
showed the presence of a potential HFD in
yTAFII47. Further support for this comes
from Sullivan et al., who also identified
yTAFII47 as a histone-like protein using an
alternative algorithm (32). The positions of the helices, notably the 1 helix, proposed by these authors differ
significantly from that shown in Fig. 1. This is due mainly to the
absence of gaps in the loops in their alignments. Previous alignments
of the hTAFII135 sequence with that of H2A, whose
structure has been determined, favor the alignment shown in Fig. 1A.
Furthermore, mutation of the conserved RI pair in
yTAFII47 abolishes interaction with
yTAFII25 and the same mutation in
hTAFII135 abolishes interaction with
hTAFII20 (our unpublished data). Both
observations point to these amino acids being located in the 1
helix, as indicated in our alignments. A definitive assignment of the
precise 1 helix boundaries will require that the structure of this
molecule be determined. The presence of the highly conserved D(I, V, L)
pair allows the position of the 3 helix in each of the proteins
described here to be determined based on the alignment with histone
fold proteins of known structure.
Interestingly, these database searches reveal the existence of
potential metazoan homologues of yTAFII47. As the
metazoan homologues of yTAFII25
(hTAFII30 and dTAFII24/16)
(12, 18) are known TFIID components, it is only to be
expected that metazoan TFIID will contain a homologue of their
heterodimerization partner, yTAFII47. It will be
interesting to determine whether the human and mouse proteins revealed
in these database searches are TFIID components. These observations
reinforce the idea that the structure and function of TFIID have been
strongly conserved throughout evolution. Hence, it is likely that
metazoan TFIID also contains a homologue of
yTAFII65 and, as yTAFII25
and hTAFII30 are components of SAGA, TFTC, and
PCAF, we would expect these complexes to contain a homologue of ySPT7.
yTAFII47-yTAFII25, a novel histone-like
pair in yTFIID.
Our results show that the
yTAFII47 HFD is necessary and sufficient for the
function of this protein in vivo. The growth of the
yTAFII47 null strain can be complemented by
expression of the HFD alone, and its function is abolished by mutation
of the well-conserved RI pair in the 1 helix. Interestingly, this
same mutation is not lethal in the context of the full-length protein but rather results in a temperature-sensitive phenotype. One
interpretation of this result is that other regions of
yTAFII47 missing in the 1-81 mutant act to
stabilize the interactions of the mutant protein at permissive
temperatures. This is also suggested by the observation that
heterodimerization with yTAFII25 is abolished by
mutation m1 in yTAFII47 in the context of the
minimal HFD, yet in the context of the full-length
yTAFII47 mutation m1 does not abolish
heterodimerization. It is therefore likely that heterodimerization is
necessary for function in vivo. In addition to the three helices
which constitute the minimal HFD, C and N helices can often be
found. In the yTAFII47 family, a short region
downstream of the 3 helix is conserved, and computer algorithms
predict that this conserved sequence may form an helix. Therefore,
this C helix or another, as-yet-undefined domain in
yTAFII47 may act to stabilize interactions with
yTAFII25 or with other components of yTFIID, but
their function becomes evident only when the HFD is mutated.
Our results show that the temperature-sensitive phenotype of the
yTAFII47(1-353)m1 allele can be suppressed by
overexpression of several other histone-like
yTAFIIs. At 34°C, suppression is most efficient
with the direct heterodimerization partner
yTAFII25. A weaker suppression is seen with
yTAFII40 and yTAFII19,
suggesting that this pair makes close contact with the
yTAFII47-yTAFII25 pair in
native yTFIID. Overexpression of yTAFII60,
but not its heterodimeric partner yTAFII17, can
suppress the temperature-sensitive phenotype of this allele, suggesting
that this pair may also in some way contact the
yTAFII47-yTAFII25 pair. At
higher temperatures, however, only the direct heterodimerization
partner yTAFII25 can suppress the
temperature-sensitive phenotype of the
yTAFII47(1-353)m1 allele.
We also tested the ability of overexpressed
yTAFII65 to suppress the
TAFII47 temperature-sensitive mutation.
