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Molecular and Cellular Biology, May 2000, p. 3608-3615, Vol. 20, No. 10
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Roles for Non-TATA Core Promoter Sequences in
Transcription and Factor Binding
Branden S.
Wolner and
Jay D.
Gralla*
Department of Chemistry and Biochemistry and
the Molecular Biology Institute, University of California, Los
Angeles, Los Angeles, California 90095-1569
Received 31 January 2000/Accepted 22 February 2000
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ABSTRACT |
Sequence blocks within the core region were swapped among RNA
polymerase II promoters to explore effects on transcription in vitro.
The pair of blocks flanking TATA strongly influenced general
transcription, with an additional effect on promoter activation. These
flanking elements induced a change in the ratio of activated to basal
transcription, whereas swapping TATA and initiator sequences only
altered general transcription levels. Swapping the flanking blocks
influenced binding by general transcription factors TBP and TFIIB. The
results suggest that the architecture of the extended core sequence is
important in determining promoter-specific effects on both general
transcription levels and the tightness of regulation.
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INTRODUCTION |
Accurate initiation of transcription
by RNA polymerase II (pol II) requires the formation of a large,
multiprotein complex at the gene promoter (reviewed in references
11, 26, and 29). A typical core promoter is
approximately 60 bp long and extends to just downstream of the
transcription start site. Recognition elements include the TATA box
element located 25 to 30 bp upstream of the start site and an initiator
(Inr) element spanning the start site (reviewed in references
11 and 33). These two elements, either singly or in combination, are thought to be sufficient to drive
basal levels of transcription (11, 19, 33, 34). Sometimes a
downstream promoter element exists just beyond the core (2).
Virtually all cellular promoters are thought to be associated with
binding sites for activator proteins. These activators can be
constitutive or inducible and typically bind upstream to help recruit
the transcription machinery to the core promoter elements (27,
35). The core promoter region contains the interaction sites for
several of the general transcription factors (11, 33). A
weak, sequence-specific TFIIB binding site exists immediately upstream
of the TATA box element (21). Other factors collectively contact nearly the entire extent of the core promoter sequences (5, 20, 22, 23, 28). With the possible exception of the
upstream TFIIB site, which may influence basal transcription levels,
these non-TATA and non-Inr sequences have not been implicated in
control of transcription. The sequences between the TATA box and
transcription start site are thought only to provide appropriate spacing (11).
Although activation determinants have recently been found within Inr
(3), the specificity of activation has been largely associated with distal regions. That is, removal of activation sites
leads to core promoters that function in basal transcription with RNA
levels depending on the "strength" of TATA and/or Inr (11, 27,
35).
Nonetheless, prior studies are consistent with a role of the core
sequences in setting the quantitative response to activators. This is
suggested most prominently by comparing properties of the two prototype
adenovirus promoters, the major late (ML) and the E4. Both in vivo and
in vitro studies have shown that ML is the stronger basal promoter
while E4 is more responsive to activators (7, 8, 10, 12, 13, 14,
15-17, 37). One possibility is that this is a simple consequence
of activators compensating for the low basal transcription specified by
the "weaker" E4 TATA and Inr sequences. Both promoters resemble the
consensus TATA and Inr elements, with ML displaying a superior match to
the consensus. Inspection of the core sequences (see Fig. 1) shows that
the two promoters are by far the most different outside the
TATAAAA and pyrimidine-rich Inr elements. Remarkably, only 5 of 39 positions have an identical base pair in these other promoter
regions. The nucleotides of the ML promoter closely resemble sequences
that appear most often in promoters collected in the EPD database (see Fig. 1, majority). The influence of these similarities and differences is not known.
In this report, we explore the influence of the entire core promoter on
the level of basal transcription and on the response to activation. The
approach involves swapping sequence blocks between the ML and E4
promoters and between these and several promoters of human origin.
