From the Typically the internal promoter
elements of tRNA genes are necessary and sufficient to support
transcription. Here a sequence element preceding a Xenopus tRNA
gene is shown to be required for transcription in late stage, but not early
stage oocyte extracts. The constitutive tyrD gene is expressed in
both early and late oocyte extracts, whereas the early oocyte-specific
tyrCooc gene is only expressed in early extracts. An upstream
promoter element (URR), between positions -42 and -14 of the
tyrD gene, mediates this differential expression. The URR is
required for tyrD transcription in late oocyte extracts. Placing
the URR upstream of the tyrCooc gene allows this gene to be
transcribed in late extracts. The URR is irrelevant to transcription in
early extracts; transcription of tyrD or tyrCooc requires
only the internal promoter sequences. This indicates the polymerase III
transcriptional machinery changes during oogenesis, resulting in a
stringent upstream sequence requirement. Mutations within the URR are shown
to alter the preferred site of initiation by RNA polymerase III. Shifting
the position of the URR upstream by one-half helical turn also repositioned
the site of initiation, suggesting the URR directs the placement of the
initiation factor complex or polymerase itself. The core promoter of tRNA
genes consists of two highly conserved internal elements termed the A and B
boxes; however, the regulated expression of tRNA genes is in many cases due
to upstream sequences (1, 2, 3, 4, 5, 6,
7, 8, 9, 10) . Upstream elements have been found to increase
transcriptional activity by facilitating binding of transcription factors
or interaction by RNA polymerase III (2, 3, 4) . In some cases, a tissue-specific (6) or species-specific (7) requirement
for upstream promoter elements suggests that the polymerase III factor
complement can be different in different cell types. This study concerns
the promoter sequences which regulate two Xenopus tyrosine tRNA
genes: tyrD, which is constitutively expressed in adult somatic
cells, and tyrCooc, which is expressed in early oocytes and
gradually inactivated as the oocytes mature (9, 10) . Transcription of tRNA genes requires at least two
multisubunit factors, TFIIIC and TFIIIB (for reviews see Refs. 1, 11, and
12). Studies in yeast have provided the most detailed information as to the
structure-function relationships of the various subunits of TFIIIC and
TFIIIB. The principle binding sites for TFIIIC are the internal A and B
boxes. Yeast TFIIIB is comprised of two subunits in association with
TATA-binding protein (13) . This TFIIIB complex appears
to lack independent DNA binding capability and is recruited through contact
with TFIIIC to bind to a Higher eukaryotes have
requirements for tissue-specific expression of particular tRNA genes. There
are in Xenopus large families of oocyte-specific tRNA (10, 15) and 5 S RNA genes (16) , presumably to allow for large scale production of the RNA
products for use in early development. As oocytes mature, these oocyte-type
gene families are selectively and stably inactivated, whereas constitutive
or somatic-type tRNA and 5 S RNA genes remain active (10, 16, 17, 18, 19) . Partial purification and
reconstitution of the polymerase III factors from early and late stage
oocytes has indicated that a change in TFIIIC properties is responsible for
the selective loss of oocyte type tRNA gene expression (19) . Late stage TFIIIC loses the ability to activate or form
stable complexes with oocyte type tRNA promoters. This change in TFIIIC
activity can be brought about by the cdc25 phosphatase which is
instrumental in initiating the final stages of oocyte maturation (20) . This study delineates the promoter sequences which are
responsible for the regulation of the oocyte type tyrCooc gene and
the lack of regulation for the constitutive tyrD gene. The findings
show that transcription in late stage oocyte extracts requires a positive
sequence element preceding the tyrD gene (between -42 and
-14), including the probable binding site for the TFIIIB complex. This
upstream regulatory element is developmental stage specific; it has no
effect on transcription in early oocyte extracts. The tyrD maxigene construct was made by insertion of a
200-bp vector DNA fragment at the MspI site 18 bp preceding the
T-stretch termination signal. The plasmids containing the Xenopus
somatic and oocyte 5 S genes have been described previously (18, 21) . The extracts were fractionated by phosphocellulose
chromatography to separate TFIIIB and TFIIIC activities according to
previously described procedures (20, 23) , and these fractions combined to form a reconstituted
