From the Division of Gene Expression, Department of Biochemistry, Wellcome Trust Building, University of Dundee, Dundee DD1 5EH, United Kingdom
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ABSTRACT |
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The general transcription factor TFIIB plays a
crucial role in selecting the transcription initiation site in yeast.
We have analyzed the human homologs of TFIIB mutants that have
previously been shown to affect transcription start site selection in
the yeast Saccharomyces cerevisiae. Despite the distinct
mechanisms of transcription start site selection observed in S. cerevisiae and humans, the role of TFIIB in this process is
similar. However, unlike their yeast counterparts, the human mutants do
not show a severe defect in supporting either basal transcription or
transcription stimulated by an acidic activator in vitro.
Transient transfection analysis revealed that, in addition to a role in
transcription start site selection, human TFIIB residue Arg-66 performs
a critical function in vivo that is bypassed in
vitro. Furthermore, although correct transcription start site
selection is dependent upon an arginine residue at position 66 in human
TFIIB, innate function in vivo is determined by the charge
of the residue alone. Our observations raise questions as to the
evolutionary conservation of TFIIB and uncover an additional function
for TFIIB that is required in vivo but can be bypassed
in vitro.
Transcription of a gene by RNA polymerase II (pol
II)1 requires the assembly of
the general transcription factors (GTFs) at the promoter to form a
preinitiation complex (PIC; reviewed in Ref. 1 and 2). The GTFs
assemble in an ordered fashion, beginning with the binding of TFIID to
the TATA element present at most promoters. TFIID is itself a
multiprotein complex of which one component mediates binding to the
TATA element (TATA-binding protein; TBP). TFIIA joins the forming PIC,
followed by TFIIB. The complex is then able to incorporate TFIIF/pol
II, and assembly is completed by further GTFs, including TFIIE and
TFIIH. This pathway of PIC assembly has been challenged by evidence
that some GTFs may exist as a partially preformed entity with pol II (a holoenzyme) that is recruited to the promoter in a single step (reviewed in Ref. 3).
TFIIB plays a pivotal role in the PIC, providing a bridge between
promoter-bound TFIID and pol II/TFIIF. Consistent with this, TFIIB has
been found to interact with several other GTFs including TBP, TFIIF,
and pol II (4). TFIIB is a 33-kDa peptide that contains a core
C-terminal domain composed of two imperfect direct repeats and an
N-terminal region that chelates zinc. Although intact TFIIB has evaded
structural analysis, structural data have been obtained for the
C-terminal domain in isolation (5, 6). The core C-terminal region is
mainly Biochemical and structural evidence suggests that, in native TFIIB, the
N- and C-terminal domains are engaged in an intramolecular interaction
(10-12). It is envisaged that to participate in a productive PIC,
TFIIB must undergo a conformational change that disturbs this
intramolecular interaction, exposing binding sites that are required
for further assembly of the PIC. Deletion analysis of human TFIIB
demonstrated that TFIIF interacts with the N terminus of TFIIB and pol
II with the core C-terminal region (4). Thus, a conformational change
in TFIIB may facilitate the interaction between TFIIB and pol
II/TFIIF.
Underlying the central role of TFIIB in PIC assembly, TFIIB has been
proposed as a target of transcriptional activator proteins (reviewed in
Ref. 1 and 2). At the adenovirus E4 promoter, TFIIB assembly is a
limiting event that can be facilitated by an activator protein (13).
Moreover, several activation domains can interact with TFIIB, and
in vitro evidence suggests that this interaction is required
for transcriptional activation (14). Significantly, the acidic
activation domain of the herpes simplex virus VP16 protein can induce a
conformational change in TFIIB that disrupts the intramolecular
interaction (12). This provides a possible mechanism by which
activators could stimulate further assembly of the PIC.
Once a PIC has assembled, the region around the transcription
initiation site melts to provide a template strand for pol II. The
actual site at which transcription initiation occurs is approximately 25-30 base pairs downstream of the TATA box in most eukaryotes (reviewed in Ref. 1). Where present, the TATA element is the sole
determinant of the transcription initiation site, and initiation will
occur at the distance set by TATA regardless of the sequence around the
site of initiation (15). One well studied exception to this is the
yeast S. cerevisiae, in which the transcription initiation
site can occur anywhere between 40 base pairs and 120 base pairs
downstream of the TATA element (reviewed in Ref. 1). Even so, open
complex formation occurs at a distance downstream of TATA similar to
that seen in other organisms. A scanning polymerase model has been
proposed in S. cerevisiae in which the PIC translocates downstream of the melted DNA and initiates transcription at distant sites (16). Schizosaccharomyces pombe exhibits a start site position akin to that seen in human cells, 25-30 base pairs downstream of the TATA element. Factor-swapping experiments between S. pombe and S. cerevisiae found that TFIIB and pol II
play a key role in determining the transcription initiation site (17).
