Received 11 October 1999/Returned for modification 8 November
1999/Accepted 23 November 1999
We have investigated the contribution of specific TATA-binding
protein (TBP)-TATA interactions to the promoter activity of a
constitutively expressed silkworm tRNACAla gene and
have also asked whether the lack of similar interactions accounts for
the low promoter activity of a silk gland-specific tRNASGAla gene. We compared TBP binding,
TFIIIB-promoter complex stability (measured by heparin resistance), and
in vitro transcriptional activity in a series of mutant
tRNACAla promoters and found that specific TBP-TATA
contacts are important for TFIIIB-promoter interaction and for
transcriptional activity. Although the wild-type
tRNACAla promoter contains two functional TBP binding
sequences that overlap, the tRNASGAla promoter lacks
any TBP binding site in the corresponding region. This feature appears
to account for the inefficiency of the tRNASGAla
promoter since provision of either of the wild-type TATA sequences derived from the tRNACAla promoter confers robust
transcriptional activity. Transcriptional impairment of the wild-type
tRNASGAla gene is not due to reduced incorporation of
TBP into transcription complexes since both the
tRNACAla and tRNASGAla promoters form
transcription complexes that contain the same amount of TBP. Thus, the
deleterious consequences of the lack of appropriate TBP-TATA contacts
in the tRNASGAla promoter must come from failure to
incorporate some other essential transcription factor(s) or to
stabilize the complete complex in an active conformation.
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INTRODUCTION |
The silkworm Bombyx mori
provides a clear example of regulated tRNA gene expression. The demand
for fibroin, the principal protein of silk, requires highly efficient
transcription and translation of the fibroin gene in cells of the silk
gland. Translational efficiency in these cells is maximized by the
quantitative adaptation of the tRNA population to the composition of
fibroin: 44% glycine, 29% alanine, and 12% serine (5, 29, 30,
42). In the case of tRNAAla, enrichment is achieved
both by increasing the level of the constitutive type of
tRNAAla (tRNACAla) and by synthesizing an
additional, silk gland-specific, type (tRNASGAla)
(31, 44). In vitro studies of representative
tRNACAla and tRNASGAla genes have
revealed transcription properties consistent with the patterns of
tRNACAla and tRNASGAla accumulation in
vivo. That is, in a variety of extracts from non-silk gland cells, the
tRNACAla gene directs transcription much more
efficiently than does the tRNASGAla gene, but in
concentrated extracts from silk gland, the two genes are equally
efficient (60). To understand how these two genes are
differentially regulated, we have investigated the basis of the
transcriptional impairment of the tRNASGAla gene that
is observed under typical in vitro conditions.
Transcription of silkworm tRNAAla genes is driven by both
internal and external promoter elements (26, 36, 56). The
critical difference between the tRNACAla and
tRNASGAla genes is in the interaction of their 5'
flanking promoter elements with the transcription factor complex,
TFIIIB (47). Although the tRNASGAla gene can
direct the addition of TFIIIB to a TFIIIC/D-promoter complex, as judged
by band shift, upstream extension of the TFIIIC/D footprint is not
observed when TFIIIB binds the tRNASGAla promoter as it
is when TFIIIB binds the tRNACAla promoter
(59). TFIIIB is a multiprotein complex containing a general
transcription factor, TATA-binding protein (TBP), and its RNA
polymerase III (Pol III)-specific associated factors (reviewed in
references 10, 41, and 54). In
yeast, TFIIIB is thought to consist of three components, TBP, BRF, and
B" (19, 38). In higher eukaryotes, the human homologue of
BRF has been cloned and shown to function with TBP in transcription of
VAI, tRNA, and 5S RNA genes (32, 53), and a putative
Drosophila BRF homologue has been identified by association
with TBP (50). No homologue of yeast B" has yet been
identified in another system.
TFIIIB is the initiation factor for Pol III (18) and is
analogous to the Pol II-specific TBP-containing factor, TFIID. The means by which these two factors are recruited to promoters are not the
same, however. TFIID is recruited to classical, TATA-containing, Pol II
promoters by direct interaction between the TBP subunit and an upstream
TATA element, and the promoter activity in such cases is strongly
affected by mutation of the TATA element (57). The mechanism
of TFIIIB association with Pol III promoters that drive 5S or tRNA
transcription is not so well understood, largely because these
promoters typically lack an obvious TBP binding site. It has been
suggested, therefore, that TBP either contacts such Pol III templates
through interactions that are not sequence specific or is incorporated
into the transcription complex entirely through protein-protein
interactions (46). This interpretation is supported by the
fact that a mutation in the DNA binding domain of yeast TBP that
renders it deficient for Pol II transcription does not impair either 5S
or tRNA transcription (40).
On the other hand, silkworm Pol III promoters frequently contain
AT-rich sequences that resemble TBP binding sites (36), although they vary in sequence and location with respect to the transcription initiation site. To determine whether the different abilities of tRNACAla and tRNASGAla
promoters to interact with TFIIIB result from differences in their
interaction with TBP, we analyzed the effects of promoter mutation on
TBP binding, on the stability of TFIIIB-promoter complexes, and on
transcriptional activity. For the tRNACAla promoter, we
find that TBP binds within an AT stretch between 
32 and 23, and
optimal interaction increases both TFIIIB-promoter complex stability
and transcriptional activity. In contrast, although the wild-type
tRNASGAla promoter binds TBP with reasonable affinity,
the TBP binding site is not optimally positioned and does not stabilize
the TFIIIB-promoter complex or contribute to promoter activity. Our
results indicate that specific TBP-TATA contact is a key determinant of
tRNACAla gene promoter strength and that the lack of
this contact at the required location explains the relative
inefficiency of the tRNASGAla promoter.
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MATERIALS AND METHODS |
Cloned genes used in this work.
The wild-type
tRNACAla and tRNASGAla promoter
constructs were described previously (59), and mutants were
created either by PCR with mutagenic primers or by recombinant PCR
(11).
Recombinant silkworm TBP.
Recombinant silkworm TBP was
expressed in Escherichia coli and purified as previously
described (35).
Crude extracts and Pol III transcription fractions.
Transcription was catalyzed by extracts derived from B. mori
ovaries (33). Fractionated transcription machinery was
derived from silk gland extracts. Pol III was fractionated as described in reference 34, and TFIIIC/D was fractionated as
described in reference 47. TFIIIB was isolated from
the 300 mM KCl elution step from the heparin-Sepharose column used to
isolate Pol III, and fractions containing TFIIIB activity were pooled
and dialyzed against buffer H containing 50 mM KCl.