Interestingly, we found that even at permissive temperatures,
yTAFII65 overexpression was toxic in the
yTAFII47 temperature-sensitive background but not
in the yTAFII47 wild-type or other backgrounds.
One interpretation of this result is that at 30°C, the
TAFII47-TAFII25 interaction is already sufficiently weakened that when
yTAFII65 is overexpressed it competes with
yTAFII47(1-353)m1 and titrates
yTAFII25. Consequently, there is no longer
enough of the
TAFII47-TAFII25 heterodimer
for the yeast to survive, providing evidence that formation of the yTAFII47-yTAFII25
heterodimer is essential for viability.
In addition to a genetic interaction, our results show that the
yTAFII47 HFD interacts physically with the
conserved C-terminal domain of yTAFII25 (and
hTAFII30) both in yeast two-hybrid assays and by
coexpression in E. coli. The minimum
yTAFII25 region necessary for interaction with
yTAFII47 is located between amino acids 84 and
199. Comparison with the sequences of other families of histone-like TAFIIs revealed a similarity with the
hTAFII28 family in the 1-L1-2 region, but
the 3 helix of yTAFII25 shows higher homology
to those of other known histone fold proteins. Like
yTAFII40 and ySPT3,
yTAFII25 contains a large insertion in the L2 loop.
This alignment predicts the existence of a possible N helix in
yTAFII25 and hTAFII30. The
presence of an helix at this position is also predicted by
secondary structure computer algorithms (our unpublished data).
Deletion of this helix, however, does not lead to a loss of interaction
with yTAFII47 or yTAFII65.
Interestingly, the putative LN loop is highly conserved in the
yTAFII25/hTAFII30 family
(12), and its deletion results in a loss of interaction with yTAFII47 and yTAFII65,
but not with ySPT7. This strongly suggests that the LN region plays a
critical role in heterodimerization by making direct contacts with
yTAFII47 and yTAFII65 as is
seen in the
hTAFII28-hTAFII18 pair
(5), while in the yTAFII25-ySPT7 heterodimer this interaction either does not take place or is not
essential for complex formation.
Introduction of stop codons truncating the 2 helix abolishes
yTAFII25 function. Moreover, in a screen for
yTAFII25 temperature-sensitive mutants, several
alleles with substitutions at G101 were found (29). This
position corresponds to the G residue in the L1 loop, highly conserved
in both the yTAFII25 and
hTAFII28 families. A more detailed functional
analysis of yTAFII25 reveals a tight correlation
between its ability to interact with its heterodimerization partners
and its function (D. Kirschner and L. Tora, unpublished data).
It has previously been suggested that
yTAFII25 and hTAFII30
may homodimerize (18, 19). Klebanow et al. reported
two-hybrid interactions using full-length
yTAFII25 fusions. Our two-hybrid experiments show that this potential homodimerization requires only the
HFD of yTAFII25. As it is possible that the
two-hybrid interaction is indirect, we wished to visualize
homodimerization directly. However, while the
GST-yTAFII25(74-206) fusion was relatively soluble in E. coli when expressed alone, no homodimerization
was seen when it was coexpressed with native
yTAFII25(74-206). Therefore, under the
conditions used to observe heterodimerization via the HFD, we
detected no yTAFII25 homodimerization.
Consequently, it is unlikely that the observed
yTAFII25 oligomerization represents the formation
of a histone-like homodimer. This does not, however, exclude
the possibility that oligomerization of yTAFII25
may occur in some other way, involving for example loop-loop
interactions or via the exposed hydrophilic faces of the helices.
yTAFII65-yTAFII25, a novel
histone-like pair in yTFIID.
Here we have identified an HFD
in the N terminus of yTAFII65. This HFD shows
high homology to the
yTAFII17/hTAFII31 family. Both two-hybrid analysis and coexpression in E. coli
indicate that the yTAFII65 HFD mediates a
selective heterodimerization with the same
yTAFII25 domain that is required for interaction with yTAFII47. The yTAFII65
HFD alone is sufficient to allow interaction with
yTAFII25, and interaction is abolished by
mutations within this domain. These results strongly
suggest that
yTAFII25-yTAFII65 form an
additional histone-like pair in TFIID. Unlike
yTAFII47, yTAFII65 does not
interact with the hTAFII30 HFD despite their high
homology. We have previously reported that
hTAFII20, but not
yTAFII68, interacts with
hTAFII135. In this case, an important determinant
for specificity was mapped to the L2-3 region of the
hTAFII20/yTAFII68 HFD
(10). This observation together with those reported here
indicate that there is a strict specificity code which determines the
choice of a heterodimerization partner.