Transcription studies are then used to define the influence of
individual blocks of sequences on both basal and activated
transcription. The results indicate that the nature of the sequence
blocks surrounding the TATA element has a very significant effect on
how responsive a promoter is to activation. This leads to the view that
one must consider the architecture of the core promoter as a whole in
understanding how promoters are designed to produce appropriate levels
of RNA before and after induction.
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MATERIALS AND METHODS |
Promoter alignments.
Promoter sequences were obtained from
the Eukaryotic Promoter Database
(http://www.epd.isb-sib.ch/seq_download.html). A "representative set
of not closely related sequences" was requested. The 180 human promoters in this set were loaded into MegAlign (DNAStar, Inc.). TATA-less promoters were eliminated, and the remaining 122 promoters were manually aligned by their TATA boxes. "Consensi" were defined per instructions.
Nucleic acids, cloning, and proteins.
Promoters were cloned
as described previously (37). Oligonucleotides (Operon) were
made such that they were complementary at their 3' ends. They were
annealed and the ends were filled with Klenow enzyme. The ends were
trimmed with EcoRI and BamHI and then ligated
into a pSP72 vector (Promega) that had two Gal4 binding sites cloned
into the BglII site. Ligation mixtures were transformed into
strain DH5
, and the DNA sequences were verified. TBP (40)
and TFIIB (14) were purified as previously described. Gal-AH
and Gal-VP16 were kind gifts of Michael Carey (University of
California, Los Angeles). All promoters were in an identical sequence
context outside of the nucleotides indicated in the figures.
Human promoter sequences were obtained from GenBank
(http://www.ncbi.nlm.nih.gov/). Accession numbers are as
follows: thymidine kinase (TK), M13643 (31); lymphotoxin
(tumor necrosis factor beta [TNF-
]), X02911 (25);
immunoglobin H (IgH), L07386 (36); dihydrofolate reductase
(DHFR), K01612 and M10235 (4); and terminal deoxynucleotidyl
transferase (TdT), S70315 (1). The 1 subunit of the
Na/K-ATPase promoter is not on deposit. The sequence was obtained from
Kawakami et al. (18).
In vitro transcription.
In vitro transcription was performed
as previously described (37). Generally, 100 ng of
supercoiled plasmid was mixed with 20 µl of HeLa nuclear extract (6 mg of protein/ml [see reference 6], 8 mM
MgCl2, 500 ng of pGEM as carrier, and a 500 mM
concentration of each nucleoside triphosphate [NTP]) in a total of 40 µl. Reactions were performed for 30 min at 30°C. A 100-µl volume
of stop buffer (10 mM EDTA, 0.3 M sodium acetate [pH 5.5], 0.2%
sodium dodecyl sulfate, 50 µg of yeast tRNA/ml, 20 µg of proteinase
K) was added followed by incubation at room temperature for 30 min. RNA
was isolated and copied by using reverse transcriptase (Promega)
extension of 5'-32P-labeled Inr primer (37). The
labeled cDNA products were resolved on urea-6% polyacrylamide gels
(19:1 ratio of acrylamide to bisacrylamide, 8 M urea) and visualized
and quantitated by using a PhosphorImager (Molecular Dynamics). Gal-AH
was included where indicated.
For single-round assays (
37,
39), the above protocol was
modified as follows: only 25 ng of plasmid was used, NTPs were
omitted
in the first step, and preinitiation complexes were formed
on the DNA
for 45 min. NTPs were then added at a final concentration
of 500 mM
each. Reaction mixtures were incubated at 30°C for 2
min. Human
promoters were transcribed as follows: single-round
assays were
performed as described above by using 500 ng of supercoiled
template.
The activator Gal-VP16 was included where
indicated.
Band shifts.
Electrophoretic mobility shift assays (EMSAs)
were performed essentially as previously described (40).
