From the Departments of Molecular Pharmacology and
Biochemistry, Schools of Pharmacy and Medicine, University of
Southern California, Los Angeles, California 90033 and the
§ Institute of Molecular Biology, the
Department
of Biology, and the ¶ Department of Physics, University of Oregon,
Eugene, Oregon 97403
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In contrast to yeast and mammalian systems, which
depend principally on internal promoter elements for tRNA gene
transcription, insect systems require additional upstream sequences. To
understand the function of the upstream sequences, we have asked
whether the Bombyx mori tRNACAla and
tRNASGAla genes, which are absolutely dependent on
these sequences in vitro, also require them for
transcription in vivo. We introduced wild-type and mutant
versions of the Bombyx tRNAAla genes into
Drosophila Schneider-2 cells and found that the
tRNACAla gene is efficiently transcribed and that its
transcription depends strongly on the distal segment of its upstream
promoter. In contrast, the tRNASGAla gene is
inefficiently transcribed, and this inefficiency results from
lack of a specific sequence within the distal
tRNACAla upstream promoter. This sequence,
5'-TTTATAT-3', is sufficient to increase the activity of the
tRNASGAla promoter to that of the
tRNACAla promoter. Moreover, promoters containing
the 5'-TTTATAT-3' element are stimulated by increased levels
of cellular TATA-binding protein. Together these results
indicate that, in insect cells, a TATA-like element is
specifically required to form functional TATA-binding protein-containing complexes that promote efficient transcription of
tRNA genes.
Transcription of cloned genes in cell-free extracts has been
widely used to define the promoters of tRNA genes. This approach has
established the contribution of gene-internal promoter elements (reviewed in Refs. 1 and 2) and has revealed the effects of 5'-flanking
sequences (both positive and negative) in a variety of organisms (2).
In vivo assays to determine the biological relevance of the
class III promoter elements defined in vitro have not been
employed widely, but they suggest that at least some promoter elements
identified in vitro are also required in vivo.
For instance, the canonical A and B boxes function in vivo in yeast (3) and trypanosomes (4), and 5'-flanking sequences appear to
modulate promoter function in several organisms (5-10).
There has been no systematic analysis of the function of upstream
flanking sequences in vivo in any system however. Such an analysis of silkworm tRNAAla genes could be particularly
informative because transcription of these genes in vitro by
homologous transcription machinery is strongly dependent on upstream
sequences (2). Moreover, the two AT-rich sequence blocks that provide
most of the upstream promoter function for the silkworm
tRNACAla also occur in other silkworm RNA
polymerase III (pol III)1
templates (11-15). These sequences thus have the potential to comprise
a class of general upstream promoter elements for polymerase III-dependent genes, something that has not been recognized
in any other system (16-18). Upstream promoter elements are also
responsible for the differential transcription in vitro of
silk gland-specific (SG) and constitutive (C) silkworm
tRNAAla genes (19).
To determine whether upstream sequences contribute to promoter function
in vivo, we have transiently introduced wild-type and mutant
tRNAAla genes into Drosophila Schneider-2 cells.
Although they are heterologous, these cells can be transfected
efficiently (>10% (20)),2
and the upstream sequence dependence of the Drosophila pol
III transcription machinery suggests functional similarity to the Bombyx machinery (reviewed in Refs. 2 and 21). In addition, because Drosophila S2 cells are not derived from silk
glands, they provide an opportunity to ask whether C and SG upstream
promoters are differentially active in non-silk gland cells as they are in non-silk gland extracts. In this paper we show that the activity of
the tRNACAla upstream promoter in
Drosophila S2 cells depends on a subset of the sequences
required in vitro. Specifically, a TATA-like sequence
located from positions Plasmid Constructs--
The parental wild-type constitutively
expressed tRNAAla gene used in this work
(tRNACAla) contained sequences from
To distinguish the products of introduced genes from endogenous
Drosophila alanine tRNA, maxigene derivatives were made by cutting the genes at the unique internal SphI site and
inserting a self-annealing, XhoI-containing oligonucleotide
(5'-GCTCGAGCCATG-3') to yield maxigenes with 12 additional bp (Cwt
maxi). An additional Cwt maxi+8 was made by restricting a Cwt maxi
construct with XhoI, filling in the XhoI ends
with the Klenow fragment of DNA polymerase I, and re-ligating. To make
antisense probes for RNase protection assays, we cloned a Cwt maxi gene
and a Cwt maxi+8 gene into pBluescript (pSK+), linearized
these clones with BamHI, and used T7 RNA polymerase to
generate antisense RNA (see below).
