(Received for publication, December 28, 1995; and in revised form, January 17, 1996)
From the
The transcription factor TFIID is a multimeric protein complex
containing the TATA box-binding polypeptide (TBP) and TBP-associated
factors. We have previously reported that the N-terminal regions of
dTAF62 and dTAF
42 have sequence similarities
with histones H4 and H3. Here, we demonstrate that the
histone-homologous regions of dTAF
62 and
dTAF
42 form a heteromeric complex both in vitro and in a yeast two-hybrid system. Neither dTAF
62 nor
dTAF
42 forms a homomeric complex, in agreement with a
nucleosomal histone character. Moreover, circular dichroism
measurements show that the heteromeric complex is dominated by
-helical secondary structure. These results strongly suggest the
existence of a histone-like surface on TFIID.
The transcription factor TFIID plays a central role in the
assembly of the basic transcriptional machinery into a preinitiation
complex (for review, see (1) ). TFIID binds to the core
promoter and thus provides a foundation for association of the other
initiation factors and RNA polymerase II. Biochemical studies of
eukaryotic transcriptional activation demonstrate both physical and
functional interactions of activators with TFIID that in turn
facilitate preinitiation complex assembly and function (for review, see (1) ). TFIID is a multimeric protein complex, comprising the
TATA box-binding protein (TBP) ()and numerous tightly
associated factors called TAFs (for review, see (2) and (3) ). To date, nine TAF subunits of Drosophila TFIID
(dTAF
230, -150, -110, -85, -62, -42, -28
, -28
,
and -22) have been cloned (reviewed in (3) ). In addition,
several homologs have been isolated from both human (hTAF) and yeast
(yTAF), suggesting that TAF-mediated activation pathways are conserved
from yeast to man (reviewed in (3) ).
A central question in
eukaryotic transcriptional regulation is how TFIID gains access to a
chromatin template and how stable association is maintained within the
chromosome. A potentially relevant finding is that the N-terminal
regions of the dTAF62 and dTAF
42 proteins
have sequence similarities with the C-terminal core domain of histones
H4 and H3, respectively(4) . More recently, it has been shown
that the histone-like regions are well conserved in both the yeast (5) (
)and human (6, 7, 8, 9) counterparts to
dTAF
62 and dTAF
42.
The nucleosome core
consists of 146 bp of DNA wrapped around an octameric complex of two
copies of each of histones H2A, H2B, H3, and H4. Despite a low sequence
similarity, all histones contain a long helix flanked on either side by
a loop segment and short helix, termed the ``histone fold''
(reviewed in (10, 11, 12) ). The histone fold
is involved in formation of the stable H2A-H2B and H3-H4 heterodimers.
H3-H4 dimers further associate to form the (H3-H4) tetramer. The (H3-H4)
tetramer alone is responsible
for organizing the core 120 bp of DNA into the arrangement found in the
octameric complex(13) .
Recently, numerous proteins
potentially containing the histone fold have been identified by data
base search(14) . This group includes TAF62 and
TAF
42, plus many DNA-binding proteins and multimeric
proteins. The data base search also identified transcription initiation
factor TFIIB as a protein that has the histone fold. However, both NMR (15) and x-ray crystallography (16) show that TFIIB
does not have the histone fold. Thus, it is crucial to confirm the
prediction using biochemical and physical techniques. To investigate
the significance of the sequence similarities between
dTAF
62-dTAF
42 and histones H4-H3, we have
tested the ability of the histone-like regions in dTAF
62
and dTAF
42 to form a stable heteromeric complex. Here, we
demonstrate that the histone-homologous regions of dTAF
62
and dTAF
42 form a heteromeric complex both in vitro and in a yeast two-hybrid system. We further demonstrate that this
heteromeric complex, like the H3-H4 complex, is predominantly
-helical. The results described here strongly suggest that TFIID
has a histone-like surface.
CD measurements were
performed at 25 °C using a Jasco J700 spectropolarimeter. The
spectra are presented as the average of eight scans in units of mean
residue ellipticity (). The concentration of the protein was 15.7
µM in 20 mM sodium phosphate (pH 7.0). Protein
concentration was determined by measuring absorbance at 280 nm and
using a calculated extinction coefficient. The protein was denatured by
the addition of guanidine hydrochloride to a concentration of 6 M. Protein secondary structure was estimated using the
software supplied with the Jasco J700 spectropolarimeter.