Assays. (i) TBP-TATA binding.
Binding reactions and analysis
by gel retardation were performed as previously described
(35). For competition assays, the DNA fragments used were
PCR amplified products containing either the adenovirus major late
promoter (Ad MLP) (145 bp) or derivatives of the
tRNACAla (131 bp, extending from 91 to +40) or the
tRNASGAla (138 bp, extending from 98 to +40)
promoter. In each reaction mixture, radioactively labeled Ad MLP
fragment at a concentration of 1 nM was incubated with increasing
amounts of nonradioactive competitor fragments in the presence of an
amount of recombinant TBP (8 nM total protein based on Bradford assay)
that was found empirically to be limiting. From at least three
experiments, the data points obtained in the presence of competitor
were normalized to the points obtained in the absence of competitor,
and the mean and standard deviation for each point were calculated. The
most probable straight line (based on a least squares fit) was drawn through the data points plotted as the reciprocal of the fraction bound
versus the molar ratio of competitor to labeled fragment. Relative
affinity of different competitors was then obtained from the relative
slopes of these lines.
(ii) Heparin-resistant TFIIIB-promoter complexes.
The
interaction between TFIIIB and promoter DNA was examined by
quantitating heparin-resistant TFIIIB-promoter complexes. A 262-bp
tRNAAla gene-containing DNA fragment (extending from 91
to +171) was amplified by PCR, using radioactively labeled primers. A
transcription factor complex was assembled on this fragment in a
20-µl reaction mixture that contained 5 fmol of labeled DNA, 4 µg
of dG-dC, 5 µl of TFIIIC/D, and, if included, 7.5 µl of TFIIIB. The
final concentrations of buffer components were 70 mM KCl, 30 mM
Tris-HCl (pH 7.5), 4 mM MgCl2, 13% glycerol, and 3 mM
dithiothreitol. After incubation for 1 h, the complex was
separated from unbound components by native gel electrophoresis as
described in reference 59. Components other than
TFIIIB were removed by adding heparin to a final concentration of 100 ng/µl for 20 to 30 s before loading the gel.
(iii) Quantitative binding analysis.
Band shift gels were
scanned on a Storm 860 PhosphorImager at 200-µm resolution and
analyzed by ImageQuant v1.1 software (Molecular Dynamics). The number
of heparin-resistant TFIIIB-promoter complexes formed on each template
was first normalized to the number of unstripped complexes
(TFIIIB/C/D-promoter complexes) formed on the same template and then
expressed as a percentage of the number of heparin-resistant complexes
on a wild-type tRNACAla promoter measured in the same experiment.
(iv) Footprinting.
Footprints of bound transcription factors
were obtained by DNase I as described previously (59) or
hydroxyl radical cleavage (4).
(v) In vitro transcription.
Transcription reactions were
performed as previously described (36). Each 20-µl
reaction mixture contained 5 µl of oocyte extract, 2 ng of
gene-containing plasmid, and nonspecific DNA (pUC13M
[36]) to bring the total amount of DNA to 200 ng.
Transcripts were detected autoradiographically after resolution by gel
electrophoresis as described elsewhere (33).
(vi) Transcription complex isolation and Western blotting.
Binding reaction mixtures assembled and incubated as described above
for heparin-resistant TFIIIB-promoter complexes were loaded onto a
1.5% agarose gel in 50 mM Tris-borate (pH 8.0)-5 mM EDTA-5%
glycerol. To optimize resolution, the agarose gel was prepared in a
vertical apparatus containing a layer of 5% acrylamide at the bottom
to retain the agarose. The gel was prerun at 150 V for 60 min, and the
samples were fractionated at 150 V for 60 to 90 min. Band shift
complexes were detected autoradiographically and excised from the gel.
Agarose was removed by digestion with Gelase (Epicentre Technologies)
at 42°C overnight according to the manufacturer's directions. The
samples were concentrated by precipitation with 10% trichloroacetic
acid (Sigma) and resuspended in the sodium dodecyl sulfate-containing
sample buffer used for standard protein gels (39). The
amount of complex was standardized by quantitating the labeled DNA
fragment, and proteins in the complexes were resolved by sodium dodecyl
sulfate-11% polyacrylamide gel electrophoresis for Western analysis.
Generally 2 to 3 fmol of complex was obtained from a standard 20 µl
band shift reaction. Western analysis was performed by using a Bio-Rad
modular mini-protein II system following the standard procedure
(39). TBP was detected by incubating with antibodies against
silkworm TBP, followed by chemiluminescence detection of goat
anti-rabbit immunoglobulin G (heavy plus light chain)-horseradish
peroxidase conjugate (Bio-Rad) (51).
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RESULTS |
Recombinant silkworm TBP is able to bind both
tRNACAla and tRNASGAla upstream
promoters but at different positions.
To understand the role of
TBP-TATA contacts in tRNACAla and
tRNASGAla upstream promoters, we first asked whether
silkworm TBP by itself is able to bind these AT-rich, but different,
sequences (Fig. 1A). Band shift assays
revealed that recombinant silkworm TBP binds both wild-type promoters
(Fig. 1B, lanes 2 and 4). To determine the relative affinities of
tRNACAla and tRNASGAla promoters for
TBP, we compared the ability of these promoters to compete with the Ad
MLP TATA element for TBP binding. Silkworm TBP was previously
determined to bind Ad MLP with the same affinity as does yeast TBP
(2 × 109 M
1) (12, 35). As
shown in Fig. 1C, silkworm TBP binds both tRNA promoters with high
affinity, about twofold (tRNACAla) and sixfold
(tRNASGAla) below its affinity for Ad MLP.
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FIG. 1.
Cloned silkworm TBP binds both tRNACAla
and tRNASGAla promoters. (A) Diagram of the DNA
fragments used for TBP binding assays. The tRNACAla and
tRNASGAla promoters are designated as C and SG,
respectively, in all figures. Vector
( ) and
wild-type tRNACAla
( ) or
tRNASGAla
( ) DNA
are shown. Upstream promoter sequences are in capital letters, and
positions relative to the transcription initiation site (arrow) are
numbered. The D (distal), TAT (TATAT), I (intervening), and AT (AATTTT)
regions of the tRNACAla promoter are delineated by
brackets at the top. (B) Cloned silkworm TBP binds specifically to both
tRNACAla and tRNASGAla promoters.
