From the
Transcription of yeast class III genes requires the sequential
assembly of the general transcription factors TFIIIC and TFIIIB, and of
RNA polymerase III, into an initiation complex composed of at least 25
polypeptides. The 70-kDa subunit of TFIIIB (TFIIIB70) is central in
this network of interactions as it contacts both TATA-binding protein
and a subunit of polymerase III. We show here that the TATA-binding
protein interacts with the carboxyl-terminal part of TFIIIB70. TFIIIB70
also contacts TFIIIC (factor
The cascade of interactions leading to class III gene activation
and the key components involved in this process have been much
investigated(1, 2, 3, 4) . The yeast
factor TFIIIC or
We have shown previously that
TFIIIB70 interacts with TBP (7) and with the C34 subunit of pol
III(8) . It also interacts with TFIIIC
Single-stranded pCK14 (23) or pNC1
DNA was used to mutagenize the TFC4 gene. pNC1 plasmid was
constructed by introducing a BamHI site at -9 relative
to the TFC4 ATG codon and by destroying the BamHI
site at +2310. pNC8 was constructed by modifying the BamHI site of pNC1 from -9 to -8 and destroying
the EcoRV site at +2710. pNC11 to pNC31 were obtained by
deletion of nucleotides 4-369, 4-292, 382-483,
484-585, 586-687, 688-789, 790-801,
1294-1398, 1396-1503, 1500-1605, 1606-1707,
1810-1881, 1830-1881, 1882-1920, 1921-2034,
1921-1977, 1977-2034, 2035-2094, 2077-2163,
2623-2724, and 2975-2986 of TFC4, respectively.
Single-stranded pNC1 DNA was used to construct pNC13, pNC14, pNC22,
pNC26, pNC27, and pNC29. The KpnI/SpeI fragments from
plasmids pNC11 to pNC31 were cloned into pUN45.
Single-stranded
pRMS3 DNA (22) was used to mutagenize the PCF4 gene.
pRMS3-50 was created by introducing a NcoI site at -8
relative to the PCF4 ATG codon. pRMS3-50,52 and pRMS3-52,54
were created by introducing a NcoI site at -8 and
+107, respectively, and a stop codon at +859. pRMS3-53 and
pRMS3-54 were created by introducing a NcoI site at +752
and +107, respectively. The EcoRI, BglII/EcoRI, and NdeI/SalI
fragments of pRMS3 were cloned in pET28b (Novagen), leading to plasmids
pCC-7, pCC-8 and pCC-9, respectively.
The GAL4-
During these experiments,
we noted that when fused to the GAL4 DNA binding domain,
We reasoned that wild type levels of
interaction could be restored by using truncated versions of
Using a yeast interaction system, we have mapped regions of
TFIIIB and TFIIIC that interact within the TFIIIB
A direct interaction between TBP and
TFIIIB70 was previously reported (7, 33). We found here that TBP
interacts with the CTE moiety of TFIIIB70, whereas no binding to the
TFIIB-like domain could be detected. In contrast, Khoo et al.(33) recently found by affinity chromatography, using
glutathione S-transferase fusions with wild type or deleted
forms of TFIIIB70, that
The protein
domains of
Our results demonstrated an interaction between the
NH
As in
the case of
The present interaction
studies extend the functional similarities, previously
noted(20, 21, 22) , between TFIIB and TFIIIB70.
The role of
The symbols + and - are attributed to
blue coloration and no coloration on X-gal plates, respectively, when
the TFIIIB70 or
The two-hybrid system was used to study protein-protein interactions
between
We greatly thank Christian Marck who initiated the
mutagenesis analysis, Olivier Lefebvre and Laurence Damier for
assistance with the two hybrid experiments, Jochen Rüth for the
gift of pAS-TBP and pACTII-JR and for help with the suppressor
analysis, and Janine Huet for her contribution to the FarWestern
analysis. We acknowledge Stephen Elledge for providing us with the two
hybrid strains and plasmids, James Hopper and Colleen Lebo for
providing the anti-GAL4 antibodies, and Michael Hampsey for the SUA7
plasmids.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
) via its
131 subunit. The
protein domains of
131 and TFIIIB70 involved in this interaction,
either positively or negatively, were mapped using the two-hybrid
system. We provide evidence that intramolecular interactions mask
functional domains in both polypeptides.