TFIIB and pol II are not individually interchangeable between the two yeast species. However, when exchanged together, not only does this
result in full transcriptional activity in both species, but the start
site use reflects the species from which the TFIIB and pol II were
derived. Thus, TFIIB and pol II in concert determine the differences in
transcription initiation site between S. cerevisiae and
other eukaryotes.
A genetic screen in S. cerevisiae recovered mutations in
TFIIB that confer a bias toward the use of downstream transcription initiation sites (18). Two critical residues in yeast TFIIB were
analyzed; Glu-62 and Arg-78. Substitution of residue Glu-62 with an
amino acid of opposing charge (Lys) resulted in a yeast strain that
exhibited cold sensitivity and a slow growth phenotype at 30 °C
(19). Transcription at the CYC1 and ADH1 genes in this strain showed a
preference for the use of downstream initiation sites. The mutation of
residue Arg-78 to cysteine resulted in a similar phenotype to E62K, but
the mutant R78E resulted in a loss of viability. Because a double
mutant (E62K/R78E) was phenotypically similar to E62K and not R78E, it
was suggested that Glu-62 and Arg-78 may form a salt bridge. Glu-62 and
Arg-78 mutants of TFIIB are transcriptionally defective in a yeast
in vitro transcription system (10, 20-22). This can be
explained, at least in part, by their failure to interact with pol II
in several different assays.
As discussed above, the mechanism of transcription start site selection
in S. cerevisiae is distinct from that observed in most
other eukaryotes. In this study we analyze human TFIIB mutants homologous to the S. cerevisiae mutants described above both
in vitro and in vivo. Surprisingly, we find that
all the mutants are able to support both basal transcription and the
response to an activator protein. However, the mutants do exhibit a
downstream shift in transcription start site selection in a
promoter-specific manner. In vivo analysis confirmed the
importance of these residues in human TFIIB for correct transcription
start site selection. However, we demonstrate that mutation of a
particular residue in human TFIIB affects a function in addition to
transcription start site selection that manifests itself in
vivo but is bypassed in vitro. We discuss these
observations in light of current models of the role of TFIIB in both
PIC formation and transcription start site selection.
Plasmids--
The promoter DNA template G5E4T has been described
previously (23). G5ML contains nucleotides
Polymerase chain reaction-mediated site-directed mutagenesis was
performed to produce the E51R, E51D, E51A, R66E, R66K, R66A, and
E51R/R66E mutants. All clones were sequenced to ensure only the
intended mutations were present. cDNAs encoding wild type TFIIB and
the mutants (E51R, R66E, R66A, R66K, and E51R/R66E) were cloned into
the vector pCDNA3 for expression in 293 cells. The in
vivo vector expressing GAL4-RII has been described previously (24).
Protein Purification--
TFIIB and TFIIB mutants and GAL4-AH
were purified as described (4, 23). Polyhistidine-tagged TBP was
purified by nickel chelate affinity chromatography as described by the
manufacturer (Qiagen). The HeLa fraction containing RNA polymerase II,
TFIIF, TFIIE, and TFIIH was purified as described previously (25). Briefly, 20 ml of HeLa nuclear extract (10 mg/ml; Computer Cell Culture
Center, Mons Belgium) in buffer A (40 mM Tris, pH 7.9, 20%
(v/v) glycerol, 0.5 mM EDTA, 10 mM
In Vitro Transcription--
Anti-TFIIB antiserum was produced
against full-length recombinant TFIIB (Scottish antibody production
unit). HeLa nuclear extract was depleted of TFIIB by immunoaffinity
chromatography over a protein-G-Sepharose column containing covalently
linked TFIIB antibodies. Transcription assays using HeLa nuclear
extract or TFIIB-depleted nuclear extracts were performed as described (13) using the amounts of recombinant TFIIB indicated in the figure
legends. The reconstituted transcription assay was performed similarly,
except it contained 20 µl of the HeLa RNA pol II/TFIIF/TFIIE fraction, 20 ng of recombinant TBP, and the indicated amounts of TFIIB.