Silkworm TBP (5 nM) was incubated with radioactively labeled wild-type
(C WT; SG WT) or mutant tRNAAla (C mut.,
29CGGC26; SG mut.,
10CGGC7) promoter fragments (0.2 nM), and
the complexes were resolved on a nondenaturing gel. The locations of
the mutations on both promoters are within the protected sequences
bracketed in Fig. 2. The positions of TBP-bound fragments (TBP-DNA) and
unbound fragments (unbound DNA) are marked on the left. (C) Relative
affinities of TBP for the Ad MLP and the tRNACAla and
tRNASGAla promoters, determined by competition band
shift assays. Silkworm TBP and radioactively labeled Ad MLP fragments
were incubated with increasing amounts of unlabeled DNA fragments
corresponding to the Ad MLP or the tRNAAla promoters
diagrammed in panel A. Data points corresponding to the reciprocal of
the fraction of labeled DNA bound from seven experiments were
normalized to the same origin, and the mean and standard deviation for
each point were calculated. The relative affinities of
tRNACAla and tRNASGAla promoters to Ad
MLP were estimated by comparing the slopes of least-square-fitted
straight lines: tRNACAla/Ad MLP  1/2 and
tRNASGAla/Ad MLP 1/6.
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To determine the location of bound TBP, we used both hydroxyl radical
and DNase I footprints. As shown in Fig.
2, TBP binds the two promoters in
different places, ~32 to 23 in the tRNACAla
promoter and 10 to 3 in the tRNASGAla promoter, and
at each site generates both protection from and hypersensitivity to
cleavage. Loss of TBP binding upon mutation of these sequences
confirmed that they are specifically required for binding (Fig. 1B,
lanes 3 and 5). The footprints in Fig. 2 are typical in showing
ill-defined boundaries for the tRNACAla binding site.
The inefficiency of DNase I cleavage of AT-rich sequences is partly
responsible, but even hydroxyl radical cleavage, which is not base
specific, does not display a sharp boundary between protected and
unprotected sequences. In contrast, the tRNASGAla
sequence protected from hydroxyl radical cleavage is sharply defined,
and the boundaries are unambiguous.
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FIG. 2.
TBP binds tRNACAla and
tRNASGAla promoters in different positions. (A) Diagram
of the DNA fragments used for TBP binding assays. Diagrammatical
symbols are the same as Fig. 1A. Footprints obtained on both strands by
hydroxyl radical (light gray) or DNase I (black) cleavage are
summarized by bars (protection) and dots (hypersensitivity). (B) Gels
showing the cleavage of the noncoding strand of the two promoters
either by DNase I or by hydroxyl radicals (HR). Lanes: G, partial
chemical cleavage at G residues; Free and TBP, cleavage of unbound and
TBP-bound fragments, respectively. The extent of each promoter is shown
by the solid line on the left with the initiation site labeled with an
arrow, the extent of the primary transcript indicated by a rectangle,
and vector sequences shown by dashed lines. Protected sequences are
delineated by brackets on the right.
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Silkworm TBP can bind alternative overlapping sites in the
tRNACAla promoter.
Since the 10-bp protected
region in the tRNACAla promoter (Fig. 2) is longer than
the sequence of 7 specific bp defined by mutational and
crystallographic data (15), we wondered whether this region might contain more than one TBP binding site. Heterogeneity in the
location of bound TBP would account both for the longer protected region and for the fuzziness of the footprint boundaries. Inspection of
all of the 7-bp sequences within the TBP footprint revealed three
candidates, based on TBP binding preferences inferred from Pol II
transcription (57) or direct binding measurements
(45, 58). Two candidates read on the top strand are
31TTTATAT25 (TL [top left])
and 29TATATTA23 (TR [top
right]), and one read on the bottom strand is
26TATAAAG32 (B
[bottom]). We considered the other 7-bp segments unlikely because
they contain either C or A in the first position or A in the third
position, and these departures from a canonical TATA are known to
reduce TBP binding (45). We tested the binding ability of
the three strongest candidates by using each of them to replace the
wild-type TATA sequence in Ad MLP. Band shift assays verified that TBP
is able to bind each of these sequences with nearly equal affinity,
although we consistently observed more binding to TL than to TR or B. Mutant sequences confirmed the specificity of the observed binding
(Fig. 3A).
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FIG. 3.
Specific TBP-TATA interaction contributes to wild-type
tRNACAla promoter activity. (A) Cloned silkworm TBP is
able to interact with at least three TATA sequences within the
footprinted region of tRNACAla promoter. Three
potential binding sites, two
(31TTTATAT25 [TL] and
29TATATTA23 [TR]) read on the
top strand and one (26TATAAAG32
[B]) read on the bottom strand, were cloned into Ad MLP to
replace its wild-type TATAAAA (ML-WT) sequence and tested
for TBP binding. For each construct, patterns of TBP binding to
wild-type and mutant versions (ML mut., TGTAAAA; ML-TL mut.,
TGTATAT; ML-TR mut., TGTATTA; ML-B mut.,
TATAAAC) were compared. Silkworm TBP (5 nM) was incubated
with radioactively labeled fragments (0.2 nM), and the complexes were
resolved on a nondenaturing gel. The kind of labeled fragment is marked
at the top, and the positions of TBP-bound fragments (TBP-DNA) and
unbound fragments (unbound DNA) are marked on the left. The percent
labeled DNA bound, shown below each lane, is an average of multiple
experiments. (B) Effects of promoter mutations on TBP binding. The
positions of the TBP binding sequences within the
tRNACAla promoter are diagrammed at the top, and a
representative band shift gel illustrating the effect of all the
promoter mutations on TBP binding is shown below. All symbols are the
same as in previous figures. The percent labeled DNA bound is the
average of multiple experiments. (C) Quantitative comparison of the
effects of mutation on affinity for TBP and transcriptional activity.
The wild-type (WT) promoter is represented by a dark gray bar, and
mutant sequences are shown in capital letters on a lighter gray
background. The two top-strand TATA sequences are delineated by black
and gray brackets above each promoter. The relative affinity for TBP
was determined by at least three competition assays as described in
Fig. 1C. (D) Quantitative comparison of the effects of mutation on
affinity for TBP and transcriptional activity for promoter constructs
containing an isolated TATA sequence. In the constructs, sequences of
the isolated wild-type or mutant TATA elements are shown in capital
letters. The G-C base pairs used to isolate each TATA element are shown
against a medium gray background, and asterisks indicate positions
within the TATA that were mutated.