Transient Transfections--
Transient cotransfections were
performed by a calcium phosphate precipitation technique (20).
Drosophila Schneider 2 cells were maintained at 25 °C in
Schneider medium (Life Technologies, Inc.) containing 10% fetal bovine
serum (Gemini Bioproducts). For transfections, cells were plated at
2.5 × 106 to 5 × 106 cells/25
mm2 in Corning brand tissue culture flasks. For each
transfection, 6 µg of reporter plasmid DNA (tRNAAla gene
constructs) and 2 µg of a transfection efficiency control plasmid
(pActCAT or pZIL, a luciferase-containing plasmid (23)) were used. The
final DNA concentration was brought to 20 µg with pBluescriptSK (pSK,
Stratagene) or pUC DNAs. All transfections were performed 3-6 times.
Medium was changed 24 h post-transfection, and cells were
harvested the following day. Approximately 2 × 106
cells (one-fifth of the total cells) were used to make extracts to
assay for chloramphenicol acetyltransferase or luciferase activity. Four-fifths of the cells (8 × 106 cells) were used
for RNA extraction to measure the transcription activity of the tRNA
gene promoter.
Ribonuclease Protection Assay--
RNA was extracted using
TriZOL (Life Technologies, Inc.) following the protocol provided by the
vendor. The RNA yield was determined by measuring the absorbance at 260 nm. A ribonuclease protection assay was performed on the RNAs isolated
from transiently transfected cells, using an RPA II kit from Ambion.
Briefly, a linearized plasmid containing the gene of interest served as
a template for antisense transcript labeled with
[ CAT Assay--
The transfected cells were harvested and
centrifuged, and the cell pellets (2 × 106 cells)
were resuspended in 90 µl of 0.25 M Tris, pH 7.5, followed by repeated freeze-thaw cycles. The extracts were then diluted 1:50 to 1:500 in 0.25 M Tris, and 5 µl of the diluted
extracts were used to assay for CAT activity. The assay was performed
as described previously (24). Products were analyzed by thin layer chromatography (TLC) and quantitated by scanning the autoradiograms in
a Bioimage Scanner.
Luciferase Assay--
The transfected cells were harvested and
centrifuged, and the cell pellets (~2 × 106 cells)
were resuspended in 1 × cell lysis buffer (Promega) and assayed
with the luciferase assay system (Promega) using a Flow Tech Model 3010 Luminometer.
S100 Extracts and in Vitro Transcription
Reactions--
Cytoplasmic S100 extracts were made from
Drosophila S2 cells as described (25). Briefly, cells were
grown in T65 cm2 tissue culture flasks in
Drosophila Schneider medium (Life Technologies, Inc.)
containing 10% fetal bovine serum (Gemini Bioproducts). Cells were
grown to confluence (~2 × 108 cells/flask) and
harvested by scraping with a rubber policeman. The cells were washed
once with ice-cold phosphate-buffered saline, and the packed cell
volume was determined. The cells were resuspended in 2 to 2.5 times the
packed cell volume with hypotonic buffer (10 mM HEPES-KOH,
pH 8, 10 mM KCl, 1.5 mM MgCl2, 0.5 mM dithiothreitol) and allowed to swell for 30-90 min.
Cells were homogenized in a Dounce homogenizer, using 20 strokes with
the tight pestle. Isotonic buffer was added (1/10 the total cell
volume) (0.3 M HEPES-KOH (pH 8), 1.27 M KCl, 40 mM MgCl2, 50 mM dithiothreitol), and the extract was centrifuged at 42,000 × g in a
Ti60 rotor at 4 °C for 60 min. Glycerol was added to the supernatant
to a final concentration of 20-25%, and the extracts were stored at
In vitro transcription reactions were carried out using
60-100 µg of protein/reaction mixture. Standard reaction mixtures contained 0.4 µg of DNA template, 0.5 mM each of ATP,
UTP, and CTP, 0.1 mM GTP, 1-5 µCi of
[ The Distal Segment of the Silkworm tRNACAla
Upstream Promoter Is Critical for Transcription in Drosophila
Extracts--
To determine whether the tRNACAla
gene upstream promoter elements are recognized by the
Drosophila transcription machinery, we first asked whether
tRNACAla transcription depends on these elements
in vitro in Drosophila extracts. In these and
subsequent experiments we used a set of mutants that systematically
dissect the region between
The series of mutants was designed to delineate promoter function with
increasing resolution, starting with a mutant that alters most of the
upstream promoter. Substitutions in the three contiguous sequence
blocks, TAT, I, and AT, eliminate promoter activity in
Drosophila extracts. Two of the mutants with substitutions in two adjoining blocks of sequence (D-TAT or TAT-I) eliminate transcription entirely, whereas the third such mutant (I-AT) reduces promoter activity by only ~30% (Fig.