Figure 1:
Histone-like
regions in dTAF62 and dTAF
42 form a
heteromeric complex in vitro. A, schematic overall
structures of dTAF
62 and dTAF
42. The regions
having sequence similarities with nucleosomal core histones H4 and H3 (4) are shaded. The regions expressed in E. coli for interaction study are depicted by thick bars. B, interaction between histone-like regions in vitro.
GST-dTAF
62 (aa 1-91) and His-dTAF
42 (aa
1-100) were expressed independently as inclusion bodies and
co-renatured (lanes 1-5). Control extracts without
His-dTAF
42 (aa 1-100) (lanes 6-8) or
GST-dTAF
62 (aa 1-91) (lanes 9-11)
were also prepared. Complexes were purified by
Ni
-NTA-agarose chromatography followed by
glutathione-Sepharose chromatography (lanes 2 and 3; lanes 7 and 8 for control). Similarly, complexes were
also purified by glutathione-Sepharose chromatography followed by
Ni
-NTA-agarose chromatography (lanes 4 and 5; lanes 10 and 11 for
control).
The far-UV (250-200 nm) CD spectrum of
the dTAF62 (aa 1-91)-dTAF
42 (aa
1-100) heterocomplex is shown in Fig. 2. The overall shape
of the spectrum, with minima in the mean residue ellipticity at 208 and
222 nm, is characteristic of polypeptides dominated by
-helical
secondary structure. In support of this, we used manufacturer-supplied
software to obtain the following estimates for the secondary structure:
41.4%
-helix, 8.4%
-strand, 15.8% turn, and 34.4% random.
Figure 2:
Circular dichroism spectra of
dTAF62 (aa 1-91)-dTAF
42 (aa
1-100) in native (solid line) and denatured (dashed
line) conditions.
In summary, we have demonstrated that
the N-terminal regions of dTAF62 and dTAF
42,
similar in sequence to nucleosome core histones H4 and H3, form a
heteromeric complex both in vitro and in a yeast two-hybrid
system. Like histones H3 and H4, neither dTAF
62 nor
dTAF
42 forms a homomeric complex, and CD measurements
suggest that the heteromeric dTAF
62-dTAF
42
complex is dominated by
-helical secondary structure.
In some
promoters, including the adenovirus major late and human gfa promoters(20, 21, 22) , the TFIID
footprint extends from the TATA box through to 35 bp downstream from
the initiation site, whereas the TBP footprint extends only over the
TATA box region. These results indicate that one or more TAFs
contribute to the downstream interaction. Recent studies with
recombinant TAFs demonstrated that dTAF150 alone can bind
to the initiator element(23) . Moreover, the
dTAF
150-TBP complex gives a footprint from the TATA box
through to 35 bp downstream from the initiation
site(23, 24) , like native TFIID. However, the
dTAF
150-TBP complex does not produce DNase
I-hypersensitive sites as observed for native TFIID(24) . Thus,
it is likely that other TAFs are also involved in the downstream
interaction. We predict that the histone-like region in
dTAF
62-dTAF
42 is bound to the downstream
sequence because, similar to histone core, TFIID produces DNase
I-hypersensitive sites about every 10 bp. The important question is
whether the dTAF
62-dTAF
42 complex is a
heterodimer or heterotetramer in the TFIID complex; the N-terminal
histone-like regions of dTAF
62-dTAF
42 do form
heterotetramers under physiological conditions, (
)suggesting
that these TAFs could be present as a tetramer in the TFIID complex.
The downstream interaction plays an important role in both basal and activated transcription. TFIID binds to the downstream region in a DNA sequence-dependent fashion, at least in the human gfa promoter, contributing to both stable TFIID binding and effective transcription in vitro(22, 25) . In contrast, in the adenovirus E4 promoter, TFIID does not bind to the downstream region and produces very low basal transcription activity. Importantly, activators induce the downstream interaction and facilitate preinitiation complex formation(26, 27) . Furthermore, the histone-like domains may have a more critical role in a natural chromatin environment, e.g. stimulation of the displacement of nucleosomal histones near the transcription start site and/or maintenance of histone exclusion from the transcription start site.