The yTAFII65 HFD is not sufficient for growth and
in fact is not essential at 30°C. Nevertheless, the
yTAFII65 HFD contributes to function, since its
deletion or mutation results in a temperature-sensitive phenotype. It
has previously been shown that introduction of proline residues in the
2 helix of yTAFII60 and
yTAFII17 results in a temperature-sensitive
phenotype (24). This is also true for yTAFII65, since the m1 mutation which introduces
two prolines generates a temperature-sensitive phenotype. This same
mutant abolishes interaction with yTAFII25. In
yTAFII47 and yTAFII68 the
HFD is necessary and sufficient for growth. In contrast, however, yTAFII65 must contain another essential domain(s)
located between amino acids 103 and 510. Further complementation
analysis reveals that this essential domain(s) is located between amino
acids 161 and 406 (our unpublished data). In this context, it is
interesting that, as with yTAFII47, we attempted
to suppress the temperature-sensitive phenotype of this mutation by
overexpression of other yTAFIIs. In this case,
however, the only genetic interaction detected was with
yTAFII68, whose overexpression suppressed the
temperature-sensitive phenotype not only of
yTAFII65(1-510)m1 but also of
yTAFII65(103-510), in which the HFD is deleted
(our unpublished data). There is therefore a genetic interaction
between yTAFII68 and
yTAFII65, but this involves a functional
domain(s) of yTAFII65 other than the HFD.
yTAFII25-ySPT7 a novel histone-like pair in ySAGA.
We have shown here that yTAFII25 can
heterodimerize with yTAFII47 and
yTAFII65. Both of these heterodimerization
partners are TFIID specific and are not present in SAGA.
yTAFII25 is, however, a SAGA subunit, and
therefore, an additional heterodimerization partner for
yTAFII25 must exist in this complex. Our results, and those of Sullivan et al. (32), identified an HFD in ySPT7, and we
show that this HFD heterodimerizes with the
yTAFII25 HFD in both the two-hybrid and bacterial
coexpression assays. This HFD is found in the C-terminal half of the
protein, which has been reported to partially rescue the phenotype of
the SPT7 null strain (11). Genetic studies have also shown
that mutations in SPT7 have the same severe phenotype as mutations in
ADA1 and SPT20. Mutation of each of these proteins completely disrupts the SAGA complex, showing that they are critical for its structural integrity (31). We have previously reported a direct
interaction between the TAFII and ADA families of
proteins through the heterodimerization of
yTAFII68 with yADA1 (10). Here we
show that the histone fold is also the interface between the
TAFII and the SPT families in SAGA. Together with
the genetic studies, this suggests that the yTAFII68-yADA1 and
yTAFII25-SPT7 pairs are both key structural elements of SAGA.
Implications for TFIID structure.
In summary, our present
results show yTFIID to comprise at least nine histone-like
yTAFIIs rather than the three originally described. These yTAFIIs assemble into five
heterodimeric pairs (yTAFII60-yTAFII17,
yTAFII40-yTAFII19,
yTAFII68-yTAFII48,
yTAFII25-yTAFII47, and
yTAFII25-yTAFII65). Amongst
these, yTAFII25 appears to be a key player
which can form two distinct heterodimers in TFIID. Previous results
have indicated that yTAFII47 can be
coimmunoprecipitated with yTAFII65 in extracts
from yeast strains harboring an epitope-tagged yTAFII65 (30). This excludes the
possibility that the
yTAFII25-yTAFII47 and
yTAFII25-yTAFII65 pairs are
present in distinct populations of yTFIID complexes; instead, it
indicates that the two pairs coexist in the same population of yTFIID.