1A). These results contrast with those observed in Bombyx extracts in which mutation of
D-TAT, TAT-I, and I-AT yields promoters with ~40, ~5, and 2%
wild-type activity, respectively. In the Drosophila system,
mutation of the TAT region alone abolishes transcriptional activity
(Fig. 1A), and mutation of the distal D region, by itself,
reduces promoter activity 5-fold. In contrast, mutation of the AT
region alone causes only about a 2-fold reduction. The observed mutant
phenotypes were independent of DNA concentration (Fig. 1B),
and variations in template concentration did not reveal additional
mutant phenotypes. Taken together, these results suggest that in the
Drosophila system, promoter function is largely confined to
the distal part of the promoter (TAT, plus its neighbors), whereas in
the Bombyx system, it is distributed between the distal TAT
and the proximal AT regions.
Transient Expression of the Bombyx tRNACAla
Gene in Drosophila S2 Cells--
For promoter analysis in
vivo, we constructed derivatives of wild-type and mutant
tRNACAla genes that were marked with unique
internal sequences (described under "Experimental Procedures") to
distinguish their transcripts from those of endogenous tRNA genes. The
introduced sequences had no effect on the transcriptional activity of
the genes in vitro in either Bombyx or
Drosophila
extracts.3
Drosophila S2 cells were cotransfected with the marked
derivative of either a wild-type or a mutant
tRNACAla gene, together with a transfection
efficiency standard in the form of a CAT reporter driven by the
Drosophila actin 5C promoter (26). As shown in Fig.
2A, when the wild-type
tRNACAla gene marked by a 12-base pair insertion
(Cwt maxi) was introduced into S2 cells, two new transcripts of ~110
and 90 nucleotides were detected. To verify the identity of the
protected RNAs, we introduced a different tRNACAla
maxigene derivative containing a longer inserted sequence (20 bp
instead of 12 bp) and showed that the two transcripts migrated more
slowly, as expected for the addition of 8 nucleotides.
The Distal Segment of the tRNACAla Upstream
Promoter Is Important in Vivo--
The set of promoter mutants that
had been tested in vitro (Fig. 1A) was introduced
into Drosophila S2 cells. Fig. 2B shows the
primary data from representative assays, and Fig. 2C shows the quantitative results, based on averages of at least three independent experiments. Simultaneous loss of the three sequence blocks, TAT, I, and AT, reduces promoter activity nearly 20-fold. Loss
of only two regions is less deleterious. Removal of either the D-TAT or
the TAT-I region reduces transcriptional activity 3- to 5-fold, and
removal of the I-AT region has no detectable effect. The impact of
losing individual smaller regions is more modest. As shown in Fig.
2C, mutation of the TAT region has the largest effect,
reducing transcription 2-fold (to 44% of the wild-type level).
Mutation of any of the other short sequence blocks has a smaller effect
or none at all.
tRNACAla and tRNASGAla
Genes Are Differentially Transcribed in Vitro and in Vivo--
To
compare tRNACAla and
tRNASGAla promoter activity in vivo, we
used constructs that yield identical tRNACAla
primary transcripts in order to avoid differences in transcript processing or stability. The two promoters were first tested in vitro in extracts from S2 cells. Fig.
3A shows that the
tRNASGAla promoter is at least 100-fold less
active than the tRNACAla promoter, as expected
from previous studies in other non-silk gland extracts. When these two
promoters were tested in vivo, they also directed
transcription with different efficiencies. As shown in Fig.
3B, the signal from the tRNACAla
promoter is 50-fold higher than that from the
tRNASGAla promoter. This difference was
independent of the amount of template used over the range tested (2-8
µg).