transcription system (19) . The tyrD upstream regulatory region (URR) is a positive
effector of transcription. In contrast, the tyrCooc upstream
region has no effect on transcription, either positive or
negative. Replacing the tyrD sequences preceding position -14
with vector sequences virtually abolished transcription (Fig. 1, B and D, lanes 5 and 6),
while replacing the corresponding tyrCooc upstream region with
vector sequences did not rescue transcription ( lanes 1 and
2). This is a stage-specific transcriptional enhancement; the
upstream sequences have no effect on transcription in early oocyte
extracts, yet are essential for transcription in late oocyte extracts,
suggesting a change during oogenesis in the transcriptional machinery which
interacts with the upstream region. The Positive Regulatory Element Is
Situated between -42 and -14 of the tyrD Gene-To determine more
precisely which sequences are important for transcription, deletions and
mutations were introduced (Fig. 2 A). Deletion to
position -42 did not reduce transcriptional activity (Fig. 2, B and D, lanes 1 and 2),
indicating the positive element is located between positions -42 and
-14. Deletion to position -30 reduced tyrD transcription
by 10-20-fold ( lanes 2 and 6), whereas deletion to
position -14 reduced the tyrD signal still further, to
nondetectable levels ( lane 7), an additional decrease of
5-10-fold. This indicates that sequences extending between positions
-42 and -14 are essential for high levels of expression in late
stage extracts. The effect is specific to late stage oocytes; deletion to
-30 or -14 reduced transcription by no more than 2-3-fold in early
oocyte extracts (Fig. 2
D). To
investigate the significance of the AT-rich-asymmetric sequence preceding
tyrD, a series of triplet base substitutions were introduced between
-40 and -32 (Fig. 2 A); AAC centered at
-39 was substituted by TTG, the corresponding sequence upstream of
tyrCooc; AAG centered at -36, common to both genes, was
substituted by pyrimidines, TCC; AAG centered at -33 was substituted by
TCC, the corresponding tyrCooc sequence. Each of these triplet
substitutions resulted in a 2-3-fold decrease in transcription level (Fig. 2 D, lanes 3-5), indicating this entire
region extending from -40 to -32 contributes to the transcriptional
enhancement. The AT-rich/asymmetric regions ends in a motif AAGTYA in the
case of both the tyrD and somatic 5 S genes (Fig. 2 A, underlined). An earlier study reported the related
motif AAAGT as being present in the 5`-flanking sequences of a number of
Xenopus tRNA and 5 S genes and implicated for interaction with a
novel stimulatory factor (27) . This motif is present
upstream of both the somatic and oocyte 5 S genes (but not tyrD or
tyrCooc) and overlaps with the AAGTYA motif (AAAGTYA). The triplet
substitution centered at -33 disrupts the AAGTYA motif and results in a
2-3-fold drop in transcription, suggesting this motif may be important for
transcription in late oocytes. As another point of interest, the site of
initiation by RNA polymerase III is altered by the -36 and -33
triplet substitutions. This is seen as a change in the relative proportions
of two transcript sizes. The -42tyrD gene produced
equal proportions of two transcripts corresponding to the two previously
reported initiation sites at -3 and -5 (Fig. 2
B, lane 2) (9, 10) . The
-36 triplet mutation results in only the larger transcript ( lane
4), whereas the -33 triplet mutant produces the smaller transcript
( lane 5). Since these sequence changes are located at least 25 bp
distant from the actual initiation sites, these changes are unlikely to
directly affect the choice of initiation residue by RNA polymerase III and
instead may be affecting the positioning of the TFIIIB initiation
complex. It is important to note that these sequence changes alter the
initiation site both in early and late extracts (Fig. 2
B), thus this positioning effect is not specific to late stage
extracts. These results demonstrate that
the differential expression of the oocyte-specific tyrCooc and
constitutive tyrD genes is due to a defined upstream sequence
element. This upstream regulatory region or URR is situated between
positions -42 and -14 of the tyrD gene and is required for
assembly of a functional transcription complex in late stage oocyte
extracts; deletion of the URR abolished transcription of the tyrD
gene, whereas placing this sequence element upstream of tyrCooc
restored transcription in late extracts. The tyrD upstream element
had no effect on transcription of either gene in early oocyte extracts,
indicating the regulatory effect is stage-specific, apparently reflecting a
change in the polymerase III transcription machinery during