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To determine whether TBP has a preference for one of
these sequences in the context of the tRNACAla
promoter, we examined the effects on binding of block and single base
pair mutations in and around the TBP footprint. As shown in Fig. 3B,
binding to all three potential binding sites should be affected by
substitution of the TAT region, and the strong deleterious effect of
the TAT mutation supports this idea. In contrast, only
two sites (TL and B) in D (distal region) mutants and only one
(TR) in I (intermediate region) mutants should be affected. Figure 3B
shows that neither a D nor an I mutation reduces affinity for TBP.
These results are consistent with the possibility that TBP is capable
of binding multiple sites within the tRNACAla promoter
and that binding is not significantly impaired in mutants that retain
at least one functional site. Since the block mutations vary in the
extent to which they alter different binding sites, however, we could
not exclude the possibility of a single strongly preferred site.
Therefore, we dissected the region more systematically with single G-C
or C-G base pair substitutions at positions 32, 31, 30,
28, 25, 24, and 23. As shown in Fig. 3B and C, mutations at
32, 24, and 23, which affect either site B
(26TATAAAG32) or site TR
(29TATATTA23) but not site TL
(31TTTATAT25), have no effect on
TBP binding. Mutations at 31 and 30, which affect sites TL and B
but not site TR, reduce TBP binding, but only modestly (to ~60% of
the wild-type level). Similarly, a mutation at 25, which affects both
TR and TL but not B, reduces TBP binding somewhat (to ~50% of the
wild-type level). In contrast, a mutation at position 28, which
affects key residues in all three binding sites, reduces TBP binding
dramatically (to 5% of the wild-type level). Taken together, the data
argue that TBP is capable of binding alternative sites within the
tRNACAla promoter and that binding is greatly impaired
only by mutations that alter all of the sites. The modest reduction
caused by mutations at 31, 30, and 25 is consistent with the
idea, suggested in Fig. 3B, of inherent affinity differences among the
three sites. Specifically, for these mutants, the remaining unimpaired
site (TR or B) is expected to be slightly weaker than the TL site
available in a wild-type promoter.
Specific TBP-TATA interaction contributes to wild-type
tRNACAla promoter activity.
We next wanted to know
whether TBP interaction with any or all of these sites matters for
transcription. If so, mutations that reduce TBP binding should also
reduce transcription. As shown in Fig. 3C, the overall patterns of
mutant effects on TBP binding and transcription are similar, although
in most cases, the effects on transcription are smaller. For instance,
the TAT mutant essentially eliminates TBP binding and
reduces transcription to ~20% of the wild-type level. Single base
pair substitutions at 32, 24, or 23, which do not affect TBP
binding, do not affect transcription, and three of the substitutions
that reduce TBP binding (30, 28, and 25) also reduce
transcription. Of these, the mutation at 25 is most deleterious to
transcription. Since position 25 is outside the bottom-strand TATA,
this result suggests that transcription does not depend strongly on the
bottom TATA. Consistent with this interpretation, there is no
transcriptional phenotype associated with a mutation that is unique to
the bottom TATA (32). Since all of the mutations that do have
transcriptional phenotypes affect one or both of the top-strand TATAs,
it appears that the interaction of TBP with these sites is
transcriptionally important.
To test the function of the top-strand TATAs directly, we designed
constructs in which a tRNACAla promoter was provided
with only one or the other of them. As controls, we constructed mutant
versions of these isolated TATAs that were expected to have reduced
affinity for TBP because of sequence changes at the second position
(31TGTATAT25 and
29TGTATTA23). Figure 3D shows that
both of the wild-type isolated TATAs have affinities for TBP close to
that of the wild-type tRNACAla promoter, although
TBP binds slightly better to TL than to TR. Mutation of either
site reduces binding. Figure 3D also shows that both of the
isolated wild-type TATAs support efficient transcription, with a
preference for TL (96% ± 5% of the wild-type level) over TR (69% ± 5% of the wild-type level), and that mutation of either TATA reduces
transcriptional activity. These results suggest that, as part of a
transcription complex, TBP can interact productively with either
of the two top-strand TATAs.
Both specific TBP-TATA interaction and optimal geometry are
required for full stability of TFIIIB-promoter complexes.
Since
TBP functions in Pol III transcription as part of the TFIIIB complex,
the role of specific TBP-TATA contacts may be to ensure proper
association of TFIIIB with the template. If so, mutations that disrupt
the sites contacted by TBP in the transcription complex should weaken
the TFIIIB-template interaction. As a sensitive probe of this
interaction, we wanted to analyze complexes consisting simply of TFIIIB
and DNA, without the potentially stabilizing influence of the remainder
of the transcription complex. We had to circumvent the problem,
however, that silkworm TFIIIB does not bind to tRNA promoters by
itself. As in all other systems tested, prior binding by TFIIIC
(TFIIIC/D in the silkworm system) is required (reviewed in references
6, 10, and 54).
We therefore generated TFIIIB-promoter complexes by using heparin or
KCl to strip complete complexes of the other transcription factors, an
approach that was pioneered with yeast (18). Figure 4A shows the generation of
heparin-stripped complexes containing silkworm transcription factors
bound to the tRNACAla promoter. DNase I footprints of
these complexes (Fig. 4B) demonstrate that the binding of TFIIIC/D
alone protects sequences downstream from the tRNACAla
transcription initiation site and causes hypersensitivity at the
initiation site (+1), whereas the addition of TFIIIB extends protection
to at least 35, as previously reported (59). The complexes
that remain after stripping contain TFIIIB only, since upstream (10
to 40) but not downstream sequences are protected. The stripped
complexes are also hypersensitive to DNase I at three new sites, 2,
5, and 6, and their overall patterns of protection and
hypersensitivity strongly resemble those of yeast TFIIIB-promoter complexes (18, 20).
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FIG. 4.
Heparin treatment of tRNACAla
transcription complexes yields TFIIIB-promoter complexes. (A) Diagram
of the procedure and the heparin resistance of TFIIIB-promoter
complexes. Radioactively labeled tRNACAla promoter
fragments (5 fmol) were incubated with a fraction containing TFIIIC/D
or with this fraction plus TFIIIB for 1 h. These mixtures were
either loaded directly on a nondenaturing gel (lanes 2 and 4) or
treated with heparin (100 ng/µl) for 20 to 30 s prior to loading
(lanes 5 and 6). After electrophoresis, the complexes were visualized
by autoradiography. The identities of the major protein-DNA complexes
(indicated at the left) were determined by DNase I footprinting. (B)
DNase I footprints of the transcription factor-promoter complexes shown
in panel A. The identities of the protein-DNA complexes are shown at
the top. Lanes marked G show partial chemical cleavage at G residues.