What sequences in the upstream promoter are key discriminators between
C and SG? The critical distal portion of the C promoter contains
several overlapping AT-rich sequences that resemble binding sites for
the TATA-binding protein. TATA boxes known to bind TBP are located in
this position in pol II-transcribed genes (27) as well as in the pol
III-transcribed gene, U6 (28). Interestingly, these TATA-like sequences
are absent from the corresponding region of the SG promoter. Extensive
point mutation indicates that the TATA-like sequence TTTATAT from Overexpression of Drosophila TBP Differentially Affects
tRNACAla and tRNASGAla
Upstream Promoters--
The analysis of mutant
tRNACAla promoters, as well as the chimeric
tRNACAla/tRNASGAla promoter,
indicates the functional importance of the TATA sequence in
vitro and in vivo. Since this sequence resembles an
optimal TBP binding site (29), and since TBP is required for
transcription of tRNA genes in this and other systems (28), a plausible
role for the sequence is to provide the C promoter with DNA contacts for TBP. Lack of the sequence in the wild-type SG promoter might prevent proper interaction with TBP. To test this idea, we performed transient transfections in an S2 cell line that was stably transformed with epitope-tagged Drosophila TBP under the control of a
metallothionein promoter (26). Previous experiments had demonstrated
that TBP is limiting for class III promoter activity in this cell type (26). As shown in Fig. 5, the wild-type C
and SG promoters respond differently to TBP overproduction. TBP
concentrations sufficient to increase the activity of the C promoter
~2.5-fold do not stimulate the SG promoter. In contrast, the chimeric
TATA-containing SG promoter responds to increased TBP concentrations
just as the wild-type C promoter does.
We have shown that sequences upstream of the Bombyx
tRNAAla gene are functional as promoter elements in
vivo in Drosophila S2 cells. Within this region, the
TAT segment ( Certain promoter mutants are differentially impaired in the
Drosophila and the Bombyx systems (see Fig.
6). Since in Drosophila these
mutants have similar effects in vitro and in
vivo, the observed differences most likely reflect true functional
divergence between the Drosophila and Bombyx
transcription machineries. The most obvious example is the strong
dependence of the Drosophila transcription machinery on the
distal portion of the promoter that includes the TAT region. This
contrasts with the dependence of the Bombyx machinery on
sequences in both the distal and proximal portions of the promoter, in
particular on the TAT and AT sequences.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
31 to
25 is the key element. Moreover, the
wild-type C and SG upstream promoters are differentially active
in vivo, and introduction of the TATA element raises SG promoter activity to the level of the C promoter. Overproduction of TBP
in vivo stimulates promoter activity only if the TATA-like element is present. Thus, it is likely that in insect systems, the
TATA-like element functions to facilitate the formation of active
TBP-containing transcription complexes on tRNA genes.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
34 to +215
(with respect to the transcription initiation site) cloned into a
derivative of pUC13 in which the HindIII site had been
replaced by an MluI site (pUC13M). Mutant derivatives of the
upstream promoter from
11 to
34 were made in portions spanning the
"D," "TAT," "I," and "AT" regions using oligonucleotide-directed mutagenesis as described previously (13). The
wild-type silk gland-specific upstream promoter used here was fused to
a fully wild-type tRNACAla gene coding and
downstream region. It was derived from a previous chimera (SCC (19)) by
using recombinant PCR (22) to convert the
tRNASGAla start site (+1 to +3 is AAC) to the
tRNACAla start site (+1 to +3 is GTT). The
chimeric tRNACAla/tRNASGAla
upstream promoter derivative in which the sequence
31TTTATAT
25 from the C promoter replaced
the corresponding positions in the SG promoter was also constructed
using recombinant PCR.