oogenesis. Template exclusion assays indicate the tyrD URR is
important for stable complex formation. The only factors as yet known to
participate in stable complex formation are TFIIIB and TFIIIC (23, 28, 29, 31) . By analogy to the yeast system, the URR may include the
binding site for the TFIIIB initiation complex (14, 33) . As evidence that the tyrD URR affects
positioning of the initiation complex or polymerase III, the insertion of a
6-base pair sequence at -15 shifted the URR one-half helical turn
upstream and thereby shifted the site of transcription
initiation. Moreover, triplet base substitutions introduced within the URR
between positions -32 and -37 altered the preferred site of
initiation. These mutations are 27 or more bp upstream of the start site,
possibly too distant to directly affect polymerase III site selection,
suggesting these base changes alter the positioning of the TFIIIB
initiation complex. These findings are unexpected since for most tRNA
type genes, the internal promoter, including the A and B boxes, is
necessary and sufficient to direct factor binding and initiation by RNA
polymerase III, regardless of the upstream sequences (1,
11) . There are exceptions in insect systems in which
upstream sequences are required for transcription along with the internal
promoter (6, 8, 24)
; silkgland-specific expression of the Bombyx tRNA Studies in yeast have shown that TFIIIB, the initiation factor for
RNA polymerase III, binds to the region preceding tRNA genes, and it does
so largely in a sequence independent manner (14) . It is
thought that TFIIIC binds the internal promoter and directs TFIIIB, through
protein-protein contacts, to bind a specified distance upstream of the
initiation site. Consistent with those findings, the sequences preceding
the XenopustyrC and tyrD genes have no bearing on
transcription in early oocyte extracts. Replacement with vector sequences
did not affect transcription levels. It is therefore curious that these
upstream sequences become essential in late stage oocytes. One possible
explanation is that the interaction between factors TFIIIC and TFIIIB is
altered in late stage oocytes, such that TFIIIC is unable to direct TFIIIB
to bind to DNA in a nonspecific manner, and instead TFIIIB binding requires
the URR sequence or its equivalent. In support of a model invoking an
altered interaction between TFIIIC and TFIIIB, a recent study showed that a
mutation in a yeast TFIIIC subunit altered its ability to recruit TFIIIB
(36) . Our previous studies have shown that the
properties of TFIIIC change in maturing oocytes, such that late stage
TFIIIC is unable to form an active complex with early oocyte-specific tRNA
genes (19, 20) . As shown here, late
stage TFIIIC, or a component which copurifies with this activity, requires
the tyrD URR in order to support transcription, even though the URR
sequence is outside the canonical TFIIIC binding site. This may reflect an
ineffective TFIIIC-TFIIIB interaction, due to modification to either TFIIIC
(20) or TFIIIB (37) in late stage
oocytes. It is also important to point out that the phosphocellulose
fraction TFIIIC used in this study is a crude preparation and therefore
contains many components in addition to classical TFIIIC activity. It
remains a distinct possibility that components other than TFIIIC mediate
this regulation and such components might interact directly with the
URR. Certain features of the URR, other than primary nucleotide sequence,
may contribute to its positive effect on transcription. This region
exhibits high AT content and purine-pyrimidine asymmetry which might affect
flexibility or melting properties. The capacity to adopt bends may be
important for sequences preceding tRNA genes, since yeast TFIIIB is known
to produce a sharp bend in the DNA (25) . High AT
content is often associated with the 5`-flanking regions of tRNA genes (7, 24, 32) and is
characteristic of certain upstream sequences which have a positive effect
on tRNA transcription (3, 24) . In the
case of the sequence preceding a Bombyx constitutive tRNA The regulation of the tyrCooc and tyrD
genes is similar to regulation of the oocyte-type and somatic-type 5 S RNA
genes. Both 5 S genes are transcribed efficiently in early oocyte extracts,
but the somatic 5 S gene is 50-100-fold more active than the oocyte 5 S
gene in late oocyte extracts (18) . This regulation is
partly due to upstream sequences within 32 bp of the genes and partly due
to internal sequence differences (21, 26) , unlike the tyrCooc and tyrD genes in which
the internal sequence differences play no significant role in the
differential expression. The mechanisms by which the upstream promoter
elements enhance transcription of somatic tyrD and somatic 5 S