Probes were made from supercoiled plasmids by PCR amplification using
Inr (37) and T7 promoter (Promega) primers. Deep Vent (NEB)
polymerase was used to generate blunt ends. The PCR products were
resolved on 2% agarose gels, purified by using Qiagen columns, and end labeled with T4 polynucleotide kinase (NEB) and
[
-32P]ATP (NEN). A 0.5-fmol amount of labeled probe
was mixed with 2 ng of TBP, 8 ng of TFIIB, 12 mM HEPES (pH 7.9), 12%
glycerol, 60 mM KCl, 0.12 mM EDTA, 0.6 mM dithiothreitol, 8 mM
MgCl2, 5 µg of poly(dG:dC) (Boehringer Mannheim) per ml,
and 50 µg of bovine serum albumin (Sigma) per ml. Mixtures were
incubated at 30°C for 30 min and resolved on 5% polyacrylamide (59:1
ratio of acrylamide to bisacrylamide)-2 mM MgCl2-0.5×
Tris-borate-EDTA (TBE) gels (30). Gels were run at 400 V for
approximately 30 min in Bio-Rad Mini-Protean II tanks by using
prechilled running buffer (0.5× TBE, 2 mM MgCl2) submerged
in ice. The inner tank was checked every 10 to 15 min, and the buffer
was changed upon reaching 30°C. The gels were dried and exposed to
PhosphorImager plates (Molecular Dynamics). The unbound DNA runs
off the bottom of these gels. For quantitation, 10% of the amount of
probe used in binding reactions was run on a parallel gel. These probes
are unbound and run only halfway though the gel.
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RESULTS |
Promoter alignments and swaps.
Alignment of 122 TATA-containing promoters was done as described in Materials and
Methods. The only positions at which a strong consensus emerged was the
TATAAAA sequence, which was retained with 75 to 95%
conservation in every position except the final A (41% conserved).
Outside of this consensus, no nucleotide was conserved at even the 40%
level. Each non-TATA position was identified with a majority base that
appeared most often, typically only with very weak 30 to 35%
conservation. Figure 1 shows that the non-TATA majority sequence consists of 51 GC pairs and a single AT pair
within the start site region. The analysis shows that the majority
promoter architecture consists of a GC-rich background interrupted by
two AT-rich elements: the heptanucleotide TATA box and a single base
pair near the start site.
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FIG. 1.
The majority sequence and promoter blocks. The majority
sequence is shown divided into blocks according to base composition.
The adenovirus type 2 ML and E4 promoters are aligned to these blocks
by their TATA boxes. Bases in ML and E4 which match the composition
(type) of the majority promoter are shaded. Bases which match the
majority sequence by position are further underlined.
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We divided the promoter into blocks based on inspection of the majority
sequence. Blocks 1 and 2 were taken as 10-bp sequences,
i.e., one full
turn of the helix, on either side of the TATA box.
The remaining blocks
were taken as downstream segments of 9 to
13 bp. Block 2 is
distinctive, as it consists entirely of G's
on the nontemplate strand
of the majority. The Inr block was designed
to be slightly longer to
accommodate the swapping of the entire
pyrimidine-rich nontemplate
strand surrounding the ML promoter
initiator. Blocks 3 and 4 are not
clearly distinctive except that
they consist entirely of GC pairs in
the majority
promoter.
The adenovirus ML and E4 promoters were aligned to these blocks (Fig.
1). These two promoters are quite different in their
agreement with the
sequences of the majority blocks. The ML promoter
agrees very closely
in composition with the majority sequence
in all blocks. It has 22 of
29 GC pairs in blocks 1, 3, and 4;
9 of the 10 G's in block 2; all 12 pyrimidines in the Inr block;
and a perfect match to the TATA region.
The E4 promoter matches
well only in the TATA and Inr blocks, with only
12 of 29 base
pairs being GC in blocks 1, 3, and 4 and only 2 of 10 nucleotides
being G in block 2. The figure shows the block segments
that were
swapped in the experiments described
below.
E4 TATA and Inr elements influence general transcription but not
response to activator.