(six subunits, named
138,
131,
95,
91,
60, and
50), plays a primary role in the
process of transcription complex assembly by binding to the intragenic
promotor sequences of tRNA genes or to the preformed 5 S
DNA
TFIIIA complex. Once bound, TFIIIC acts as an assembly factor
to allow the binding of the initiation factor TFIIIB (three components,
TATA-binding protein (TBP),
(
)TFIIIB70 and
TFIIIB90) to an upstream gene position. TFIIIB then recruits RNA
polymerase III (pol III) and directs accurate initiation of
transcription(5, 6) . The genes encoding most of these
components are cloned(4) .
DNA complex, a weak
interaction profoundly modified by addition of TBP(6) . Thus,
TFIIIB70 appears as a central bridging factor between the basal
components of the class III transcription machinery, in a way that is
much similar to the role proposed for TFIIB in the case of class II
genes(9) . TFIIB is a target of transactivators (10, 11, 12) that facilitate its incorporation
into the early preinitiation complex; there it binds TBP (and possibly
DNA) and recruits the RNA polymerase II
TFIIF
complex(13, 14, 15, 16) . Indeed, TFIIB
and TFIIIB70 have a conserved core with a NH
-terminal
region which contains a putative zinc finger domain, followed by two
imperfect direct repeats (17-22) that, in TFIIB, harbor the
target for TBP(13, 14, 15, 16) . A major
structural difference is the existence in TFIIIB70 of a
carboxyl-terminal extension (CTE) that almost doubles the size of the
polypeptide compared to TFIIB (see Fig. 4; Refs. 20-22).
One hypothesis is that the conserved domains were retained to perform
basal functions like TBP, DNA, and polymerase binding, whereas the CTE
evolved to recognize the assembly factor TFIIIC. The interaction with
TFIIIC is of particular interest as it confers gene specificity. Using
the two-hybrid system, we have sought first to identify the subunit(s)
of TFIIIC that interacts with TFIIIB70, then to map the protein domains
involved in this interaction. Unexpectedly, it was the TFIIB-like
region that we found to interact with TFIIIC, via its
131 subunit.
We also show that the CTE of TFIIIB70 is involved in TBP binding.
Figure 4:
Schematic representation of mutant
TFIIIB70 fusion proteins. Parts of the open reading frame encoding
TFIIIB70 were amplified by polymerase chain reaction and fused in frame
with the GAL4 DNA binding or activation domains. Numbers in parentheses indicate the TFIIIB70 amino acids present in the
fusion protein. Motifs of TFIIIB70 homologous to TFIIB are
indicated.
Construction of Yeast Strains
Standard genetic
techniques and media were used. Plasmids harboring modified alleles of
the TFC4 gene were used to transform the YCK107 haploid strain
containing the TFC4 gene on a plasmid with the chromosomal
copy disrupted(23) . The modified copies of TFC4 were
substituted for wild type TFC4 by plasmid shuffling on plates
containing 5-fluoroorotic acid. Viable strains isolated at 30 °C
have also been tested for growth at 37 and 16 °C.
Site-directed Mutagenesis and Construction of
Plasmids
Oligonucleotide-mediated mutagenesis was performed as
described by the manufacturer, using a Muta-Gene kit (Bio-Rad).
Uracil-enriched single-stranded DNA was prepared from a Escherichia
coli strain CJ236.
Construction of GAL4 Fusions
pAS-CC and pACT-CC
were obtained by digestion of pAS2 (24) or pACTII (kindly
provided by S. Elledge) by NcoI, fill-in by Klenow DNA
polymerase, and religation. pACT-JR, a gift from J. Rüth, was
constructed by digestion of pACT-CC by XmaI, fill-in by Klenow
enzyme, and religation. The 3382-base pair BamHI fragment from
pNC8 harboring the TFC4 open reading frame was cloned into
pAS2 for fusion with GAL4-(1-147) or into pACTII for fusion with
GAL4-(768-881). pAS-131 was then digested by NdeI
and religated to construct the GAL4-(1-147)-
N3 fusion. The
GAL4-(768-881)-
N3 fusion was obtained by digestion of pNC1
by NdeI, fill-in by Klenow enzyme, digestion by BamHI, and cloning of the resulting fragment into pACTII. The
1065-base pair BamHI fragment from pCK14 was cloned into
pAS-CC and pACT-CC to obtain GAL4-
N4 fusions. The BamHI
fragments of pNC13, pNC14, and pNC22 were cloned into pACT-JR to obtain
the GAL4-
TPR1, GAL4-
TPR2, and GAL4-
basic2 fusions,
respectively. The TFC4 open reading frame, deleted of
nucleotides 4-292 or 586-687, was cloned into the BamHI site of pACTII to obtain the
GAL4-(768-881)-
N2 and
TPR3 fusions, respectively.