Transient Transfection Analysis--
Human embryonic kidney 293 cells were cultured as monolayers in Dulbecco's modified Eagle's
medium containing 10% fetal calf serum, 5 mM
L-glutamine, 100 mg/ml streptomycin, and 100 units/ml penicillin. Cells were transfected in 90-mm dishes at 50% confluency using calcium phosphate. Briefly, 1 µg of G5E4CAT (or G5MLCAT), 2 µg of RSV-BxGALII, and 2 µg of pCDNA3-TFIIB (or mutant) was diluted to 438 µl with water, and 61 µl of 2 M
CaCl2 was added. This was mixed with 500 µl of 2× HBS
(54.6 mM HEPES, pH 7.1, 0.274 M NaCl, and 1.5 mM Na2HPO4). The mixture was added
dropwise to a plate of cells, and the media was changed 24 h
later. Cells were harvested 48 h after transfection. For
immunoblot, equal volumes of cells were added to SDS-polyacrylamide gel
electrophoresis-loading dye, and the proteins were resolved by
electrophoresis. After transfer to Immobilon P membrane, immunoblotting
was performed with anti-T7 antibody (Novagen), which recognizes a tag
at the C terminus of the TFIIB derivatives. Detection was performed by chemiluminescence (ECL, Amersham Pharmacia Biotech). Total RNA was
prepared using an Rneasy kit as described by the manufacturer (Qiagen).
50 µg of total RNA was annealed to a 32P-labeled CAT
primer, and extension was performed as for the in vitro
transcription reactions. All transfections were performed at least
three times.
Human TFIIB Mutants E51R, R66E, and E51R/R66E Support Transcription
in Vitro--
The region of S. cerevisiae TFIIB that is
involved in transcription start site selection is very highly conserved
between species (Fig. 1A). We
have constructed single (E51R, R66E) and a double (E51R/R66E) point
mutants of human TFIIB that correspond to the S. cerevisiae
TFIIB mutants previously characterized by others to show defects in
transcription start site selection (19). The mutants were purified from
Escherichia coli and analyzed by SDS-polyacrylamide gel
electrophoresis and Coomassie staining, demonstrating that all of the
proteins were of equivalent purity (Fig. 1B). Wild type
TFIIB and the mutants were then tested in a transcription reaction
reconstituted from partially purified/recombinant factors and dependent
upon the addition of TFIIB. The promoter DNA used was the adenovirus
major late (AdML) promoter, and transcripts were detected by primer
extension. Fig. 2A shows that
the system is dependent upon the addition of recombinant TFIIB for the
production of accurately initiated transcripts (compare lanes
0 and wt IIB). E51R, R66E, and E51R,R66E were also able
to support a level of transcription equivalent to that seen with wild
type TFIIB. This is in contrast to the S. cerevisiae TFIIB
mutants corresponding to E51R and R66E, which showed a significant
defect in transcription in vitro (10, 20-22). Moreover, all
of the mutants utilized the same transcription initiation site as that
observed with wild type TFIIB.
In view of our finding that E51R, R66E, and E51R/R66E support
transcription in vitro at a level equivalent to wild type
TFIIB, we next tested the possibility that the mutants may be defective in supporting the levels of transcription required during
transcriptional activation. To monitor the high levels of transcription
in the presence of the activator GAL4-AH, we prepared a nuclear extract depleted of TFIIB by chromatography over a column containing
immobilized TFIIB antibodies. The TFIIB-depleted nuclear extract was
deficient in supporting significant levels of transcription in the
presence of GAL4-AH, but addition of wild type TFIIB restored activated levels of transcription (Fig. 2B). E51R, R66E, and E51R/R66E
also restored high levels of transcriptional activation. Thus, the human TFIIB mutants E51R, R66E, and E51R/R66E exhibit wild type transcriptional activity (with respect to level and initiation site) at
the AdML promoter both in the absence and presence of an activator protein.
TFIIB Mutants Cause a Downstream Shift in Initiation Site at the
Adenovirus E4 Promoter--
Transcription at the adenovirus major late
promoter largely occurs from a single site. We therefore tested the
adenovirus E4 promoter, at which transcription initiation naturally
occurs at multiple sites. Fig.