It has previously been shown that a temperature-sensitive allele of
yTAFII17 can be suppressed by overexpression of
yTAFII60 or yTAFII68 and
that a temperature-sensitive allele of yTAFII60 can be suppressed by overexpressed yTAFII17 and
yTAFII68 (24). Furthermore,
yTAFII48 overexpression can suppress a
temperature-sensitive mutant of yTAFII68
(28). These genetic interactions were interpreted as
providing evidence of an octamer-like substructure in TFIID (24). Our present results showing genetic interactions
between yTAFII47 and
yTAFII25, yTAFII40,
yTAFII19, and yTAFII60
suggest a more complex picture which is difficult to interpret in the context of a single octamer-like substructure. Altogether, the existing
data suggest that there may be at least two potential substructures, one composed of yTAFII68,
yTAFII60, yTAFII48,
and yTAFII17 as described by Michel et al. and
Reese et al. (24, 28) and the other composed minimally of
yTAFII47, yTAFII40,
yTAFII25, and yTAFII19 as
reported here. The suppression of the yTAFII47 temperature-sensitive allele by overexpressed
yTAFII60 further implies interplay between the
substructures via yTAFII60. The stoichiometry of
the yTAFIIs present within each substructure and
whether they interact in a way analogous to the core histones in the
nucleosome octamer remain to be determined. Similarly, it is not
clear whether the
yTAFII25-yTAFII65 pair
associates with the
yTAFII25-yTAFII47
substructure via the
yTAFII25-yTAFII25 interactions discussed above or whether it is located elsewhere within TFIID.
We thank S. Hollenberg for the generous gift of yeast strain L40,
L. Carré for technical assistance, S. Thuault for gifts of
material and critical comments, S. Werten for gifts of materials, S. Vicaire and D. Stephane for DNA sequencing, the staff of cell culture
and oligonucleotide facilities, and B. Boulay and J. M. Lafontaine for illustrations.
C.R. was supported by EMBO fellowships. This work was supported by
grants from the CNRS, the INSERM, the Hôpital Universitaire de
Strasbourg, the Ministère de la Recherche et de la Technologie, the Association pour la Recherche contre le Cancer, the Ligue Nationale
contre le Cancer, the Human Frontier Science Programme, and NIH grant GM52461.
| 1.
|
Andel, F., III,
A. G. Ladurner,
C. Inouye,
R. Tjian, and E. Nogales.
1999.
Three-dimensional structure of the human TFIID-IIA-IIB complex.
Science
286:2153-2156[Abstract/Free Full Text].
|
| 2.
|
Apone, L. M.,
C. A. Virbasius,
F. C. Holstege,
J. Wang,
R. A. Young, and M. R. Green.
1998.
Broad, but not universal, transcriptional requirement for yTAFII17, a histone H3-like TAFII present in TFIID and SAGA.
Mol. Cell
2:653-661[CrossRef][Medline].
|
| 3.
|
Apone, L. M.,
C. M. Virbasius,
J. C. Reese, and M. R. Green.
1996.
Yeast TAFII90 is required for cell-cycle progression through G2/M but not for general transcription activation.
Genes Dev.
10:2368-2380[Abstract/Free Full Text].
|
| 4.
|
Bell, B., and L. Tora.
1999.
Regulation of gene expression by multiple forms of TFIID and other novel TAFII-containing complexes.
Exp. Cell Res.
246:11-19[CrossRef][Medline].
|
| 5.
|
Birck, C.,
O. Poch,
C. Romier,
M. Ruff,
G. Mengus,
A. C. Lavigne,
I. Davidson, and D. Moras.
1998.
Human TAFII28 and TAFII18 interact through a histone fold encoded by atypical evolutionary conserved motifs also found in the SPT3 family.
Cell
94:239-249[CrossRef][Medline].
|
| 6.
|
Brand, M.,
C. Leurent,
V. Mallouh,
L. Tora, and P. Schultz.
1999.
Three-dimensional structures of the TAFII-containing complexes TFIID and TFTC.
Science
286:2151-2153[Abstract/Free Full Text].
|
| 7.
|
Brand, M.,
K. Yamamoto,
A. Staub, and L. Tora.
1999.
Identification of TATA-binding protein-free TAFII-containing complex subunits suggests a role in nucleosome acetylation and signal transduction.