-32P]CTP (ICN, specific activity, >600 Ci/mmol),
using the Maxiscript kit (Ambion). DNA was transcribed with T7 RNA
polymerase, and the transcript was labeled with
[
-32P]CTP (ICN, specific activity, >600 Ci/mmol). The
probe was treated with DNase I to digest the template and was further
purified by organic extractions and ethanol precipitation. Probe
(0.5 × 106 to 1 × 106 cpm/reaction
mixture) was hybridized with RNA (0.1-0.2 µg/reaction mixture) at
45 °C overnight and then digested with RNase T1 (200 units/reaction
mixture) and prepared for electrophoresis as described by the RPA kit
manufacturer. Electrophoresis was on 8 M urea-8% polyacrylamide gels, which were quantitated in two ways. 1) The gel was
exposed to x-ray film for 30-120 min at
80 °C, and the resulting
autoradiographs were quantitated using a Bioimage Scanner, or 2) the
gel was exposed directly to a phosphor screen (Molecular Dynamics) for
60-120 min, and the data were collected using a Model 860 STORM
PhosphorImager and quantitated using Image Quant software
(Molecular Dynamics).
80 °C.
-32P]GTP (3000 Ci/mmol), 20 mM HEPES, pH
7.9, 5 mM MgCl2, 3 mM
dithiothreitol, 100 mM KCl, and 10% glycerol in a final
reaction volume of 60 µl. Mutant phenotypes were independent of
variations in the amount of template (see Fig. 1B for a
representative experiment) and nonspecific DNA in the reaction mixture
(not shown). The amount of template ranged from 0.015 µg to 0.45 µg, and the phenotypes of all mutants were checked at a subsaturating
amount of template (0.015 µg) in the presence and absence of 0.4 µg
of nonspecific DNA. Reactions were carried out at room temperature for
1 h and stopped by the addition of sodium dodecyl sulfate to
0.1%. After the addition of proteinase K (400 µg/ml), samples were
incubated at 37 °C for 30 min, extracted once with phenol, and
precipitated with ethanol. The transcripts were fractionated on 8 M urea, 8% polyacrylamide gels, detected by exposure to
x-ray film, and quantitated with a Bioimage Scanner.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
15 and
34 of the
tRNACAla gene. Within this region, two AT-rich
sequences, "AT," located at
15 to
20 relative to the
transcription initiation site, and "TAT" at
25 to
29 had
previously been shown to be important for transcription in silk gland
extracts (13). The sequence between TAT and AT (
21 to
24) is
designated "I" (Intermediate), and the sequence
upstream of TAT (
30 to
34) is designated "D" (Distal). In all cases, the mutant sequences that replace
the wild-type sequences are GC-rich and do not alter the spacing of any
presumptive promoter elements relative to one another or to the
transcription initiation site.
View larger version (16K):
[in a new window]
Fig. 1.
The Bombyx
tRNACAla upstream promoter is active in extracts of
Drosophila S2 cells. A, the sequence of a
wild-type tRNACAla gene from 12 to
34 is shown at
the top, with the D, TAT, I, and AT regions of the upstream
promoter delineated by brackets. The mutants are listed
below, with the mutant sequences that have replaced wild-type sequences
enclosed by boxes. Phenotypes are shown graphically and
numerically at the right as means of percentages of the
activity of a wild-type promoter determined in parallel. The size of 1 S.D. from each mean is shown. B, mutant phenotype is
independent of template concentration. The transcription rates of the
wild-type C upstream promoter (
) and the D region substitution
mutant (
) are plotted as a function of the amount of template per
reaction mixture.
View larger version (45K):
[in a new window]
Fig. 2.
Bombyx
tRNACAla upstream promoter elements direct
transcription in vivo in Drosophila
S2 cells. A, RNase protection assays are specific
for the products of B. mori tRNAAla genes
expressed in Drosophila S2 cells. Shown are the products of
protection by RNA from cells transfected with the 12-bp marked version
of the Bombyx tRNACAla gene (Cwt
maxi), the 20-bp marked version of the Bombyx
tRNACAla gene (Cwt maxi +8), or pSK+
vector lacking a tRNA gene( ). Transfection efficiency was the same
(±20%)in each case, as monitored by cotransfection with the actin-CAT
plasmid described under "Experimental Procedures." Products in the
leftmost lane were derived by protection of an
antisense "maxi" probe; products in the rightmost
two lanes were derived by protection of an
antisense "maxi+8" probe. Smaller fragments that represent
protection of endogenous Drosophila tRNAAla were
near the bottom of the gel and are not shown. B, RNase
protection assays show the in vivo phenotypes of low
resolution (upper panel) and high resolution
(lower panel) mutants of the Bombyx
tRNACAla upstream promoter. The transfection
efficiency for each mutant, relative to that of the wild-type promoter,
is shown below the lane. C, quantitative data for
the mutants shown in B. The size of 1 S.D. from each mean is
shown. Symbols are defined in the legend to Fig. 1.