The full extent of promoter sequences is shown by the solid line on the
left, with the transcription initiation site labeled with an arrow and
the extent of the primary transcript indicated by a rectangle.
Sequences protected by TFIIIC/D alone (solid brackets) or by TFIIIB
(dashed bracket) in the presence or absence of TFIIIC/D are indicated
on the right. DNase I hypersensitivity induced by TFIIIC or TFIIIB is
marked by an asterisk or dots, respectively. The light gray bracket
shows the hypersensitive region of TFIIIB-promoter complexes.
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In preliminary experiments, we had observed promoter-specific variation
in the amounts of stripped TFIIIB-promoter complex obtained. Since we
started with identical amounts of complete complex, the variation had
to reflect differences in the ability of TFIIIB bound to certain
promoters to withstand challenge by heparin or gel electrophoresis. We
reasoned, therefore, that quantitation of the amounts of
TFIIIB-promoter complex that survive stripping would measure the
relative stability of the TFIIIB interaction with different promoters.
Using this assay, we carried out a systematic analysis of the effects
of tRNACAla promoter mutations on the stability of
TFIIIB-promoter complexes. Figure 5A
shows a representative sample of the primary data, and Fig. 5B quantitates all of the data and compares them to the effects of
the same mutations on transcriptional activity. Mutation of either the
TAT or the D region eliminates detectable heparin-resistant TFIIIB-promoter complexes, whereas mutation of the I region reduces the
amount only twofold. We estimate the lower limit of detection to be
~10% of the amount of complex formed on the wild-type
tRNACAla promoter. Single mutations from 31 to 25
all have strong deleterious effects on TFIIIB-promoter complex
stability (<20% of wild-type heparin resistance remains), but
mutations outside this region (32, 24, and 23) have much smaller
effects or none at all. These results suggest that the wild-type TL
TATA (31TTTATAT25) makes a major
contribution to the formation of TFIIIB-promoter complexes that are
stable to heparin. This idea is reinforced by the results with
constructs containing isolated TATAs, shown in Fig. 5C. The two
constructs containing wild-type TATAs differ significantly in the way
they bind TFIIIB. Heparin-resistant TFIIIB-promoter complexes are
detectable on promoters containing the isolated TL sequence but not on
those containing the isolated TR sequence.
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FIG. 5.
Both specific TBP-TATA interaction and optimal geometry
are required for full stability of TFIIIB-promoter complexes. (A)
Effects of tRNACAla promoter block and point mutations
on the heparin resistance of TFIIIB-promoter complexes. The formation
and resolution of transcription factor-promoter complexes were as
described for Fig. 4. The kind of promoter (wild type [WT] or mutant)
is shown at the top, and the resolved complexes (TFIIIB-DNA,
TFIIIC/D-DNA, and TFIIIB/C/D-DNA) and unbound DNA are indicated on the
left. Two mutants (30G and 28G) were not included in the experiment
shown. (B) Quantitative comparison of the effects of mutation on
affinity for TBP and on the heparin resistance of TFIIIB-promoter
complexes. The promoter constructs are the same as in Fig. 3. The
relative affinity for TBP was determined by at least three competition
assays as described for Fig. 1C. The relative heparin resistance of
TFIIIB-promoter complexes was determined by measuring the amount of
protein-DNA complex after heparin treatment. The means and standard
deviations based on at least three determinations are shown. (C)
Quantitative comparison of the affinity for TBP and the heparin
resistance of TFIIIB-promoter complexes for promoter constructs
containing an isolated TATA sequence. TL and TR are the constructs
containing the isolated sequence,
31TTTATAT25 or
29TATATTA23, respectively. TL
mut. and TR mut. are mutants in which the second position of the
wild-type TATA has been replaced by G. Mutants in which the seventh
position was replaced by C also exhibited reduced TBP binding and
TFIIIB-DNA complex stability (data not shown). (D) Quantitative
comparison of the affinity for TBP, the heparin resistance of
TFIIIB-promoter complexes, and transcriptional activity for
tRNACAla promoter constructs containing interchanged
isolated TATA sequences. TR L
(31TATATTA25) and TLR
(29TTTATAT23) are constructs
containing isolated TATA sequence with switched positions.
|
|
Based on the preferences of Drosophila Pol III
(52), we anticipated that the second-position T might give
an advantage to the TL sequence over the TR sequence. To test the
importance of sequence versus position directly, however, we
interchanged the sequences of these isolated TATAs. The results (Fig.
5D) show that position is the most important determinant of both
TFIIIB-promoter complex stability and transcriptional activity. With
either the TTTATAT or the TATATTA sequence
located at the left position (31 to 25), heparin-resistant
TFIIIB-promoter complexes are detectable, whereas with either sequence
located at the right position (29 to 23), they are not. Moreover,
although the effects on transcriptional activity are not as large, the
pattern is the same. Thus, there are at least two requirements for the
formation of heparin-resistant TFIIIB-promoter complexes: specific
TBP-TATA interactions and proper placement of the TATA relative to
other promoter elements.
As summarized in Fig. 6, we find
restriction in the use of alternative TBP-TATA interactions by
different complexes formed on the tRNACAla promoter. Of
the three sites that support binding by TBP alone, only two are used in
transcription complexes and only one is able to stabilize the
TFIIIB-promoter interaction to heparin challenge.
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FIG. 6.
All TBP-TATA interactions are not equally effective for
transcriptional activity and heparin resistance on the
tRNACAla promoter. Cloned TBP is able to interact with
three different sequences, but only two of them (TL and TR) are used in
full transcription complexes, and only one (TL) can confer heparin
resistance to TFIIIB-promoter complexes. The observation that
differently spaced sites are effective for transcription but not
TFIIIB-promoter stability suggests alternative linkages between TFIIIB
and TFIIIC/D (diagrammed as knobs and holes) and stabilization of
TFIIIB by sequences outside the TATA box (short vertical bars) (see
Discussion).
|
|
The natural TBP binding sequence does not contribute positively to
wild-type tRNASGAla promoter activity.
To
determine whether the TBP-TATA interaction contributes to
tRNASGAla promoter activity, we determined the
transcriptional consequence of mutating the TBP binding site in the
tRNASGAla promoter. The
10TTTAAAA4 binding sequence was
changed either to 10CGGCAAA4 or,
to minimize extraneous effects of altered base composition, to
10TGTAAAA4. A band shift
experiment confirmed that isolated silkworm TBP was no longer able to
bind (Fig. 1B, lane 5, and data not shown). As shown in Fig.