3A shows transcription assays
in a TFIIB-depleted extract in the presence of GAL4-AH at the AdE4
promoter. Consistent with the results we obtained with the AdML
promoter, E51R, R66E, and E51R/R66E supported a level of
transcriptional activation equivalent to wild type TFIIB. However,
there was a clear shift toward the use of downstream initiation sites
by all three of the mutants compared with wild type TFIIB.
Specifically, there was an increase in the use of distal transcription
initiation sites (indicated B and C) compared
with proximal (A). The same effect was observed when we used
GAL4-SP1 as the transcriptional activator (data not shown). To examine
this further, we resolved the different transcripts produced by wild
type TFIIB and E51R alongside a sequencing ladder of the AdE4 promoter
produced using the same radiolabeled primer (Fig. 3B).
Comparing the transcripts produced by wild type TFIIB with those
produced by E51R shows a clear shift toward the use of downstream
initiation sites. The sequence of this region of the AdE4 promoter is
shown in Fig. 3C, with arrows indicating the
sites of initiation in the presence of wild type TFIIB (shown above the
sequence) and E51R (shown below the sequence). The size of the
arrows indicates the relative use of each initiation site.
Thus, the role of human TFIIB residues Glu-51 and Arg-66 in
transcription start site selection is conserved between yeast and
mammals. However, unlike in S. cerevisiae, the human mutants
support transcription in vitro at a level equivalent to wild
type TFIIB.
Determinants of the Transcription Start Site Shift--
The TFIIB
start site mutations used above involve the substitution of charged
amino acids with residues of opposite charge. We next constructed
substitutions of Glu-51 and Arg-66 to either alanine (E51A and R66A) or
different amino acids of similar charge (E51D and R66K). The
recombinant proteins (shown in Fig.
4A) were tested in a
TFIIB-depleted nuclear extract at the AdE4 promoter (Fig.
4B). As seen with the original mutants, E51A, E51D, R66A, and R66K all supported transcription in vitro to a level
equivalent to that observed with wild type TFIIB. Furthermore, they all
caused a downstream shift in the transcription start sites. Thus, the charge per se of these residues is not the critical
determinant in correct transcription start site selection in
vitro.
Analysis of the Human TFIIB Mutants in Vivo--
The human
TFIIB start site mutants E51R, R66E, and E51R/R66E support levels of
basal and activated transcription in vitro similar to that
seen with wild type TFIIB. It would be predicted therefore that the
addition of an excess of one of the mutant TFIIB proteins to a native
nuclear extract would titrate the HeLa TFIIB and result in a downstream
shift in the transcription initiation site. We tested this possibility
and found that this was indeed the case (data not shown and Fig.
5A). We reasoned that this
effect would also occur in a living cell and therefore used a transient transfection assay to determine whether the start site shift effects we
observe in vitro could be recapitulated in vivo.
Transfection of G5E4T into living cells failed to produce a stable
message. We therefore used the same G5E4 core promoter but fused to
CAT, which allowed us to detect stable transcripts. Thus, it was first necessary to test G5E4CAT in vitro to ensure that we could
make a direct comparison with the in vivo effects. Fig.
5A shows a transcription assay using G5E4CAT and native
nuclear extract to which either wild type TFIIB, E51R, R66E, or
E51R/R66E had been added. Results comparable with those seen in a
TFIIB-depleted extract with G5E4T were obtained. Specifically, the
mutants caused a shift toward the use of distal transcription
initiation sites (B and C) with a concomitant
reduction in the proximal initiation sites (A). Next, wild
type TFIIB or the mutants (E51R, R66E, and E51R/R66E) under a
cytomegalovirus promoter were transfected into human embryonic kidney
293 cells along with the activator Gal4-RII and G5E4CAT. The cells were
harvested 48 h after transfection, total RNA was prepared, then
primer extension was performed to analyze the transcripts. A
representative assay is shown in Fig. 5B. There are several
striking features in this result. First, there is a clear difference in
the transcription start site pattern at the AdE4 promoter in
vivo compared with that seen in vitro. Essentially, the
most distal initiation sites seen in vitro (C) are not used in vivo. This cannot be explained by the
difference in cell type as nuclear extracts made from 293 cells produce
the same pattern of transcripts at the AdE4 promoter as HeLa cell nuclear extracts (data not shown). Second, in contrast to our in
vitro observations, R66E significantly inhibited transcription in
the transient transfection assay, but E51R and E51R/R66E did not. This
was not because of a difference in the levels of the various TFIIB
constructs, as immunoblotting showed that wild type TFIIB and the
mutants were of similar abundance (Fig. 5C). Finally, overexpression of E51R and E51R/R66E caused a shift toward the use of
the downstream transcription initiation sites. The intensities of the
two sets of transcripts (A and B) were quantified
and are expressed as a ratio (B/A) below each
lane. Thus, although there are greater constraints on the
pattern of transcription initiation at the AdE4 promoter in
vivo, there is a clear shift toward the use of the downstream
transcription initiation sites in vivo by the TFIIB mutants.