View larger version (34K):
[in a new window]
Fig. 3.
Bombyx
tRNACAla and
tRNASGAla upstream promoters are differentially
active in vitro and in vivo.
In vitro transcription in extracts of Drosophila
S2 cells (A) or in vivo transcription in
transfected Drosophila S2 cells (B) was directed
either by the wild-type tRNACAla promoter
(Cwt) or the wild-type tRNASGAla
promoter (SGwt). Transcripts (A) or products
protected from RNase digestion (B) are shown after gel
electrophoretic fractionation. No tRNASGAla
transcripts are detectable above a background of ~1% of C promoter
activity.
31
to
25 is the most effective of the distal AT-rich sequences for C
promoter activity in vitro in Bombyx
extracts.4 To ask whether
this sequence could rescue the activity of the SG promoter in
Drosophila cells, we constructed a chimeric C/SG promoter
that placed the sequence in the corresponding position (
31 to
25)
in the SG promoter (Fig. 4A).
The T at position
24 in the SG promoter was mutated to a C to prevent
the fortuitous introduction of additional TATA-like sequences. The data
in Fig. 4B show that the promoter activity of this chimera
is indistinguishable from that of the wild-type C promoter, both
in vitro and in vivo.
View larger version (21K):
[in a new window]
Fig. 4.
The C gene sequence
31TTTATAT
25 functionally distinguishes C
and SG promoters. A, sequences of the wild-type C
(white bar) and SG promoter (gray bar) are shown,
as well as a chimeric derivative of the SG promoter in which the region
from
31 to
25 has been replaced by the corresponding region from
the C promoter (SG+TBS). The asterisk (*) denotes
a mutation at position
24 that was introduced to prevent the creation
of additional TATA-like sequences. B, transcription activity
directed by the promoters diagrammed in A either in
vitro in extracts of Drosophila S2 cells or in
vivo in transfected Drosophila S2 cells is plotted as a
percentage of the activity of the wild-type C promoter. *, in
vitro transcripts from the wild-type SG promoter (SG)
were undetectable.
View larger version (17K):
[in a new window]
Fig. 5.
Overexpression of Drosophila
TBP differentially affects C and SG promoter activity.
Plotted are the relative activities of the wild-type C ( ) and SG
promoters (
) and the C/SG chimera described in Fig. 4A
(
) in a Drosophila S2 derivative cell line that had been
stably transformed with Drosophila TBP under the control of
the Drosophila metallothionein promoter. Cells were induced
to overproduce TBP by incubation with 250 µM or 500 µM CuSO4, and promoter activity was measured
by RNase protection assays. Results are normalized to the activity of
each promoter in the absence of copper.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
29 to
25) is the most important short sequence.
Although tRNA promoter function is similar in vivo and
in vitro, there are differences in detail. For instance, the
effect of mutating short (4-5 bp) promoter sequences is less
pronounced in vivo than it is in vitro. This
result could suggest that sequence-specific interactions between
transcription factors and upstream DNA do not occur in vivo,
but the 20-fold effect of a 15-base pair substitution argues that they
do. We think it likely that differences in the relative concentrations of particular transcription factors account for the disparity between
in vitro and in vivo data. For example, TFIIIB,
the transcription factor complex that binds the
tRNACAla upstream promoter in vitro
(30), may be less concentrated in the extracts and fractions used
in vitro than it is within cells. The phenotypic variation
observed in vitro and in vivo in
Xenopus systems has been attributed to differences in the
concentrations of various transcription factors (31-33). Moreover,
quantitative Western blot analysis has shown directly that the
components of yeast TFIIIB are at least 100-fold more concentrated
in vivo than they are in typical yeast transcription
extracts (23). If a similar relationship exists for
Drosophila cells and extracts, efficient incorporation of
TFIIIB into transcription complexes could require a higher affinity
TFIIIB binding site in vitro than it does in
vivo, and, therefore, depend more heavily on a complete set of
protein-DNA contacts.
View larger version (29K):
[in a new window]
Fig. 6.