In order to determine the roles these
sequences may play in transcription, a series of "block-swapped"
promoters was constructed. We started with the wild-type ML promoter
and systematically substituted the corresponding E4 sequences block by
block. This series of promoters was inserted into a vector containing
two Gal4 sites upstream of the cloning site and subjected to standard
in vitro transcription assays (6, 9, 12, 37-40).
We began by swapping the E4 TATA and Inr block sequences into the ML
promoter both singly and in combination (Fig.
2A). These
elements are usually thought
to determine the level of basal transcription
and would be expected to
make the ML promoter weaker. We are interested
in confirming this and
in learning whether the elements are also
associated with the known
greater response of the E4 promoter
to activators (see also Table
1).
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FIG. 2.
TATA and Inr control total transcription. (a) The
promoters used in this experiment are shown. ML sequences are in normal
type and E4 sequences are in bold. The transcription start site is
indicated by an arrow. (b) In vitro transcription was performed with
100 ng of supercoiled plasmid containing the indicated promoter
sequence. The activator Gal-AH was present where indicated. The primary
transcript is indicated with an arrow. 5'-end analyses (not shown)
confirm that these transcripts initiate at the +1 adenosine, as
indicated in panel a. Lanes M-ETI+Gal-AH, activated transcription from
100 ng of M-ETI with (+) and without ( ) -amanitin (1 µg/ml).
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These promoters were transcribed in both the presence and the absence
of the activator Gal-AH as shown in Fig.
2b. Each of
the four promoters
was transcribed in duplicate, and the bands
were quantified with
ImageQuaNT software (Molecular Dynamics).
For weaker basal signals, the
bands were more easily identified
by using higher contrast settings.
The values obtained for both
basal and activated data were corrected
for background and averaged
over several trials (Table
1).
Alpha-amanitin controls (example
in Fig.
2b, right) showed
transcription to be at least 98% pol
II specific. Transcription was
normalized to basal levels of the
parent ML
promoter.
Swapping the TATA box alone or in conjunction with the Inr block led to
a reduction in both basal and activated transcription
(compare M with
M-ET and with M-ETI in Table
1; see below for
a similar effect of the
M-EI initiator swap). The reductions were
approximately 2.5- to 5-fold.
However, these reductions were equivalent
in basal and activated
transcription, leading to no change in
the activation ratio of
approximately
5.
The data show that for these promoters the TATA and Inr blocks
influence the basal transcription level but not the response
to
activators. Substitution of the E4 TATA and Inr into ML weakens
the
promoter in general. The basal level of transcription from
M-ETI is
nearly identical to E4 (Table
1), yet the E4 promoter
has a much
greater response to activator: E4 is stimulated 15-fold
while ML and
M-ETI are stimulated only 5-fold (Table
1). We infer
that this enhanced
response to activator in the E4 promoter requires
core sequences
outside of the identical TATA and Inr sequences
in the E4 and M-ETI
promoters. These other blocks were tested
individually, as described
below.
E4 blocks flanking TATA influence both general transcription and
the tightness of regulation.
Non-TATA E4 blocks 1, 2, 3, and I
were swapped individually into the ML promoter (Fig.
3a). Both basal and activated
transcription were assayed (Fig. 3b). Each swap produces a distinctive
effect, with the most obvious ones associated with swapping the I block and block 2. I-block substitution strongly reduces both basal and
activated transcription, which is reasonable as it contains the
initiator sequence (compare M-EI with M). The most interesting of the
non-TATA non-Inr swaps was due to E4 block 2. Its substitution for the
ML block 2 sequence strongly increases activated transcription (compare
M-E2 with M). This increase is attained without any significant change
in the level of basal transcription (see Table
2 for the average of several
experiments). We infer that the sequences within E4 block 2 can
selectively mediate enhanced levels of activated transcription.
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FIG. 3.