131-0TPR, GAL4-
131-1TPR, GAL4-
131-5TPR,
GAL4-
131-9TPR, and GAL4-bHLH fusions were constructed by
polymerase chain reaction amplification of nucleotides 1-300,
1-495, 1-912, 1-1737, and 1727-2205 of pCK14,
respectively. The oligonucleotides primers were designed in order to
create a NcoI site at -8 and a BamHI site after
the stop codon at +303, +498, +915, +1740, and
+489, respectively. The resulting NcoI/BamHI
fragments were then cloned into pAS2 and pACTII. The GAL4-TFIIIB70
fusions were constructed by cloning the NcoI/SalI
fragments from pRMS3-50, pRMS3-50,52, pRMS3-53, pRMS3-54, and
pRMS3-52,54 into pAS2 and pACTII. The sequence of the fusion joint was
determined for all the constructs. The plasmids have been used to
transform a Y526 yeast strain.
(
)
GAL1-lacZ Activation Assay
Seven
independent transformants for each combination of plasmids were grown
as patches for 2 days at 30 °C on synthetic complete solid medium
containing 2% raffinose as carbon source. -Galactosidase activity
was revealed by overlaying the cells with 10 ml of X-gal agar as
described (8) and incubating the plates for 24 h at 30 °C.
-Galactosidase activity was measured in yeast extracts exactly as
described previously(8) .
Expression of TFIIIB70 Variants
Recombinant
histidine-tagged TFIIIB70 variants were expressed in E. coli cells from plasmids pSH360 (a gift from Steve Hahn), pCC-7, pCC-8,
and pCC-9, purified by chromatography on
Ni-nitrilo-triacetic acid-agarose (Qiagen) under
native conditions.
(
)pCC-7, pCC-8, and pCC-9
encoded TFIIIB70 derivatives from residues 263 to 596 (CTE), 14 to 262
(
CTE), or 141 to 596 (
N140), respectively, tagged at their
NH
-terminal end with six histidine residues and the T7-TAG
epitope (Novagen).
Interaction of TFIIIB70 Variants with
FarWestern analysis was
performed as described using in vitro synthesized S-Labeled TBP
S-labeled TBP(7) . Recombinant TFIIIB70 derivatives
were separated by SDS-PAGE and transferred on a nitrocellulose
membrane. The filters were incubated with
S-labeled TBP,
washed, and autoradiographed. TFIIIB70 polypeptides were located by
anti-TFIIIB70 antibodies (kindly provided by Steve Hahn) or by
anti-T7-TAG antibodies (Novagen). Immune complexes were visualized with
antibodies tagged with alkaline phosphatase (Promega).
TBP Interacts with the CTE Moiety of
TFIIIB70
Using a FarWestern blotting experiment, we have
previously demonstrated that TBP interacted with TFIIIB70 in absence of
DNA(7) . To delineate more precisely the protein domains
involved in this interaction, three derivatives of TFIIIB70 were
expressed in E. coli cells, as histidine fusions, and purified
under native conditions on Ni-nitrilo-triacetic
acid-agarose. After SDS-PAGE, the proteins were transferred onto a
membrane and probed with
S-labeled TBP. As shown in Fig. 1(lanes 3 and 4), the CTE moiety of
TFIIIB70, migrating as a polypeptide of 49 kDa, bound TPB. A
COOH-terminal truncated form of the CTE migrating as a 41-kDa
polypeptide (this polypeptide was recognized by antibodies directed to
the T7-TAG epitope, located at the NH
-terminal end of the
recombinant CTE construct, not shown) bound very weakly to TBP, as
judged from the relative band intensity in the Western blot and in the
autoradiogram, suggesting that the integrity of the CTE was important
for TBP binding. In contrast, the integrity of the TFIIB-like moiety
did not seem necessary, since a derivative of TFIIIB70 deleted at the
NH
-terminal end (
N140, lacking the zinc finger motif
and half of the first repeated domain) was able to bind TBP (lanes
5 and 6). In fact, no binding of TBP could be detected
with the TFIIB-like domain of TFIIIB70 (
CTE, lane 8),
migrating as a polypeptide of 33 kDa (lane 7). These results
indicated that the CTE moiety of TFIIIB70 was involved in TBP binding.