J. Biol. Chem.
274:18285-18289[Abstract/Free Full Text].
|
| 8.
|
Burley, S. K., and R. G. Roeder.
1996.
Biochemistry and structural biology of transcription factor IID (TFIID).
Annu. Rev. Biochem.
65:769-799[CrossRef][Medline].
|
| 9.
|
Burley, S. K.,
X. Xie,
K. L. Clark, and F. Shu.
1997.
Histone-like transcription factors in eukaryotes.
Curr. Opin. Struct. Biol.
7:94-102[CrossRef][Medline].
|
| 9a.
| Fribourg, S., C. Romier, S. Werten, Y. G. Gangloff, A. Poterszman, and D. Moras. Dissecting the interaction network of
multiprotein complexes by pairwise coexpression of subunits in E. coli.
J. Mol. Biol., in press.
|
| 10.
|
Gangloff, Y. G.,
S. Werten,
C. Romier,
L. Carre,
O. Poch,
D. Moras, and I. Davidson.
2000.
The human TFIID components TAFII135 and TAFII20 and the yeast SAGA components ADA1 and TAFII68 heterodimerize to form histone-like pairs.
Mol. Cell. Biol.
20:340-351[Abstract/Free Full Text].
|
| 11.
|
Gansheroff, L. J.,
C. Dollard,
P. Tan, and F. Winston.
1995.
The Saccharomyces cerevisiae SPT7 gene encodes a very acidic protein important for transcription in vivo.
Genetics
139:523-536[Abstract].
|
| 12.
|
Georgieva, S.,
D. B. Kirschner,
T. Jagla,
E. Nabirochkina,
S. Hanke,
H. Schenkel,
C. de Lorenzo,
P. Sinha,
K. Jagla,
B. Mechler, and L. Tora.
2000.
Two novel Drosophila TAFIIs have homology with human TAFII30 and are differentially regulated during development.
Mol. Cell. Biol.
20:1639-1648[Abstract/Free Full Text].
|
| 13.
|
Grant, P. A.,
D. Schieltz,
M. G. Pray-Grant,
D. J. Steger,
J. C. Reese,
J. R. Yates, and J. L. Workman.
1998.
A subset of TAFIIs are integral components of the SAGA complex required for nucleosome acetylation and transcriptional stimulation.
Cell
94:45-53[CrossRef][Medline].
|
| 14.
|
Green, M. R.
2000.
TBP-associated factors (TAFIIs): multiple, selective transcriptional mediators in common complexes.
Trends Biochem. Sci.
25:59-63[CrossRef][Medline].
|
| 15.
|
Hampsey, M.
1998.
Molecular genetics of the RNA polymerase II general transcriptional machinery.
Microbiol. Mol. Biol. Rev.
62:465-503[Abstract/Free Full Text].
|
| 16.
|
Hoffmann, A.,
T. Oelgeschlager, and R. G. Roeder.
1997.
Considerations of transcriptional control mechanisms: do TFIID-core promoter complexes recapitulate nucleosome-like functions?
Proc. Natl. Acad. Sci. USA
94:8928-8935[Abstract/Free Full Text].
|
| 17.
|
Hoffmann, A., and R. G. Roeder.
1996.
Cloning and characterization of human TAF20/15. Multiple interactions suggest a central role in TFIID complex formation.
J. Biol. Chem.
271:18194-18202[Abstract/Free Full Text].
|
| 18.
|
Jacq, X.,
C. Brou,
Y. Lutz,
I. Davidson,
P. Chambon, and L. Tora.
1994.
Human TAFII30 is present in a distinct TFIID complex and is required for transcriptional activation by the estrogen receptor.
Cell
79:107-117[CrossRef][Medline].
|
| 19.
|
Klebanow, E. R.,
D. Poon,
S. Zhou, and P. A. Weil.
1996.
Isolation and characterization of TAF25, an essential yeast gene that encodes an RNA polymerase II-specific TATA-binding protein-associated factor.
J. Biol. Chem.
271:13706-13715[Abstract/Free Full Text].
|
| 20.
|
Kokubo, T.,
D. W. Gong,
J. C. Wootton,
M. Horikoshi,
R. G. Roeder, and Y. Nakatani.
1994.