E4 sequences alter the transcription properties of the
ML promoter. (a) The promoters used in these experiments are shown as
in Fig. 2a. (b and c) Representative transcription gels using the
E4-swapped ML promoters. Transcription was performed as in Fig. 2.
Gal-AH was included as indicated (+).
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The quantitative analysis of these data is presented in Table
2. Each
of the block substitutions has some effect on transcription,
either
basal, activated, or both. Taken together with the data
of Table
1, a
common pattern can be seen that distinguishes blocks
1, 2, and 3 from
the TATA and Inr blocks. Substitution of the
E4 TATA or Inr blocks led
to comparable reductions in basal and
activated transcription, leaving
the activation ratio unchanged
(compare M with M-EI in Table
2 or with
M-ET in Table
1). In
contrast, substitution of E4 blocks 1, 2, and 3 always leads to
an increase in activation ratio (compare M with M-E1,
M-E2, and
M-E3 in Table
2). Similar effects were seen whether
continuous
transcription or single-round assays were used (data not
shown).
These effects were consistently strongest with substitution of
blocks 1 and 2. They were not specific to the activator used as
comparable increases in activation ratio were seen with CREB-mediated
activation (Table
2). We constructed a double substitution involving
these E4 blocks and assayed for basal and activated transcription
(Fig.
3c). This promoter has an activation ratio that approached,
but did not
quite reach, that of the parent E4 promoter (compare
M-E12 with E). We
infer that blocks 1 and 2 of the E4 promoter
have a very strong
influence on the promoter activation ratio.
This result is in strong
contrast to that obtained with the double
substitution of the TATA and
Inr blocks (Table
1). In that case
(M-ETI), both basal and activated
transcription were reduced drastically,
leading to no change in the
tightness of regulation as judged
by an unchanged activation ratio. It
appears that one pair of
blocks, TATA and Inr, functions to direct
general transcription
levels, whereas the other pair, blocks 1 and 2, can function to
direct the tightness of
regulation.
The flanking ML blocks also influence transcription
regulation.
The data have shown that replacing blocks 1 and 2 of
the adenovirus ML promoter with corresponding sequences from the
adenovirus E4 promoter can alter the tightness of regulation. Next, the
reciprocal swaps of these ML blocks into the E4 promoter were
investigated. These blocks are shown in Fig.
4a, with their transcription shown in
Fig. 4b. They include both the single and double swaps.
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FIG. 4.
ML sequences alter the properties of the E4 promoter.
(a) The promoters used in these experiments are shown as in Fig. 2a and
3a, except that E4 sequences are in normal face and ML sequences are in
boldface. The expected E4 transcription start sites are indicated by
arrows. (b) A representative transcription gel. The major transcripts
are indicated by arrows. 5'-end mapping confirms that these transcripts
initiated at the expected sites (not shown).
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The results are consistent with expectations. The double swap of ML
sequences reduces the E4 activation ratio from 11 to 3
(Table
3) and increases basal transcription.
This activation
ratio is comparable to that exhibited by the parental
ML promoter
(5.5-fold). Each individual block swap also reduces the
ratio,
although to a lesser extent than the double swap. Thus, ML
blocks
1 and 2 are associated with a low activation ratio whether in
the context of the E4 or ML promoter. As shown above, this is
analogous
to the E4 promoter blocks but with the expected opposite
effect; E4
blocks are associated with high activation ratios whether
in the
context of the E4 or ML promoter. It appears that these
two sequence
blocks play a critical role in setting the activation
ratio,
independent of ML or E4 context.
Response of human promoters to block 1 and 2 swaps.
We wished
to learn if these block 1 and 2 sequences could affect a wide variety
of promoters. Few promoters have been tested systematically in vitro,
and their sensitivity to activation is rarely known. Six human
promoters were chosen based on their being widely studied in the
literature. The set represents a wide diversity of sequences throughout
the promoter region (Fig. 5). Four
contain clear TATA elements with matches to the consensus ranging from four of seven for 1 to six of seven for TNF-. Two promoters usually considered to be TATA-less (DHFR and TdT) were included (32, 33).