genes may be related; in the case of yeast 5 S and tRNA genes, TFIIIC binds
initially to the internal promoter of both types of genes and thereafter
recruits TFIIIB to bind to the upstream regions (14)
. Earlier findings have indicated that sequences preceding the
Xenopus somatic-type 5 S gene increase binding efficiency for
transcription factors (21) , as shown here for
tyrD (Fig. 4). These two genes also have common
AT-rich and asymmetric sequences in their upstream regions, as shown here
(Fig. 2). In summary, a sequence element preceding the
constitutive tyrD gene and encompassing the probable binding site
of the TFIIIB initiation complex is required for transcription in late
stage oocyte extracts. The absence of this upstream sequence, or a sequence
with equivalent properties, is responsible for the transcriptional
inactivity of the early oocyte type tyrCooc gene. This apparently
reflects changes in the polymerase III transcriptional machinery during
oocyte maturation such that a particular upstream sequence becomes
essential for formation of an active complex. A suggested model is that
modification to TFIIIC alters the interaction with TFIIIB, or other
component of the preinitiation complex, such that the complex loses the
ability to interact with the upstream DNA in a sequence-independent
manner. We thank Stuart
Clarkson for kindly providing plasmid DNAs and Richard Maki for critical
comments on the manuscript.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
40-bp
(
)
region preceding tRNA genes (14) . Within this complex,
TFIIIB forms a tight association with the DNA, which is largely
sequence-independent. Following high salt removal of TFIIIC, TFIIIB is able
on its own to direct initiation by RNA polymerase III, designating TFIIIB
as the initiation factor, whereas TFIIIC serves to position that complex
upstream of the gene (14) .
Xenopus DNAs
Plasmid tyrC
is previously described pTyrC* (9) which contains the
oocyte-type tRNAtyrC gene and 5`-flanking sequences joined to the
3`-flanking sequences of the tyrD gene. Plasmid pTyrD contains the
tRNAtyrD gene as described previously (9) . The
tyrD gene with tyrCooc upstream sequence was described
previously as p5`C-3`D, and the tyrC* gene with tyrD
upstream sequence as p5`D-3`C* (9) . The -42,
-30, and -14 deletion mutants of tyrD were made by
polymerase chain reaction using synthetic primers starting at the
designated positions, combined with a downstream primer starting 20 bp 3`
of the tyrD T-stretch termination signal. Polymerase chain
reaction primers were also used to make the -42tyrD
constructs with triplet base substitutions centered at -39, -36,
and -33. Primers started at position -and extended past the triplet
mutation sites. For the -14tyrCooc construct, primers
were used to place CCTGCACCCACCGG upstream of the tyrC*
gene. This sequence is tyrD-type at -14 to -9 (underlined)
and tyrC type at -8 to -1 ( bold). All of the
polymerase chain reaction-derived constructs are ligated in the same
orientation into Bluescript KS+ (Stratagene) at the EcoRV
site. Oocyte Extract Preparation
For
preparation of early oocyte extracts, ovaries were removed from
intermediate sized frogs (3-4 inches) and contained a mixture of oocytes at
all stages of maturation with relatively few fully mature stage (Dumont
stage VI). The ovaries were homogenized and S100 extracts prepared as
described previously (20) . For preparation of late
stage oocyte extracts, ovaries were removed from large, adult frogs and
treated with collagenase. The mature (stage VI) oocytes were isolated and
S150 extracts prepared as described previously (20, 22) . Transcription
Reactions
Transcription assays were performed for 3 h (unless
otherwise stated in the text) at 22 °C in a reaction volume of 25
µl containing 20 µl of oocyte extract, 5 mM
MgCl, 1 mM dithiothreitol, 0.6 mM each ATP, CTP,
and UTP, 20 µm GTP, 10 µCi of [
-
P] GTP, and 100 ng of template
plasmid DNA. Reactions were terminated by addition of 5 volumes of 20
mM EDTA, 0.5% sodium dodecyl sulfate (SDS), and
carrier RNA. The transcription products were purified by extraction with
phenol/chloroform (1:1), followed by ethanol precipitation. The precipitate
was redissolved in 95% formamide, 0.05% bromphenol blue, 1
TBE (90 mM Tris base, 90 mM boric acid, 2.5
mM EDTA). The samples were heated at 95 °C for
3 min, and an aliquot was loaded onto 8% polyacrylamide gels containing 1
TBE
and 8 M urea. Following electrophoresis, the gel was
dried and exposed to x-ray film. Typical exposure times were between 1 and
4 h. Quantitation of the relative amounts of label incorporated was
determined by excising and counting the corresponding regions of the
gel.