Upstream promoter mutants are differentially
impaired in Drosophila and Bombyx
extracts. The sequence of the wild-type C promoter from 34
to
15 is shown at the bottom with the D, TAT, I, and AT
regions delineated by brackets and the mutant replacement
sequences shown below each region. Phenotypes are plotted as
percentages of the activity of a wild-type C promoter determined in
parallel. Shown are the means ± S.D. of at least three
determinations. The size of 1 S.D. from each mean is shown. The
asterisk (*) denotes a mutant region with undetectable
levels of transcript in Drosophila extracts.
The different predilections shown by the Drosophila and
Bombyx systems in these experiments fit with previous
indications that the Drosophila transcription machinery is
particularly sensitive to mutation of Drosophila pol III
templates at about 25 (a position equivalent to Bombyx
TAT), but is relatively indifferent to mutation closer to the
transcription initiation site. Specifically, alteration of the natural
sequences between 30 and 20 bp upstream of the transcription start site
reduces the capacity of three different Drosophila tRNA
genes (tRNAArg (34), tRNAVal (18),
tRNAAsn (16)) and a Drosophila 5 S RNA gene
(35) to direct transcription in Drosophila extracts.
Although these effects are pronounced, a common sequence motif
responsible for the activity of the wild-type
30 to
20 regions is
not apparent. It is striking, however, that part of the critical region
of the Drosophila 5 S RNA gene (
31 to
27: TATAA)
strongly resembles the Bombyx TAT region (
29 to
25:
TATAT). Mutation of this sequence alone eliminates 5 S transcriptional activity (35). In contrast, mutation of the region equivalent in
position to the AT element (
20 to
15) has a much smaller effect
(35). These results suggest that the transcriptional enhancement due to
the TATA-like sequence in Bombyx tRNA genes may reflect a
general mechanism used in insect cells to increase the activities of
specific 5 S and tRNA genes.
The Drosophila S2 assay system revealed that the C and SG
promoters are differentially active in vivo, as they are
in vitro. The lack of the sequence TTTATAT (31 to
25) in
the SG promoter appears to account for the activity difference, since
addition of this sequence endows an otherwise inactive SG promoter with an activity indistinguishable from that of a wild-type C promoter. What
function does this sequence provide? Preliminary results indicate that
it can be bound by purified silkworm TBP. Does this TBP-DNA interaction
contribute to C promoter activity? The geometry of yeast transcription
complexes suggests that it could, since the preferred geometry for
transcription complexes formed on the wild-type SUP4
tRNATyr gene is one that places the upstream edge of TBP
near the base pair at
30 (36). By analogy, the location of the
TTTATAT sequence in the C promoter should allow it to be contacted by
TBP whose position is established by protein-protein contacts within
the transcription complex. Thus, the primary role of this sequence may
be to supply the C promoter with DNA contacts that add to protein
contacts to create a high affinity TBP binding site. Lack of a
comparable sequence may enfeeble the SG promoter.
In contrast to our results, previous studies have indicated that
specific TBP-DNA contact is not required for transcription of 5 S and
tRNA genes by pol III. In all systems tested, TFIIIB recruitment to
these templates does not occur through DNA binding alone but depends on
protein-protein interactions with TFIIIC (28). Moreover, there is
direct evidence in yeast that specific interaction of TBP with a TATA
element does not make a major contribution to TFIIIB recruitment, since
mutation of the DNA binding domain of TBP has no effect on 5 S and tRNA
transcription (37). Thus our results provide new evidence that, at
least in insect cells, direct contact of TBP with DNA through a TATA
element may be necessary for productive association of TFIIIB with the promoter.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Nancy Ahnert for technical assistance and the Institute of Molecular Biology DNA Sequencing Facility for sequence analysis.
![]() |
FOOTNOTES |
---|
* This work was supported by United States Public Health Service Grants CA74138 (to D. L. J.) and GM25388 (to K. U. S.) from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
** To whom correspondence should be addressed. Tel.: 541-346-6094; Fax: 541-346-5891; E-mail: ksprague{at}molbio.uoregon.edu.
2 A. Trivedi, unpublished results.
3 Data not shown.
4 C. Ouyang, M. J. Martinez, L. S. Young, and K. U. Sprague, manuscript in preparation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: pol III, RNA polymerase III; S2, Schneider-2; SG, tRNASGAla; C, tRNACAla; CAT, chloramphenicol acetyltransferase; TBP, TATA-binding protein; PCR, polymerase chain reaction; bp, base pair(s).
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|