7A, however, neither mutation had a
deleterious effect on promoter activity. In fact, the activity of the
10TGTAAAA4 mutant was slightly
elevated. Thus, the TBP binding site in the tRNASGAla
promoter does not play the same positive role as it does in the tRNACAla promoter. To determine whether this site
stabilizes the TFIIIB-promoter interaction, we subjected transcription
factor complexes on the tRNASGAla gene to heparin
challenge. We anticipated that these complexes might not resist this
challenge because we already knew that although they are stable to gel
electrophoresis, the TFIIIB within them does not protect upstream
sequences from DNase I cleavage (59). Indeed, as shown in
Fig. 7B, the wild-type tRNASGAla promoter does not
support a heparin-resistant complex.
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FIG. 7.
Specific interaction of TBP with the natural TATA
element at 10 does not contribute positively to
tRNASGAla promoter activity. (A) Transcriptional
activity of mutant tRNASGAla promoters containing
substitutions in the TBP binding site (Fig. 2) is plotted relative to
the activity of a wild-type tRNASGAla promoter. The
wild-type (WT) tRNASGAla promoter is represented by a
light gray bar, and its natural or mutant (mut.) TATA sequences are
shown in capital letters delineated by a bracket, with asterisks
indicating positions altered in the mutants. (B) The wild-type
tRNASGAla promoter does not support a heparin-resistant
complex. Transcription factor-promoter complexes were formed on the
tRNASGAla (SG WT) and tRNACAla (C WT)
genes and challenged with heparin as described for Fig. 4A. The
identities of the major protein-DNA complexes are indicated at the
left.
|
|
Lack of a properly positioned TBP binding site causes the low
activity of the wild-type tRNASGAla promoter.
Since the natural TBP binding site does not contribute to
tRNASGAla promoter activity and does not confer heparin
resistance, we wondered whether an optimally positioned site would do
so. To investigate this possibility, we designed a series of chimeric constructs in which segments of the tRNACAla gene
upstream promoter replaced the corresponding parts of the tRNASGAla promoter. As shown in Fig.
8 (constructs a through c), replacement of the D region alone has no significant effect on either heparin resistance or transcription. However, replacement of both D and TAT
elements increases the heparin resistance to ~30% and
transcriptional activity to ~70% that of the wild-type
tRNACAla promoter, and addition of the I element brings
heparin resistance to ~80% and transcriptional activity to 100% of
wild-type levels. The construct that includes simply both TATAs
(construct d) also displays ~80% of the heparin resistance and 100%
of the transcriptional activity of a wild-type tRNACAla
promoter. In contrast, the four constructs that each contain a single
isolated TBP binding site (constructs e through h) show that either
TTTATAT or TATATTA, positioned as it is in the
wild-type tRNACAla promoter (either 31 to 25 or
29 to 23), can dramatically stimulate transcription but confers
little or no resistance to heparin. Transcriptionally, three of the
four constructs are as active as the wild-type tRNACAla
promoter, and the other one is ~70% as active, but heparin resistant complexes are undetectable on three constructs and are only ~20% of
the wild-type level on the other. Overall, the results are similar to
those for isolated TATAs within the context of the tRNACAla promoter. The difference is that the isolated
TATAs within the tRNASGAla promoter confer less heparin
resistance and, as judged by their transcriptional activities, exhibit
less position preference than they do in the tRNACAla
promoter.
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FIG. 8.
Introduction of a TATA element within the 32 to 23
region increases tRNASGAla promoter activity. The
heparin resistance of TFIIIB-promoter complexes and transcriptional
activities of wild-type (WT) tRNACAla,
tRNASGAla, and tRNAC/SGAla chimeric
promoters are shown. In the diagrams of promoter constructs, wild-type
tRNACAla and tRNASGAla promoters are
shown by dark and light gray bars, respectively. The locations of the
two functional TATA elements of the wild-type tRNACAla
promoter are shown by dark or light gray top brackets. The sequences of
these elements introduced into the tRNASGAla promoter
(constructs d to h) are shown in capital letters, and the G-C base
pairs used to isolate each TATA element are shown against a medium gray
background. The heparin resistance of TFIIIB-promoter complexes was
determined as described for Fig. 5.
|
|
The same amount of TBP is present in transcription complexes formed
on wild-type tRNACAla and tRNASGAla
genes.
Why does the lack of a properly positioned TBP binding site
reduce the efficiency of tRNASGAla transcription? There
are at least two possibilities. The probability of incorporating TBP
during transcription complex assembly might be reduced, generating a
large proportion of incomplete, hence inactive, complexes.
Alternatively, protein-protein interaction might suffice for TBP
incorporation, but the absence of TBP-TATA contacts might preclude
incorporation of another factor or allow a conformation that is
incompatible with transcription. To distinguish between these two
possibilities, we used antibodies to compare the TBP content of
transcription complexes formed on tRNACAla and
tRNASGAla genes. In each case, complexes were formed at
subsaturating (1.5 µl) or saturating (7.5 µl) TFIIIB concentrations
in the presence of 5 µl of TFIIIC/D. The resulting complexes were
separated from unbound DNA and proteins by electrophoresis through
agarose (Fig. 9A) and were then retrieved
by enzymatic digestion of the gel. The amount of TBP in the complexes
was determined by quantitative Western analysis. The amount of complex
analyzed in each case was standardized by reference to the
radioactively labeled template present in the complexes.
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FIG. 9.