Moreover, the low level of E4 transcription remaining in cells
transfected with R66E also exhibited the downstream shift. We next
performed a set of transfections identical to those above but using
G5MLCAT as a reporter (Fig. 5D). As seen with the E4
promoter, R66E significantly inhibited transcription, but E51R and
E51R/R66E did not. Consistent with our in vitro data, only a
single transcript was produced from the AdML promoter, and the mutants
did not cause the use of an alternative site.
In view of the difference we observed in the properties of hTFIIB R66E
in vivo versus in vitro, we next
compared R66E with the mutants R66A and R66K in a transient
transfection assay (Fig. 6A).
As before, R66E inhibited transcription, with residual activity showing
a downstream shift in transcription initiation site. The mutant R66A
was also inhibitory, although we consistently found this mutant to be
less inhibitory than R66E. In agreement with our in vitro
observations, R66A also caused a downstream shift in the site of
transcription initiation. Interestingly, R66K did not significantly
inhibit transcription transcription in vivo, although it did
cause a downstream shift in the transcription initiation site. All of
the mutants were expressed at similar levels in the transfection assay,
as assessed by immunoblotting (Fig. 6B). Thus, although the
charge of hTFIIB residue 66 is critical in determining the innate
transcription function of TFIIB in vivo, it is not
sufficient for correct transcription start site utilization either
in vivo or in vitro.
In this study we have demonstrated that, despite the distinct
mechanisms in transcription start site selection observed in S. cerevisiae and mammals, the role of TFIIB in this process is highly conserved. The mutation of two highly conserved charged residues
in the N terminus of human TFIIB, homologous to those previously
described in the S. cerevisiae, caused a downstream shift in
transcription start site selection both in vitro and in vivo. The transcription start site shift caused by E51R,
R66E, and E51R/R66E was promoter-specific, as it occurred at the AdE4 promoter and not the AdML promoter. Unlike the AdML promoter, the E4
promoter intrinsically exhibits a more flexible transcription initiation site pattern. This observation is similar to previous findings with the S. cerevisiae TFIIB mutants, which caused
a transcription start site shift only at promoters that normally exhibit multiple transcription start sites (22, 26). It is likely that
alterations in transcription initiation site are not tolerated at the
AdML promoter. Thus, as the mutants are competent for innate
transcription function in vitro, they would appear to
function similarly to wild type TFIIB at the AdML promoter.
There are several charged residues in this highly conserved region of
TFIIB, suggesting that they may form a functional domain that interacts
with a similarly charged surface in another protein. Indeed,
alterations to the charge of this region invariably results in TFIIB
mutants that confer downstream shifts in the transcription initiation
site (10, 22)2. Interactions
involving complementary electrostatic charge are energetically neutral
as each domain is able to form a favorable interaction with water (27).
Once removed from water, charged/polar groups need to form ion pairs
and hydrogen bonds with a complementary surface. The net result does
not generally contribute to the affinity of the interaction but can
have effects on the specificity and, therefore, the shape of the
resulting complex. This raises the intriguing possibility that the
mutants we describe in this study cause a shift in the transcription
initiation site not because the interaction with pol II/TFIIF (or other
factors) is ablated but because of an alteration in the alignment of
the complex. Such a model would be consistent with our finding that the
human TFIIB start site mutants support transcription to a level
equivalent to wild type TFIIB in vitro. Structural analysis
of this region of TFIIB will shed light on the role of the charged
cluster domain of TFIIB in transcription start site selection.