A Sequence Preceding the tyrD Gene Is
Essential for Transcription in Late Oocyte Extracts, but Has No Effect in
Early Oocyte Extracts
Earlier studies utilized distinct intron
sequences to demonstrate that tyrCooc and tyrD are
differentially regulated in vivo in early versus late
stage oocytes and in somatic cells (9, 10) . A similar pattern of regulation is observed in vitro
using oocyte extracts; tyrCooc is transcribed in early, but not
late, oocyte extracts, whereas the constitutive tyrD gene is
transcribed at high levels in both (20) . An
investigation was initiated to identify the DNA sequences responsible for
the differential expression in late stage oocytes. The two genes differ in
their upstream sequences, at one position within the B box promoter
element, and by distinct introns of 10-12 bp situated between the A and B
boxes (9) . Previous studies indicated that sequence
differences upstream, but within 12 bp of the genes, resulted in 6-fold
higher transcriptional efficiency for tyrD, relative to
tyrCooc, in somatic cell extracts (9) . To
determine which sequences are responsible for the more pronounced
50-100-fold difference in transcription levels in late oocyte extracts, a
series of deletion and exchange mutants were compared (Fig. 1 A). These included the tyrCooc gene with 65
bp of native 5` sequence, the tyrD gene with 95 bp of native 5`
sequence (constructs 1 and 5), those genes with exchanged upstream
sequences (constructs 3 and 7), both genes having 14 bp of tyrD
upstream sequence (CCTGCATGAACATC) (constructs 4 and 6), or 8 bp of the
tyrCooc immediate upstream sequence (cctgcaCCCACCGG)(constructs 2
and 8). This tyrCooc upstream region (underlined) is highly
GC-rich and surrounds the site of transcription initiation at position
-5. The position designated +1 represents the first nucleotide of the
mature tRNA sequence, rather than the actual site of transcription
initiation, in accordance with previous studies. As reported previously,
transcription initiation for tyrD occurs at positions -5 and
-3, whereas in the case of the tyrCooc gene, initiation occurs
at position -5 (10) . All eight constructs have the
same termination signal and 3` sequences derived from the tyrD
gene.
Figure 1: Sequences preceding the tyrD and
tyrCooc genes cause differential expression in late but not early
oocyte extracts. A, the following constructs were assayed by in
vitro transcription in oocyte extracts: the tyrCooc gene with
65 bp of native upstream sequence (1), 14 bp of tyrD-tyrCooc
upstream sequence (2), 95 bp of tyrD upstream sequence (3), or 14
bp of tyrD upstream sequence (4). The tyrD constructs
contained 95 bp (5) or 14 bp (6) of native upstream sequence, 65 bp of
tyrCooc upstream sequence (7), or 14 bp of tyrD-tyrCooc
upstream sequence (8). For constructs 2 and 8, the upstream 14 bp are the
tyrD sequence from -14 to -9 (CCTGCA) and the
tyrCooc GC rich sequence from -8 to -1 (CCCACCGG). For
constructs 4 and 6, the 14 bp upstream are tyrD type
(CCTGCATGAACATC). The relative transcriptional activities shown were
quantified from the data shown in B. B, in vitro
transcription assays were carried out in S150 extracts of early ( upper
panel) and late stage oocytes ( lower panel) as described under
``Materials and Methods.'' The reactions were performed for 3 h at 25
°C, and the radiolabeled RNAs were purified, resolved in 8%
sequencing gels, and exposed to film. An autoradiograph is shown. The
numbers and construct names refer to constructs 1-8 shown
in A. C, diagrammatic representation of the data showing
the relative contributions of the upstream versus coding sequences
to the tyrD transcriptional advantage in late stage extracts.