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FIG. 5.
Block swaps affect many promoters. (a) The set of six
human promoters used are shown divided into blocks. E-swap and M-swap
illustrate which blocks were substituted in each promoter. (b) Examples
of transcription from TATA-containing promoters. P, parent promoter; E,
E-swapped promoter; M, M-swapped promoter. The parent of each set is
indicated: , no Gal-VP16; +, Gal-VP16 added. The single transcript
from each promoter is indicated by an arrow and corresponds to a start
site predicted from ML transcription. (c) Transcription from TATA-less
promoters. The multiple start sites indicated are clustered around the
site predicted from ML transcription.
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Each of the six core promoter regions was cloned into the same vector
used above. Each promoter was also constructed in two
swapped versions.
In one set, E4 blocks 1 and 2 replaced the two
equivalent wild-type
blocks, and in another set, the two ML blocks
were substituted (Fig.
5a). This yields a set of 18 promoters:
six parents and two double
swaps of each. Because in vitro transcription
of these promoters had
not always been optimized previously, conditions
were altered slightly
from those described above (see Materials
and
Methods).
Figure
5 shows transcription from the TATA-containing promoters (Fig.
5b) and the TATA-less promoters (Fig.
5c). The TATA-containing
promoters (parents shown in lanes P) all initiated predominantly
at a
single site and yielded transcripts of the expected length
(Fig.
5b).
The TATA-less promoters (Fig.
5c) initiated at several
sites clustered
around the predicted start site. We observe that
in the context of the
TATA-less promoters, the swaps can alter
the start site preference
(Fig.
5c, compare lane 1 to lane 3 and
lane 10 to lane 11). The source
of this phenomenon is not clear,
and so all the bands indicated in Fig.
5c were collectively
quantified.
Inspection of the autoradiographs shows two consistent effects of the
block 1 and 2 swaps. First, at all six promoters, replacement
of the
wild-type blocks with the ML blocks reduced the amount
of activated
transcription (lanes M versus P in each of the six
activated
comparisons). Second, in five of six cases (IgH was
the exception), the
E4 swaps decreased the amount of basal transcription
(lanes E versus P
in each of the six unactivated comparisons).
We infer that blocks 1 and
2 can influence the level of transcription
in a wide variety of
contexts.
These substitutions also had the effect on the activation ratio
predicted from the experiments with the adenovirus parents
(Table
4). The ML double swap reduced the
activation ratio of
all six human promoters, reflecting the low
activation ratio of
the ML parent. The E4 double swap increased the
activation ratio
in five of six cases (again, the exception was IgH),
reflecting
the high activation ratio of the E4 parent. These are the
same
effects seen in the adenovirus contexts, demonstrating that blocks
1 and 2 can influence the tightness of regulation in the context
of a
wide variety of promoters.
Altered factor assembly levels by sequence blocks flanking
TATA.
In these human promoters, the E4 block 1 and 2 substitutions
have reasonably consistent effects on basal transcription and on the
activation ratio. The source of the effect on the activation ratio
could be quite complex, as the interactions between perhaps dozens of
polypeptides need to be considered. However, basal transcription may
rely on a more simple set of interactions, perhaps primarily on the
association of TBP and TFIIB with the DNA. Thus, we explored whether
the substitutions alter the assembly of these components by using an EMSA.
In this experiment, the ML promoter was used as the parent.
Substitution of E4 blocks 1 and 2 yields a 2.5-fold reduction
in basal
transcription (see above; Table
2). The EMSA results
(Fig.
6 and Table
5) show that assembly of the
TBP-TFIIB-DNA
complex is also reduced 2.5-fold by this substitution.
The source
of this reduction is a lessening of binding by TBP alone,
which
accounts for most or all of the effect. We infer that blocks 1
and 2 can influence TBP binding strongly and that this is a likely
influence on basal transcription levels.