Amounts of TBP contained in tRNACAla and
tRNASGAla transcription complexes are the same. (A)
Transcription factor-promoter complexes were formed on the
tRNACAla gene (as shown in Fig. 4A) and were resolved
on a 1.5% agarose gel. The resolved complexes (TFIIIC/D-DNA and
TFIIIB/C/D-DNA) and unbound DNA are indicated at the sides. (B) Western
analysis of TBP in TFIIIC/D and TFIIIB/C/D complexes. The TFIIIC/D and
TFIIIB/C/D complexes were formed on wild-type tRNACAla
(C WT), mutant tRNACAla
(TAT-AT) or wild-type
tRNASGAla (SG WT) with either 0 (), 1.5 (+), or 7.5 (++) µl of TFIIIB in the presence of 5 µl of TFIIIC/D. The
complexes were resolved on an agarose gel as shown in panel A, detected
autoradiographically, and isolated from the gel by digestion with an
agarose-digesting enzyme. After concentration, the amount of complex
was standardized by quantitating the labeled DNA fragment, and the
proteins of the isolated complexes were examined by Western analysis
using antibodies to silkworm TBP and a chemiluminescence detection
method. The amount of TBP signal was compared to known amounts of
cloned His-tagged TBP run in parallel (lane 3 to 5). The positions of
wild-type (WT) TBP and His-tagged TBP are indicated on the left. (C)
Transcription reactions were performed under the same buffer conditions
as used for binding but with the addition of 5 µl of Pol III and
nucleotides (33). Templates (200 ng of plasmid) were those
used in panel B, and their parental plasmid (pUC13M) was used as a
control. Transcription products were fractionated on an 8%
polyacrylamide denaturing gel; autoradiography and quantitation of the
transcripts were performed as previously described (33). The
percentage of transcription rate relative to the wild-type
tRNACAla gene is shown below each lane. "O" denotes
the origin; transcript position is marked with a bracket.
|
|
We analyzed complexes formed on three different templates: the
wild-type tRNACAla gene, a tRNACAla
upstream promoter mutant, and a wild-type tRNASGAla
gene. The result is shown in Fig. 9B. Known amounts of recombinant His-tagged silkworm TBP were used as quantitative standards (lanes 3 to
5). Each Western analysis was performed with 12 fmol of complex, and
complexes formed with saturating input TFIIIB contain about 12 fmol of
TBP, as judged by comparison with the His-tagged standards. The small
amounts of TBP found in complexes formed without input TFIIIB are
likely to be from the TFIIIB that contaminated the TFIIIC/D fraction,
based on assays of complementing transcription activity (data not
shown). Comparison of lanes 9, 12, and 15 shows that the same amounts
of TBP are incorporated into transcription complexes formed on the
three different templates. Thus, a quantitative difference in TBP
incorporation into transcription complexes does not explain the
difference between tRNACAla and
tRNASGAla promoter activity, which, under these
conditions, differed at least 50-fold. As shown in Fig. 9C, the
transcription rate from tRNASGAla complexes was only
1.7% of the rate from the same number of tRNACAla complexes.
| |
DISCUSSION |
The TBP-TATA interaction stabilizes TFIIIB binding and enhances
promoter function.
Our results show that specific interaction
between TBP and a TATA element is key to tRNAAla promoter
function. In the naturally robust tRNACAla promoter,
TATA mutations that reduce TBP binding also reduce transcription, and
in the naturally weak tRNASGAla promoter, provision of
a properly positioned wild-type TATA element confers high-level
promoter activity. In both cases, the transcriptional effects appear to
be mediated through the TFIIIB complex since the stability of
TFIIIB-promoter complexes is decreased by mutations that weaken the
natural TATA region in the tRNACAla promoter and is
enhanced by addition of that region to the tRNASGAla promoter.
Quantitatively, the effects on transcription and TFIIIB-promoter
complex stability are quite different. Single base pair changes do not
reduce transcription more than ~2-fold, but they destabilize TFIIIB-promoter complexes at least 5- and as much as 50-fold. The
different sizes of the functional units probed by these two assays
probably accounts for the difference. Transcriptional activity is
tested with the full transcription complex, which in the silkworm system is bound to a stretch of at least 150 bp of DNA (56, 59). In contrast, TFIIIB complexes contain only a subset of transcription factors (three in yeast [17, 19, 38])
and contact only ~30 bp of template DNA. Thus, the loss of a few
protein-DNA contacts within the TATA element is expected to have a
greater impact on the stability of complexes that contain TFIIIB by
itself than on the stability of the full transcription complex.
Interestingly, the effects of mutations in the I region
(I, 24C, and 23C), as well as the behavior of
constructs containing isolated TATAs, suggest that the TBP-TATA
interaction, though necessary, is not sufficient for full stability of
TFIIIB-promoter complexes. Additional ability to resist heparin
challenge is provided by sequences 3' to TL. This additional
stabilization is represented schematically in Fig. 6, and current
experiments are aimed at defining the limits of sequences responsible
for it. Deformability may be an important characteristic of these
sequences, given the increase in stability of yeast TFIIIB-DNA
complexes that is caused by greater DNA flexure downstream of the TATA
element (8). Both BRF and B" are candidates for proteins
that provide stabilizing function. BRF is known to stabilize TBP
binding to a wild-type Pol III promoter through sequences surrounding
the TATA element (3, 28), and B" confers heparin resistance
when added to a TBP-BRF-DNA complex (24).
Our finding that Bombyx transcription requires specific
TBP-TATA interactions differs from the main idea that has emerged from
earlier work with yeast. There, the absence of 5' flanking mutations
among tRNA gene mutants selected for reduced expression (25), as well as the observed insensitivity of promoter
activity to deliberate alteration of upstream tRNA sequences
(22), argued against sequence-specific upstream promoter
elements in this organism. On the other hand, our results fit with the
observation that in yeast, the presence or absence of an ectopic TATA
element alters the efficiency of an artificial tRNA promoter
(13). What accounts for the differences among these
observations? One possibility is that specific TBP-TATA interactions
occur in all systems but are more apparent in the Bombyx
system because of its unusually strong dependence on upstream promoter
elements. In yeast, transcription complex formation relies more heavily
on interactions with the B box than with 5' flanking sequences, whereas
in the Bombyx system, the reverse is true (7,
43). Consequently, changes in the interaction of yeast
transcription machinery with upstream promoter elements must be drastic
to be manifest as transcription defects. Changes having the required
impact in yeast may be more easily created in the context of an
artificial promoter consisting of a single TATA embedded in GC-rich DNA
than in natural tRNA promoters whose AT richness within the TFIIIB
binding site could provide redundant TATA function (20).
Another apparent difference between the yeast and silkworm systems is
in the sensitivity of TFIIIB binding to changes in promoter sequence.