D, graphic display of the data shown in B.
The eight constructs were tested in
transcription reactions in extracts of early versus late stage
oocytes. In early extracts, the upstream sequences had no significant
effect on transcription; all eight constructs were transcribed with similar
high efficiency (Fig. 1 B, upper panel, and
D). Deletion to position -14 did not alter the level of
transcription of either gene, indicating the internal promoter is the
primary determinant of transcriptional activity in early
oocytes. Surprisingly, this is not the case in late oocyte extracts; only
two of the eight constructs produced strong transcriptional signals, those
being either gene with 95 bp of tyrD upstream sequences (Fig. 1 B, lower panel, lanes 3 and 5, and
D). Deletion of the tyrD sequences between -95 and
-14 reduced transcription to nearly undetectable levels ( lanes
4 and 6), indicating this region contains an essential promoter
element. The 14-bp sequence immediately preceding the tyrD gene
provided a relatively small, 2-3-fold advantage ( lanes 1, 4, 6, and
8). Substitution with the 8 bp of GC-rich tyrCooc sequence
surrounding the initiation site abolished that 2-fold advantage ( lanes
2 and 4). These results indicated that tyrD sequences
further upstream than -14 are essential for high levels of
transcription in late oocyte extracts, with a smaller contribution from
sequences within 14 bp of the tyrD gene. These results are in
contrast to previous findings using somatic cell extracts which showed the
tyrD gene to be only 6-fold more active than tyrCooc and
that advantage was shown to be due entirely to the 12 bp of sequence
immediately preceding the gene (9, 10)
.
Figure 2: The sequence between -42 and -14 of the
tyrD gene is required for transcription in late but not early
oocyte extracts. A, the sequences preceding the tyrD and
tyrCooc genes are shown. tyrD mutant constructs designated
-39, -36, and -33 have 42 bp of tyrD upstream sequence
with triplet base substitutions indicated by underlined,
nonbold type. The -39 and -33 mutants have the corresponding
tyrCooc sequence inserted at those positions. The -36 mutant
abolishes a conserved AAG sequence present upstream of both tyrCooc
and tyrD. The -30 deletion construct has sequences preceding
-30 replaced by vector sequences. The -14 deletion construct has
sequences preceding -14 replaced by vector sequences. All constructs
are inserted in the same orientation and so have the same vector sequences
preceding the tyrD sequences. For all constructs, nucleotides in
bold are identical to the tyrD upstream sequence. Below is
shown the 32 bp of sequences preceding the somatic-type 5 S and oocyte-type
5 S genes. The lines above the sequence indicate features common to
tyrD and somatic 5 S, including a purine-pyrimidine asymmetry
followed by an AAGTYA motif. B, transcription reactions were
carried out in early and late stage oocyte extracts. The constructs used in
the reactions are the tyrD gene with 95 bp of upstream sequence,
and the constructs shown in A, as listed below each lane. C,
a diagrammatic representation of results shown in B indicating the
relative importance of the tyrD upstream sequences to
transcription in late stage extracts. D, a graphic representation
of the data shown in B. The signals obtained with the
-95tyrD gene ( lanes 1) for each extract was
assigned a relative transcriptional activity of 100%. The relative
transcriptional activity shown for lanes 2-8 is relative to
that of -95tyrD in the same extract.