Molecular cloning of Drosophila TFIID subunits.
Nature
367:484-487[CrossRef][Medline].
|
| 21.
|
Komarnitsky, P. B.,
B. Michel, and S. Buratowski.
1999.
TFIID-specific yeast TAF40 is essential for the majority of RNA polymerase II-mediated transcription in vivo.
Genes Dev.
13:2484-2489[Abstract/Free Full Text].
|
| 22.
|
Luger, K.,
A. W. Mader,
R. K. Richmond,
D. F. Sargent, and T. J. Richmond.
1997.
Crystal structure of the nucleosome core particle at 2.8 A resolution.
Nature
389:251-260[CrossRef][Medline].
|
| 23.
|
Martinez, E.,
T. K. Kundu,
J. Fu, and R. G. Roeder.
1998.
A human SPT3-TAFII31-GCN5-L acetylase complex distinct from transcription factor IID.
J. Biol. Chem.
273:23781-23785[Abstract/Free Full Text].
|
| 24.
|
Michel, B.,
P. Komarnitsky, and S. Buratowski.
1998.
Histone-like TAFs are essential for transcription in vivo.
Mol. Cell
2:663-673[CrossRef][Medline].
|
| 25.
|
Moqtaderi, Z.,
M. Keaveney, and K. Struhl.
1998.
The histone H3-like TAF is broadly required for transcription in yeast.
Mol. Cell
2:675-682[CrossRef][Medline].
|
| 26.
|
Natarajan, K.,
B. M. Jackson,
E. Rhee, and A. G. Hinnebusch.
1998.
yTAFII61 has a general role in RNA polymerase II transcription and is required by Gcn4p to recruit the SAGA coactivator complex.
Mol. Cell
2:683-692[CrossRef][Medline].
|
| 27.
|
Ogryzko, V. V.,
T. Kotani,
X. Zhang,
R. L. Schlitz,
T. Howard,
X. J. Yang,
B. H. Howard,
J. Qin, and Y. Nakatani.
1998.
Histone-like TAFs within the PCAF histone acetylase complex.
Cell
94:35-44[CrossRef][Medline].
|
| 28.
|
Reese, J. C.,
Z. Zhang, and H. Kurpad.
2000.
Identification of a yeast transcription factor IID subunit, TSG2/TAF48.
J. Biol. Chem.
275:17391-17398[Abstract/Free Full Text].
|
| 29.
|
Sanders, S. L.,
E. R. Klebanow, and P. A. Weil.
1999.
TAF25p, a non-histone-like subunit of TFIID and SAGA complexes, is essential for total mRNA gene transcription in vivo.
J. Biol. Chem.
274:18847-18850[Abstract/Free Full Text].
|
| 30.
|
Sanders, S. L., and P. A. Weil.
2000.
Identification of two novel TAF subunits of the yeast Saccharomyces cerevisiae TFIID complex.
J. Biol. Chem.
275:13895-13900[Abstract/Free Full Text].
|
| 31.
|
Sterner, D. E.,
P. A. Grant,
S. M. Roberts,
L. J. Duggan,
R. Belotserkovskaya,
L. A. Pacella,
F. Winston,
J. L. Workman, and S. L. Berger.
1999.
Functional organization of the yeast SAGA complex: distinct components involved in structural integrity, nucleosome acetylation, and TATA-binding protein interaction.
Mol. Cell. Biol.
19:86-98[Abstract/Free Full Text].
|
| 32.
|
Sullivan, S. A.,
L. Aravind,
I. Makalowska,
A. D. Baxevanis, and D. Landsman.
2000.
The histone database: a comprehensive WWW resource for histones and histone fold-containing proteins.
Nucleic Acids Res
28:320-322[Abstract/Free Full Text].
|
| 33.
|
vom Baur, E.,
C. Zechel,
D. Heery,
M. J. Heine,
J. M. Garnier,
V. Vivat,
B. Le Douarin,
H. Gronemeyer,
P. Chambon, and R. Losson.
1996.
Differential ligand-dependent interactions between the AF-2 activating domain of nuclear receptors and the putative transcriptional intermediary factors mSUG1 and TIF1.
EMBO J.
15:110-124 |
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Abstract
Introduction