Note that a COOH-terminal truncated form of TBP, lacking the last 14
amino acid residues, was not able, when labeled with
[
S]methionine, to bind to TFIIIB70 polypeptides
(data not shown).
Figure 1:
TBP interacts with the CTE domain of
TFIIIB70. Recombinant TFIIIB70 (WT) or TFIIIB70 derivatives
comprising residues 263-596 (CTE), 141-596 (N140), or 14-262 (
CTE), expressed as
hexahistidine fusions, were purified from E. coli cells under
native conditions. Eluted polypeptides (1-2 µg) were
subjected to SDS-PAGE, transferred onto a membrane, and probed with
S-labeled TBP as described under ``Materials and
Methods.'' Labeled polypeptides were revealed by autoradiography (lanes 2, 4, 6, 8). The same membrane was then incubated with
an anti-TFIIIB70 antiserum (lanes 1, 3, 5) or with antibodies
directed to the T7-TAG epitope (lane 7), and immune complexes
were visualized using antibodies tagged with alkaline phosphatase. The
molecular weight of the major polypeptides contained in wild type (left) or deleted (right) TFIIIB70 fractions is
indicated.
Genetic Deletion Analysis of
Topological
studies of TFIIIC131
DNA and TFIIIB
TFIIIC
DNA complexes
showed that only
131 subunit was located upstream of the
transcription start site. This upstream location therefore made
131 a likely candidate for assembling TFIIIB through interactions
with TFIIIB70(25, 26, 27) .
131
polypeptide, encoded by TFC4, comprises a series of notable
motifs, including a highly charged NH
-terminal domain, a
motif akin to basic-helix-loop-helix-zipper (bHLH-Zip), and 11
tetratricopeptide repeat (TPR) units divided in three
blocks(23) . Using genetic deletion analysis, we first
investigated in vivo the importance of these domains (Fig. 2). The motifs were deleted individually, and centromeric
plasmids harboring the mutant copies of the TFC4 gene were
tested for their ability to functionally replace, at different
temperatures, a chromosomally disrupted copy of TFC4. As shown
in Fig. 2, deletion of the NH
-terminal domain
(
N1,
N2), of all but two TPR units, and of the loop of the
bHLH-Zip motif (
loop), led to a lethal phenotype. Three deletions
resulted in a temperature-sensitive phenotype (
TPR8,
basic2,
H1). Deletion of the TPR9, of several domains of the bHLH-Zip
motif (
basic1,
H1,
zipper), had no effect on cell
growth. This deletion analysis suggested that most of the
characteristic motifs were important for the structural or functional
integrity of
131.
Figure 2:
Deletion analysis of 131. The motifs
noted in
131 protein were deleted individually using polymerase
chain reaction amplification or directed mutagenesis of the TFC4 gene. The positions of deleted amino acids (inclusive) are
indicated for each construct. Centromeric plasmids harboring a deleted
copy of TFC4, expressed from its own promoter, were tested for
their ability to functionally replace, at different temperatures, a
chromosomally disrupted copy of TFC4. Lethal (-), wild
type (+), and temper-ature-sensitive (ts) phenotypes are
indicated.
Extragenic Suppression of
We used
the three temperature-sensitive mutant strains (131 Mutants
TPR8,
basic2,
H1) to uncover potential genetic interactions of TFIIIC with
components of the class III transcription apparatus. Overexpression of
TFIIIB70, TBP, and
95 harbored on multicopy plasmids was found to
suppress the temperature-sensitive phenotype of the three
131
mutant strains. These results confirmed the genetic interaction of
131 with TFIIIB70 (28) and suggested a network of interactions
between
131, two components of TFIIIB and
95 (
95 subunit
is thought to interact with block
A(1, 2, 3, 4) ). However, increased gene
dosage of TFIIIB70, TBP, and
95 was also reported to suppress a
mutation in
138, the B-block binding subunit of
TFIIIC(29) . Therefore, these suppression effects do not
demonstrate direct interactions between TFIIIB and TFIIIC components.
Hence, increased concentration of TPB or TFIIIB70 could influence the
rate of TFIIIB assembly or the rate of transcription complex formation
on class III genes.