In the silkworm system, relatively subtle changes can eliminate the
heparin resistance of TFIIIB-promoter complexes, but in yeast, even
drastic changes do not do so (13, 23). This could indicate a
fundamental difference in the nature of TFIIIB-promoter interaction in
the two systems but could also simply reflect the higher threshold of
detection in yeast, where the fraction of active transcription
complexes that yields heparin-resistant TFIIIB-promoter complexes can
be close to unity (13, 18, 20). Variation in the amount of
heparin-resistant TFIIIB-promoter complex has been noted in yeast as a
function of changes in DNA sequence or conformation (1), and
in both systems, the proportion of complexes that is heparin resistant
varies among different TFIIIB preparations (13;
M. J. Martinez, unpublished data). Proteolysis during preparative
manipulations may explain the nonuniformity of TFIIIB preparations
since N-terminal truncation of BRF is known to generate TFIIIB that
retains transcriptional activity but can no longer bind DNA in a
heparin-resistant fashion (16). Thus, TFIIIB-promoter
interactions in yeast and silkworms may be similar
an idea that is
supported by a comparison of footprint data. In both cases, TFIIIB
protects sequences between ~10 and 40 but leaves the
transcription start site exposed, or even hypersensitive, to DNase
(18, 24). TFIIIB-induced hypersensitivity just upstream from
the start site is especially apparent in the silkworm system. It is
worth noting that despite the contribution of optimal TBP-TATA interaction, silkworm TFIIIB does not bind detectably to tRNA promoters
on its own (Fig. 4B, lane 3). Whether this represents a fundamental
difference from yeast TFIIIB, which does bind TATA-containing U6
promoters directly (14, 55), or is the consequence of a higher limit of detection is not clear.
Multiple protein-DNA geometries are compatible with promoter
function.
Our results argue that although specific TATA-TBP
interaction is important for transcription, there is some leeway in the geometry that is allowed. For both the wild-type
tRNACAla promoter and the TATA-containing derivatives
of the tRNASGAla promoter, TATA elements 2 bp apart are
nearly equivalent in the ability to direct transcription. Since these
two TATAs differ markedly in the capacity to stabilize stripped
transcription complexes containing only TFIIIB, their equivalence in
the context of the full transcription complex suggests flexibility in
the articulation between TFIIIB and other parts of the complex
(illustrated in Fig. 6). Our results thus fit with indications in yeast
of a flexible linkage between TFIIIB and TFIIIC (13). In
that system, when natural 5' flanking DNA is replaced by a GC stretch,
promoter activity is strongly influenced by the presence or absence of a single TATA element within the stretch but is quite tolerant of
variations in the position of the TATA. Since DNA between the TATA and
the downstream TFIIIC binding region remains protected from DNase
digestion even when it is 10 bp longer than normal, it has been
suggested that the TFIIIB/C complex is capable of stretching to
accommodate different spacing of promoter elements, probably via the
nearly equivalent interactions that can be made between BRF and
successive tetratricopeptide repeats in Tfc4, the 120-kDa subunit of
TFIIIC (2, 13, 21).
Lack of a properly placed TATA enfeebles the
tRNASGAla promoter.
In contrast to the
tRNACAla promoter, the wild-type
tRNASGAla promoter lacks a TATA element at the
appropriate location and is thereby enfeebled under typical in vitro
conditions. Why does the absence of a TATA have this effect? We have
eliminated the most obvious possibility, namely, that the efficiency of
TBP incorporation into tRNASGAla transcription
complexes is reduced. Transcription complexes formed on
tRNACAla and tRNASGAla genes contain
the same amounts of TBP, but they direct transcription at very
different rates. Under the conditions used for this comparison, the
transcription rate from tRNASGAla complexes was at
least 50-fold lower than that from the same number of
tRNACAla complexes.
Thus, we are left with two possibilities. Either the lack of TBP-TATA
interactions precludes incorporation of some other required factor, or
it allows complete complexes to adopt an inactive conformation (Fig.
10). The DNA in transcriptionally
active TFIIIB-promoter complexes in yeast is sharply bent at ~30,
the middle of the TFIIIB binding site (1, 8, 27), and our
preliminary results indicate that silkworm TFIIIB-promoter complexes
formed on the wild-type tRNACAla promoter are similarly
distorted (M. J. Martinez, unpublished data). Possibly, in the absence
of specific TBP-TATA interactions, the template in a
tRNASGAla transcription complex adopts a less distorted
conformation that is an unsuitable substrate for polymerase.
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FIG. 10.
Working model for the interaction of TFIIIB with
tRNACAla and tRNASGAla promoters.
Specific binding of TBP to the tRNACAla promoter
stabilizes TFIIIB binding, possibly by inducing a bend in the upstream
promoter. The resulting complex is transcriptionally active. In
contrast, the lack of specific interaction between TBP and the
tRNASGAla upstream promoter either prevents
incorporation of another transcription factor(s) (top) or results in a
complex with an alternative conformation that is not an effective
substrate for RNA Pol III (bottom).
|
|
Our findings suggest a mechanism for the differential activity of
tRNACAla and tRNASGAla promoters that
is observed under typical in vitro conditions. This basal state, in
which the tRNACAla promoter is on and the
tRNASGAla promoter is off, presumably corresponds to
the situation in cells other than the silk gland. If so, how is the
tRNASGAla promoter activated in silk gland cells?
Increases in TBP concentration have been seen to stimulate Pol III
transcription (48), and, in fact, tRNACAla
promoter output can be increased in Drosophila cells that
overexpress TBP (49). It is unlikely, however, that
increased TBP concentration is sufficient to rescue
tRNASGAla transcription in the silk gland, since
tRNASGAla transcription complexes contain normal
amounts of TBP but are nonetheless functionally impaired. Indeed, the
tRNASGAla promoter does not respond in vivo to elevated
levels of TBP that are sufficient to stimulate the
tRNACAla promoter (49). Moreover, the
presence of a TBP binding site in the wrong part of the
tRNASGAla promoter could potentially allow TBP to play
a negative role, by interfering with the proper binding of TFIIIB. The
increased promoter activity that results in vitro from elimination of
the site is consistent with a negative role.
Alternatively, the actual in vivo role of the resident
tRNASGAla TATA element could be positive. For instance,
the site might be capable of binding a complex that is distinct from
the traditional TBP-containing TFIIIB. In Drosophila, there
are complexes containing TBP-related factors (TRF1 and TRF2) that are
expressed in a cell-type-specific pattern and that associate with a
subset of tRNA genes (9, 37). If comparable complexes exist
in Bombyx, and if their distribution is tissue specific, the
silk gland-specificity of the tRNASGAla promoter could
be explained.
This work was supported by NIH Public Health Service grants
(GM25388 and GM32851) to K.U.S. M.J.M. is an NIH trainee supported by Graduate Training in Genetics grant GM07413.
We thank Diane Hawley, Heather Dunstan, and Fakhruddin Palida for
critically reading the manuscript, and we thank Nan Ahnert and Gusti
Zeiner for making some of the mutant promoter constructs.
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Abstract
Introduction