The transcription start site shift induced by the various mutants used
in this study was the same. Presumably, any alteration in the
positioning of pol II at the promoter would be constrained relative to
TATA and, therefore, provide a limited number of potential transcription start sites. We also note that the TFIIB mutants are
still able to direct transcription initiation at all of the upstream
sites seen with wild type TFIIB, albeit to a lesser degree. It is
therefore likely that the role of TFIIB in the selection of the
transcription initiation site may be restricted to modulating the 3'
parameter, whereas other factors determine the 5' parameter. Indeed,
mutants of the S. cerevisiae RBP9 subunit of pol II cause an
upstream shift in transcription start site selection (28, 29).
The conservative TFIIB substitution mutants E51D and R66K also caused a
downstream shift in transcription start site selection. Thus, charge
per se within this region is not the sole determinant of
correct transcription start site selection. Alterations in the size of
the side chains of key amino acids may also have effects on the
alignment of a protein-protein interaction. Significantly, the residues
corresponding to human TFIIB Glu-51 and Arg-66 are totally conserved in
all species sequenced to date. Although other charged residues are also
totally conserved, some are substituted by residues of similar charge,
and still others, where the charge is removed altogether. It is
important to note that TFIIB exhibits a high degree of species
specificity. Unlike TBP, S. cerevisiae and human TFIIB are
not functionally interchangeable, even in basal transcription (30). In
addition, it has been reported that human TFIIB can substitute for
Drosophila TFIIB only at certain promoters (31). It is
possible that the differences in the charge cluster domain of TFIIB
contribute to this species specificity.
In contrast to our results with human TFIIB, homologous yeast TFIIB
mutants are defective for transcription in vitro (10, 20-22). This defect of the S. cerevisiae mutants correlates
with their inability to stably interact with RNA polymerase II in
several different assays. Deletion mapping studies of human TFIIB have shown that the N terminus (including the region studied in this work)
is dispensable for interaction with RNA polymerase II (4). However,
deletion of the N terminus of S. cerevisiae TFIIB abolishes the interaction with pol II (22). Furthermore, evidence has been
presented that the region of S. cerevisiae TFIIB responsible for transcription start site selection and direct interaction with pol
II may constitute distinct, but overlapping, domains within the N
terminus (22). Thus, it is possible that the nature of the contacts
between TFIIB and pol II (and/or other components of the general
transcription machinery) have diverged from S. cerevisiae to
humans. Perhaps yeast TFIIB requires a more intricate interaction with
pol II as part of the scanning mechanism responsible for transcription
start site selection in S. cerevisiae. Because S. pombe exhibits a transcription start site pattern similar to that
seen in human cells, analysis of the TFIIB-pol II interaction in this
species should help resolve this issue.
The human TFIIB mutant R66E was indistinguishable from E51R or
E51R/R66E in in vitro transcription assays. However, in
transfection analysis R66E was a potent inhibitor of transcription,
whereas E51R and E51R/R66E caused effects similar to those seen
in vitro. Importantly, the low level of transcription
observed in the presence of R66E in vivo exhibited a
downstream shift in the transcription initiation site. Thus, TFIIB R66E
is severely compromised in its ability to support transcription
in vivo in a manner that is bypassed in vitro.
Furthermore, this defect can be suppressed by the additional mutation
E51R. This compensation effect in vivo is consistent with
the previous observation that the S. cerevisiae homolog of human R66E is lethal but can be rescued by a second mutation
corresponding to human E51R (19). The conclusion of the observations in
S. cerevisiae was that these two residues of opposite charge
form a salt bridge. Our data are consistent with this proposal but also
suggest that the salt bridge has a function that is not critical for
the innate ability of TFIIB to support transcription of a naked DNA
template in vitro. Substitution of Arg-66 with lysine also
results in a TFIIB mutant that causes a downstream shift in
transcription initiation site both in vitro and in
vivo. However, this conservative substitution mutant does not
significantly inhibit transcription in vivo. Taken together,
these data suggest that Arg-66 plays a role in two functions; first,
transcription start site selection that is determined by the specific
amino acid side chain, and second, a role that is required in
vivo but not in vitro and is dependent upon the charge
of the residue only.
A function that is required of TFIIB in vivo, but is
bypassed in vitro, has several implications for our
understanding of the role of TFIIB in transcription initiation.
Moreover, our findings question the minimal requirements for a
functional PIC in vivo as compared with those observed
in vitro. Several GTFs are dispensable for transcription
in vitro using supercoiled DNA templates (reviewed in Ref.