To demonstrate a
direct interaction of 131 Interacts with TFIIIB70
131 with TFIIIB components, we used the
two-hybrid system that detects in vivo interactions between
two proteins overproduced in yeast cells(30, 31) . The
proteins are fused to the DNA binding domain, or to the transcriptional
activation domain, of the yeast GAL4 protein. If the two proteins
interact, a chimeric GAL4 protein is reconstituted that activates the
transcription of a lacZ reporter gene. This method previously
revealed interactions between TFIIIB70 and the pol III subunit
C34(8) . The open reading frames encoding TFIIIB70, TBP, TFIIIA,
or TFIIIC components (
138,
131,
95) were fused to the
two GAL4 domains. All combinations of fusion proteins with GAL4 DNA
binding or activation domains were assayed. Most combinations of hybrid
proteins gave background levels of
-galactosidase activity. In
contrast, when TFIIIB70 was fused to the GAL4 DNA binding domain and
131 to the GAL4 activation domain, high levels of
-galactosidase activity were detected, suggesting that TFIIIB70
and
131 interact. The reciprocal assay gave similar results,
whereas none of the fusion protein alone activated lacZ transcription ().
The 165 First Amino Acids of
The interaction between TFIIIB70 and
131 Are Sufficient for
Interaction with TFIIIB70
131 was further characterized using deletion derivatives of
131. Some of the
131 mutants described in Fig. 2were
fused to the activation domain of GAL4 and were tested for their
interaction with TFIIIB70. We first investigated the role of the
putative bHLH-Zip motif. As shown in Fig. 3, deletion of the
basic or loop domain of the bHLH-Zip did not prevent
131 to
interact with TFIIIB70. The
-galactosidase activity measured with
these mutant
131 fusions was only half that obtained with the wild
type protein, indicating that the bHLH-Zip motif of
131 was not
involved in TFIIIB70 binding. This conclusion was supported by the fact
that the bHLH-Zip motif alone, when fused to the GAL4 activation
domain, was unable to interact with TFIIIB70 (see Fig. 3).
Figure 3:
Interaction of wild type or mutant
131 proteins with TFIIIB70. The two-hybrid system was used to
study protein-protein interactions between
131 and TFIIIB70. The
open reading frames of wild type or mutant
131 proteins were fused
in frame with the GAL4 activation domain (GAL4 768-881). TFIIIB70
was fused in frame with the GAL4 DNA binding domain (GAL4 1-147).
Transcription activation of the lacZ reporter gene was assayed
by growing the transformed cells on selective medium and overlaying
with X-gal agar. - and +, white and blue coloration of cell
patches on X-gal plates, respectively. (-) indicates a slight
blue coloration.
-Galactosidase activity was measured at 30 °C
for at least three independent transformants. Units are expressed in
nanomoles of o-nitrophenyl-
-D-galactoside
hydrolyzed per minute and per milligram of protein. Background level is
around 1-4 units. ND,
nondetermined.
To
delineate the region of 131 required for interaction with
TFIIIB70, we constructed a series of NH
- or COOH-terminal
fusions harboring the GAL4 DNA binding or activation domains. When
131-9TPR was fused to the activation domain of GAL4 and assayed
with TFIIIB70 reciprocal fusion, high level of
-galactosidase
activity was measured. In contrast, background levels of
-galactosidase activity were obtained with the
N3 or
N4
fusions, harboring the COOH-terminal moiety of
131. Even higher
levels of
-galactosidase activity were obtained with the smaller
fusions
131-5TPR or
131-1TPR. In contrast, background
-galactosidase activity was observed with only the
NH
-terminal acidic domain (
131-0TPR). These results
demonstrated that the NH
-terminal 165 amino acids of
131, comprising the highly charged domain and the first TPR unit,
were sufficient to interact with TFIIIB70 with the same efficiency as
the entire
131 protein. This conclusion was supported by the fact
that high levels of
-galactosidase activity were obtained with the
TPR2 and
TPR3 mutant fusions whereas only background levels
of
-galactosidase activity were obtained with either the
N2
or the
TPR1 deleted
131 fusions. We checked by Western
blotting analysis, using anti-GAL4 antibodies, that the fusion proteins
were normally expressed (data not shown).
131-5TPR
and
131-1TPR were by themselves strong activators of pol II
transcription. This was not the case for the entire
131
polypeptide ().