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FIG. 6.
Gel mobility shift assay: representative gel shift
result. The presence (+) or absence () of TBP and TFIIB is indicated.
Retarded complexes are indicated by arrows. The unbound DNA ran off the
bottom of the gel. The numbers below the figure indicate the percentage
of total probe that is present in the bound complexes (see Materials
and Methods).
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We also tested the ability of each block individually to influence
assembly. The data show that either E4 block 1 or 2 acting
alone
reduces TBP binding. In the case of the block 1 substitution,
this
reduction is not cured by adding TFIIB, as the TBP-TFIIB-DNA
complex
level is comparably reduced. In the case of the block
2 substitution,
addition of TFIIB can restore TBP-TFIIB-DNA complex
formation to
wild-type levels. This is probably explained by the
tighter TFIIB
binding ability of the parent block 1 sequences
that are retained only
in this
construct.
Overall, there is an excellent correlation between levels of basal
transcription and levels of TBP-TFIIB-DNA complex formation.
The
surprising aspect is that an important contribution to this
is made by
controlling TBP binding. Thus, the data indicate that
the sequences
flanking the TATA box can have a strong influence
on TBP binding, which
appears to carry through to transcription
in the appropriate
context.
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DISCUSSION |
In addition to providing core recognition sites for assembly of
transcription complexes, promoters need to include signals specifying
the responsiveness to activators and the level of mRNA. In general, the
activation signals lie in distal regions, typically upstream (11,
27, 35). The downstream core contains signals that contribute to
promoter strength. These include the well-known TATA and initiator
(Inr) elements. The TATA and Inr elements make this contribution via
recruitment of preinitiation complexes (11, 24, 37), with
the TATA box playing an additional role in specifying the rate of
continuous transcription (39, 40). In this paper, we find
that sequences flanking the TATA element are important for promoter
strength, for TBP binding, and for mediating the response to upstream
activators. The last result is particularly unexpected and contributes
to a model in which the architecture of the promoter as a whole
determines both the responsiveness to activators and the strength of
the transcription.
The origin of the work lies in the differential responsiveness of the
adenovirus ML and E4 promoters to activation (7, 8, 10, 12, 13,
15-17, 37). The former has high unactivated, basal
transcription, whereas the latter is more responsive to activation. By
swapping sequences between these two promoters, we were able to
identify core promoter regions that contributed to these properties.
The swaps showed that the sequence blocks flanking TATA played a role
distinct from that of the TATA and Inr blocks; only the flanking blocks
contributed to the responsiveness to activation. That is, substitution
of E4 sequences into ML increased its activator responsiveness, whereas
substitution of ML sequences into E4 had the opposite effect. Increases
in responsiveness were not solely due to reductions in basal
transcription, implying a more complex role for these blocks that flank
the TATA element. Moreover, swapping the TATA and Inr blocks between
the promoters altered general transcription levels without having
significant effects on activator responsiveness.
It is important to note that these TATA and Inr swaps are very
conservative. In particular, both the ML Inr and E4 Inr match the
degenerate consensus perfectly (YYAN[T/A]YY, where Y is a pyrimidine and the underlined A is the transcription start site; see
reference 33). However, another study has shown that
more dramatic changes in the Inr can affect the activation ratio
(3). Thus, we cannot conclude that TATA and Inr play no role
in activator responsiveness. In the context of these adenovirus
promoters, however, the conservative differences in these elements do
not alter activator responsiveness.
In order to assess the general significance of these observations, we
first compared the sequences of these blocks to those in the Eukaryotic
Promoter Database. Alignment of 122 TATA-containing promoters produced
no consensus outside of TATA but did reveal an unusual property of the
core promoter region. With the exception of a single nucleotide near
the start site, all non-TATA positions were most often GC base pairs
(Fig. 1). That is, this majority promoter consists of an AT-rich TATA
box embedded in a sea of GC base pairs. This majority promoter
resembles the ML promoter in this regard and is antithetical to the E4
promoter, which is unusual in being largely AT-rich.