2). TFIIF is one such GTF and is highly relevant in this case, because
both subunits (RAP30 and RAP74) can interact with TFIIB and more
specifically the N terminus of human TFIIB (4, 32). Furthermore,
suppressor mutants of the large subunit of TFIIF have been isolated
that can reverse the slow growth phenotype and transcription start site
defects of the yeast TFIIB mutant E62K (human Glu-51 (33)). However,
our preliminary data suggest that neither TFIIB E51R or R66E are
defective for interaction with RAP30 or RAP74 in
vitro.2 As discussed above, these mutations in TFIIB
may alter the specificity rather than abolish interactions with other
factors. It is possible that the RAP74 suppressor mutants restore the
alignment of the yTFIIB(E62R)-pol II/TFIIF interaction, leading to
normal transcription start site selection.
DNA topology may affect the ability of R66E to substitute the function
of wild type TFIIB in vivo. For example, R66E may be nonfunctional at a nucleosome-assembled promoter. Experiments in
vitro using nucleosomal DNA templates could be used to test this
hypothesis. It is also possible that the in vivo-specific defect of R66E involves the response to upstream transcriptional activators. Indeed, a S. cerevisiae protein (SUB1) similar
to the mammalian coactivator PC4 was isolated as a suppressor of the
yTFIIB R78H cold-sensitive phenotype (34). The prospect of an
additional function for TFIIB underscores the pivotal role that this
GTF plays in transcription. Further studies are currently in progress
to determine the function of TFIIB that is specifically required in
a living cell.
INTRODUCTION
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ABSTRACT
INTRODUCTION
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-helical, with the end of the first direct repeat forming a
basic amphipathic
helix. The second direct repeat contains a
surface cluster of hydrophobic amino acids and a helix-turn-helix motif
that interacts with a specific DNA sequence upstream of the TATA
element (7, 8). Structural information has also been obtained for the
immediate N terminus of Pyrococcus furiosus TFIIB, revealing
a
-sheet structure forming a zinc ribbon similar to that present in
the elongation factor TFIIS (9). The crystal structure of the TFIIB
C-terminal domain in a complex with TBP bound to DNA has been solved
(6). This structure suggested that the N terminus of TFIIB would
project downstream toward the region of transcriptional initiation.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
50 to +22 from the
adenovirus major late promoter cloned downstream of 5 GAL4 sites in the
vector pGEM3.
-mercaptoethanol, and 1 mM phenylmethylsulfonyl
fluoride) containing 0.1 M KCl was fractionated over a
15-ml phosphocellulose column. The 0.5 M KCl fraction (20 ml) was dialyzed against buffer A containing 0.1 M KCl.
This was applied to a 3-ml DEAE-cellulose column, which was washed
extensively with buffer A (0.1 M KCl) and then with buffer
A (0.25 M KCl). RNA polymerase II/TFIIF and TFIIE were
eluted in 5 ml of buffer A (0.3 M KCl) and dialyzed into
buffer D (20 mM HEPES, pH 8, 0.5 mM EDTA, 20%
glycerol, 1 mM dithiothreitol, and 1 mM
phenylmethylsulfonyl fluoride) containing 0.1 M KCl. This
fraction was devoid of significant levels of TFIIB as assessed by
immunoblotting, transcription, and electrophoretic mobility shift assay.
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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REFERENCES
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Fig. 1.
Mutagenesis of a highly conserved region at
the N terminus of human TFIIB. A, a diagram of human
TFIIB is shown with the two direct repeats indicated
(arrows) and a zinc-chelating structure at the N terminus
(Zn2+). Residues 40-70 of human TFIIB are shown aligned
with the homologous regions of TFIIB from the other species indicated.
Residues that are identical in all species are shaded. Human
TFIIB amino acids Glu-51 and Arg-66 are indicated by arrows.
B, recombinant wild type TFIIB (wt IIB), E51R,
R66E, and E51R/R66E were prepared from E. coli and examined
by SDS-polyacrylamide gel electrophoresis followed by Coomassie
staining. Molecular weight markers (kDa) are shown at the left.
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Fig. 2.