Next, we investigated interactions between 131 Interacts with the TFIIB-like Part of
TFIIIB70
131 and
deleted versions of TFIIIB70. The fragments of TFIIIB70 shown in Fig. 4were fused to the DNA binding or activation domains of GAL4
and assayed with the reciprocal
131 fusion. Not all combinations
could be tested as we noted that when fused to the GAL4 DNA binding
domain, the CTE moiety of TFIIIB70 was, by itself, a strong activator
of pol II transcription, although this was not the case with the whole
polypeptide (). As shown in , deletion of the
zinc finger domain of TFIIIB70 (
Zn) did not impair the
interaction. Note that the zinc finger domain of TFIIB has been
implicated in TFIIF binding (13) and as a target for
glutamine-rich activation domains(32) . The CTE moiety of
TFIIIB70 fused to the GAL4 activation domain did not detectably
interact with
131. When the NH
-terminal part of
TFIIIB70 (
CTE) was fused to the GAL4 activation domain, a small
but significant level of
-galactosidase activity was observed (), although the reciprocal combination gave background
-galactosidase levels. When the zinc finger motif and the
COOH-terminal part of TFIIIB70 were deleted (
Zn/
CTE),
background
-galactosidase activities were measured (), even though a light blue coloration was observed on
plates (data not shown). Therefore, only the NH
-terminal
part of TFIIIB70 was able to weakly interact with
131, suggesting
that the NH
-terminal and COOH-terminal moiety cooperated
for optimal interaction.
131.
Thus, the
Zn,
CTE, and
Zn/
CTE deleted TFIIIB70
proteins, fused to the GAL4 DNA binding domain, were assayed with the
131-9TPR,
131-5TPR,
131-1TPR, and
131-0TPR
reciprocal fusions described in Fig. 3. With or without the zinc
finger motif, TFIIIB70 interacted with the 165 first amino acids of
131 (). When TFIIIB70-
CTE or
TFIIIB70-
Zn/
CTE deleted proteins were assayed, the highest
level of interaction was observed with the
131-5TPR hybrid. Low
but still significant levels of interaction were observed with the
131-9TPR or
131-1TPR hybrids, whereas background
-galactosidase activity was found with
131-0TPR. These
results clearly demonstrated that the NH
-terminal part of
131 interacts with the TFIIB-like moiety of TFIIIB70.
TFIIIC
DNA
complex. We found that the NH
-terminal domain of TFIIIB70,
that is structurally similar to TFIIB, interacts with
131 subunit
of TFIIIC, whereas the COOH-terminal half binds to TBP. The
131
interacting zone was found to be confined to its
NH
-terminal end. The results support the model in which
TFIIIB70 bridges the pol III initiation complex through its
interactions with TBP,
131, and C34 subunit of pol III (this work,
Refs. 6-8 and 33).
S-labeled TBP bound to both the
CTE and the TFIIB-like domains of TFIIIB70. The discrepancy with our
own results may be due to an improper folding of the TFIIB-like domain
on the membrane preventing TBP recognition. However, the interaction
between TBP and the CTE of TFIIIB70 could also be observed using the
two-hybrid system, whereas again no interaction was detected with the
TFIIB-like part.
(
)Alternatively, the large
excess of immobilized TFIIIB70-GST fusion protein may have favored some
nonspecific binding of labeled TBP to the TFIIB homologous region. This
possibility was suggested by the fact that the full-length TBP probe
was specifically retained only by the carboxyl-terminal domain of
TFIIIB70, residues 263-596, and that mutations in TBP
specifically affected its interaction with the CTE moiety(33) .
Therefore, the presence of two distinct TBP-interacting regions in
TFIIIB70 remains an open question. We found that the integrity of the
CTE, but not of the TFIIB-like domain, was important for TBP binding.
Previous studies have indicated that deletions of NH
- or
COOH-terminal residues of TFIIIB70 disrupt its function in
vivo(20, 21) . Since a deletion of 140 residues at
the NH
-terminal end of TFIIIB70 did not impair TBP binding in vitro (Fig. 1), it seems likely that the lethality of
yeast strains bearing a derivative of TFIIIB70 deleted of 40 residues
at the NH
-terminal end (21) is not a consequence of a
TFIIIB70
TBP interaction defect. We found, instead, that this
TFIIB-like domain was involved in
131 binding.