Although promoters tend to be GC-rich, they exhibit considerable
diversity. We chose a series of six human promoters and substituted either the GC-rich ML blocks or the AT-rich E4 blocks to see the effects on transcription (Fig. 5). Transplanting the GC-rich ML sequences into human promoters had the general effect of decreasing the
responsiveness of the promoter to activation; including the E-M12 swap,
this effect occurred in seven out of seven cases (Tables 3 and 4). This
decrease in responsiveness was not due to increased basal strength, as
such an increase occurred in only three of the seven promoter swaps.
Remarkably, the level of activated transcription decreased in all seven cases.
As expected, the transplantation of the AT-rich E4 flanking blocks into
human promoters increases the activation ratio, occurring in five of
the six promoters (and six of seven when the ML parent promoter is
included [Tables 2 and 4]). This increase in responsiveness correlates with a decrease in the level of basal transcription in six
out of seven cases. The single exception is the IgH promoter, for which
basal transcription does not decrease and responsiveness does not
increase. This indicates that the E4 sequences do not universally
induce tight regulation. Whether consensus sequences exist or depend
strongly on promoter context remains to be seen. However, these data
show that the two blocks flanking TATA play a dominant role in
regulating the tightness of control. In 13 out of 14 cases, activator
responsiveness correlates directly with the types of sequences present
in these blocks.
We note that these effects are consistent with the physiological roles
of the ML and E4 promoters during the adenovirus life cycle (7,
8). The ML promoter is primarily activated by an increase in
genome copy number. Thus, it is designed to be a strong basal promoter,
weakly responsive to activators. In contrast, the E4 promoter is turned
on in response to protein activators when the copy number is low. The
above data suggest that the sequences of the blocks flanking TATA
contribute to these characteristics in a very important manner. We
expect that the diversity of sequences within these blocks of human
promoters will also contribute to the physiological design of the
promoters in terms of responsiveness to regulators.
There remains the question of how these sequence blocks function. We
consider this question from the point of view of substituting the two
AT-rich E4 blocks, which have a poor resemblance to the majority
promoter. This substitution consistently decreases the level of basal
transcription, regardless of promoter context. The data show that these
substitutions reduce the level of TBP binding (Fig. 6 and Table 5).
Thus, the sequences surrounding TATA contribute both to TATA
recognition and to basal transcription levels. TATA recognition is
optimal when the element is made most distinctive by surrounding it
with GC-rich sequences and suboptimal when the surrounding regions are
AT-rich. The typical promoter is diverse but more like the GC-rich ML
sequence, implying that it is important to generally have a distinctive
TATA sequence and the capacity to direct some basal transcription. The
band shift experiments support the idea that the block upstream from TATA may contribute to basal transcription (Table 5) through interaction of a GC-rich sequence with TFIIB (21).
The data do not indicate the source of the effects on activated
transcription. They simply suggest that the core promoter becomes more
responsive as the sequence diverges from the majority sequence.
Activation is a very complex process involving the interplay of a large
number of proteins, both activators and general transcription factors
(see the introduction). The activated transcription complex assembles
over the blocks that flank TATA so the blocks are in a position to
influence assembly, but in a manner as yet unknown. The challenge is to
understand how the diverse sequences within the promoter core interact
to create an architecture that is appropriate to the unique
physiological function of each promoter.
| |
ACKNOWLEDGMENTS |
This research was supported by USPHS grant GM49048 (J.D.G.) and
USPHS National Research Service Award GM07185 (B.S.W.).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Chemistry and Biochemistry and the Molecular Biology Institute,
University of California, Los Angeles, P.O. Box 951569, Los Angeles, CA
90095-1569. Phone: (310) 825-1620. Fax: (310) 267-2302. E-mail:
gralla{at}ewald.mbi.ucla.edu.
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Top
Abstract
Introduction