The human TFIIB mutants are functional at the
adenovirus major late promoter. A, wild type TFIIB
(wt IIB) or the indicated mutants (20 ng) were added to an
in vitro transcription reaction containing recombinant TBP
(20 ng) and a purified fraction containing RNA polymerase II, TFIIF,
and TFIIE (20 µl). Transcripts were detected by primer extension and
analyzed by denaturing electrophoresis. A diagram of the reporter
construct is shown below the autoradiogram. B, nuclear
extract (NE) depleted of endogenous TFIIB by immunoaffinity
chromatography was supplemented with either wild type TFIIB or the
indicated mutants (20 ng and 100 ng) and tested in the presence of the
transcriptional activator GAL4-AH (+).
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Fig. 3.
The human TFIIB mutants cause a downstream
shift in the transcription initiation site at the adenovirus E4
promoter. A, wild type (wt) TFIIB or the
indicated mutants were tested as in Fig. 2b but with the
adenovirus E4 promoter (shown below the autoradiogram). The three major
groups of transcripts are indicated (A, B, and
C). B, transcripts produced at the AdE4 promoter
with wild type TFIIB and E51R as shown in panel A were
resolved on a 10% sequencing gel alongside a sequencing ladder of the
same promoter. C, sequence of the AdE4 transcription
initiation region with arrows above and below, indicating
the relative intensity of each transcript produced in the presence of
wild type TFIIB and the mutant E51R, respectively.
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Fig. 4.
Determinants in TFIIB required for correct
transcription start site selection. A, recombinant
human TFIIB or the indicated mutants were purified from E. coli and analyzed by SDS-polyacrylamide gel
electrophoresis/Coomassie staining. B, wild type
(wt) TFIIB or the mutants shown in panel A were
tested in a TFIIB-depleted nuclear extract at the AdE4 promoter in the
presence of GAL4-AH as described in Fig. 3A.
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Fig. 5.
Analysis of the human TFIIB mutants E51R,
R66E, and E51R/R66E in vivo. A,
recombinant TFIIB or the indicated mutants (250 ng) were added to a
native HeLa cell nuclear extract, and transcription assays were
performed at the AdE4 promoter as described in Fig. 3. Transcript
groups A, B, and C are indicated. B, human embryonic kidney
293 cells were transfected with vectors expressing wild type TFIIB or
the indicated mutants under a cytomegalovirus promoter (2 µg)
alongside the reporter G5E4CAT and a vector driving the expression of
the transcriptional activator GAL4-RII. E4 transcripts among the total
RNA isolated from the cells was subsequently identified by primer
extension. Transcripts (A and B) are indicated,
and quantitation is expressed as a ratio B/A is shown below
each lane of the autoradiogram. C, immunoblot of
total cell lysates prepared from cells transfected as in part B with
anti-T7 antibody, recognizing an epitope engineered into the C terminus
of the TFIIB (and mutant) constructs. Molecular weight markers (kDa)
are shown at the left. D, as in panel B except
that the AdMLCAT promoter construct was used.
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Fig. 6.
Analysis of the human TFIIB mutants R66A and
R66K in vivo. A, 293 cells were
transfected with 2 µg of the indicated TFIIB expression vectors,
G5E4CAT and, where indicated, GAL4-RII. Specifically initiated
transcripts were detected by primer extension of total RNA isolated
from the transfected cells. Transcript groups A and
B are indicated, with quantitation expressed as
B/A shown below each lane. B, cell
lysates were immunoblotted to verify expression of the mutants as in
Fig. 6B. wt, wild type.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We would like to thank Lynn McKay for help with tissue culture and Neil Perkins for providing 293 cells. We are grateful to Angus Lamond, Tom Owen-Hughes, Neil Perkins, Joost Zomerdijk, and members of the lab for their critical reading of the manuscript.
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FOOTNOTES |
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* This work was funded by the Wellcome Trust (047674/Z/96/Z).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by a research studentship from the Biotechnology and
Biological Sciences Research Council.
§ A Research Career Development Fellow of the Wellcome Trust. To whom correspondence should be addressed. Tel.: 01382 344248; Fax: 01382 345783; E-mail: sgeroberts{at}bad.dundee.ac.uk.
2 N. A. Hawkes and S. G. E. Roberts, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: pol II, RNA polymerase II; GTF, general transcription factor; PIC, preinitiation complex; TBP, TATA box-binding protein; RAP, RNA polymerase II-associated protein; CAT, chloramphenicol acetyltransferase; AdML, adenovirus major late promoter.
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