131 and TFIIIB70 involved in their interaction were
mapped. Our results demonstrated that 165 residues at the
NH
-terminal end of
131 were sufficient to interact
with TFIIIB70 as efficiently as the entire protein. These are in
vivo interactions. Formally, there is the possibility that
131 and TFIIIB70 interact indirectly via a third, intervening
component (i.e. another subunit of TFIIIC). However, the
possibility of the stable assembly of TFIIIC harboring only 165
residues of
131 appears very unlikely, especially since we found
that many small deletions throughout
131 were lethal (Fig. 2). In addition, we detected no interaction of TFIIIB70
with the
95 subunit that cross-link next to
131 in
TFIIIC
DNA complexes(25, 26) . While this paper was
in preparation, Khoo et al.(33) reported that each,
the NH
-terminal, middle, and COOH-terminal thirds of
131, bound to TFIIIB70 independently. We did not detect an
interaction between the COOH-terminal part of
131 and TFIIIB70
(see Fig. 3), but our results showed that the COOH-terminal part
of
131 (past TPR5) interfered with the interaction between the
NH
-terminal part of
131 and the TFIIB-like moiety of
TFIIIB70 (). This suggests the existence of intramolecular
interactions, within
131, that shield the TFIIIB70 interaction
domain. This contention was supported by our observation that the
strong pol II activator properties of
131-5TPR or
131-1TPR
fused to GAL4 DNA binding domain were masked within the entire
131
polypeptide.
131-9TPR that interacted less efficiently with the
TFIIB-like part of TFIIIB70 also lacked activator property (see Tables
I and II). Therefore, intramolecular interactions in
131 may
possibly involve the two blocks of TPR, TPR1-5, and TPR6-9.
-terminal parts of TFIIIB70 and
131, but did not
exclude a role for the COOH-terminal part of TFIIIB70. In fact, the
COOH-terminal part of TFIIIB70 may play an active role in the
establishment of the interaction between TFIIIB70 and
131. Indeed,
the presence of the CTE was important for interaction with
131,
131-9TPR, or
131-1TPR, whereas it was dispensable for
interaction with the
131-5TPR fusion (). One could
imagine, for example, that the COOH-terminal part of TFIIIB70 enhances
131
TFIIIB70 interaction by modifying the conformation of
131, allowing the interaction between the NH
-terminal
parts of TFIIIB70 and
131. As TBP binds to the CTE of TFIIIB70,
another explanation could be that the binding of TBP to the CTE is
necessary for the interaction of the TFIIB-like part of TFIIIB70 with
131. This hypothesis is supported by the observation that TBP
stabilizes TFIIIB70
TFIIIC
DNA complexes(6) .
131, a subdomain of TFIIIB70, the CTE, displayed
strong pol II activator properties that were repressed in the entire
protein. This observation may or may not be related to the pol III
activation role of TFIIIB70. At least, the masking of this activation
domain in the entire polypeptide strongly suggests the existence of
intramolecular interactions between the TFIIB-like part of TFIIIB70 and
the CTE. We wondered if the strong pol II activator properties observed
with the CTE or some particular
131 fusions could be due to a
direct interaction with TFIIB (we have seen that
131-5TPR or
131-1TPR interacted strongly with the TFIIB-like part of
TFIIIB70). However, no interaction could be detected between TFIIB
fused to the GAL4 DNA binding domain and the reciprocal
131-5TPR,
131-1TPR, or CTE fusions. Despite their sequence homologies, TFIIB
and the TFIIB-like part of TFIIIB70 were not functionally
exchangeable.
(
)
131 appears closely related to the function of class
II transactivators that bind to TFIIB (10, 11, 12) and, as shown recently (34), induce
a conformation change in the factor molecule probably essential for the
subsequent steps of preinitiation complex assembly.
Table: Intrinsic activation properties of DNA binding
fusions proteins
131 derivatives, fused to the DNA binding domain,
were assayed with no counterpart.
Table: Interaction of wild type or mutant
TFIIIB70 proteins with 131 or COOH-terminal truncated
131
131 and TFIIIB70. Wild type or deleted derivatives of
TFIIIB70, fused in frame with the GAL4 DNA binding domain, were assayed
with the reciprocal
131 fusions. Explanations of the panel are the
same as in Fig. 3. ND, nondetermined.
-Galactosidase units in
parentheses indicate reciprocal combinations, with
131 fused to
the GAL4 DNA binding domain.
-D-galactoside; PAGE, polyacrylamide gel electrophoresis.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.