The DNA sequence between positions
-42 and -14 has features which might affect the ability to form an
active transcription complex (Fig. 2 A). The
region between -41 and -28 is relatively AT-rich, a characteristic
which has been suggested to facilitate bending of DNA (24) , and it is known that the TFIIIB initiation complex produces
a sharp bend in DNA (25) . A second feature is
purine-pyrimidine asymmetry between positions -42 and -32, in which
the upper strand is comprised of 10 out of 11 purine
residues. Interestingly, a 14-bp asymmetric sequence is similarly situated
between positions -28 and -15 preceding the somatic 5 S gene,
whereas upstream of the oocyte-specific 5 S gene, the asymmetry is
centrally interrupted at two positions (Fig. 2 A,
bottom). Like tyrD and tyrCooc, the somatic and oocyte
5 S genes are transcribed in early extracts, whereas only the somatic 5 S
gene is active in late extracts (18, 26) (Fig. 2 B, lanes 9 and
10). Previous studies have shown that 32 bp immediately preceding
the somatic 5 S gene is responsible for about half of the 100-fold somatic
5 S transcriptional advantage in late extracts (21)
. Therefore there is reason to suspect that similarities in the upstream
sequences of tyrD and somatic 5 S may be significant in terms of
the selective transcription of these genes in late oocyte extracts. Prolonged Incubation
in the Presence of Transcription Factors Does Not Rescue Templates Lacking
the Upstream Regulatory Element
The polymerase III transcription
factors form a stable complex with DNA which remains associated throughout
repeated rounds of transcription (28, 29) . Certain templates, such as oocyte 5 S, are less able to form
stable complexes with transcription factors. However, there is evidence
that increased time of incubation with factors allows eventual formation of
stable active complexes on the oocyte 5 S gene (30) . To
determine if this is the case for templates lacking the URR, the DNAs were
incubated in late extracts for increasing periods of time, prior to
addition of radiolabeled nucleotide (Fig. 3). The
templates were -65tyrC, and tyrD with 42, 30,
or 14 bp of native upstream sequence. The times of preincubation were 1 or
2 h, followed by 1 additional h with labeled nucleotide. Even the 2-h
preincubation period (3 h total incubation) was insufficient to raise the
transcription signals of templates lacking the URR ( lanes
9-12). This indicates that prolonged incubation does not result
in the eventual formation of active complexes on templates lacking the
URR.
Figure 3: Templates lacking the tyrD upstream
promoter remain inactive even after prolonged incubation periods in late
stage extracts. In the first set of transcription reactions ( lanes
1-4), the tyrD gene with 42, 30, or 14 bp of native
upstream sequences, and the tyrCooc gene were incubated for 2 h in
late stage oocyte extracts in the presence of radiolabeled nucleotide. In
the second set ( lanes 5-8), the templates were incubated for
1 h in the extract without radiolabeled nucleotide and then the labeled
nucleotide was added for 1 additional h. In the third set ( lanes
9-12), the preincubation period was 2 h, and the radiolabeled
nucleotide was added for the 3rd h. The RNA products were purified,
resolved in gels, and exposed to film as described in Fig. 1. The tyrD URR Facilitates Stable Binding
by Polymerase III Transcription Factors
Template commitment assays
provide a means to test the stability of factor complexes. In these assays,
one template is preincubated in the extract for 1 h to allow factor
binding. A reporter template is then added and incubation continued for 2 h
in the presence of labeled nucleotide. The degree to which the reporter
template is inhibited provides a measure of the ability of the first DNA to
stably sequester transcription factors. This assay relies on the
established concept that polymerase III transcription factors, once bound
to DNA, remain stably associated for many consecutive rounds of
transcription (31) . In the experiment shown in Fig. 4, tyrD constructs having 42 or 14 bp of native
upstream sequences were preincubated in intermediate stage oocyte extracts
for 1 h, followed by addition of a reporter tyrD maxigene
construct. Preincubation with -42tyrD resulted in
nearly complete inhibition of the tyrD maxigene ( lanes 1
and 2), indicating that most of the limiting factors were stably
sequestered by -42tyrD. In contrast, preincubation with
the same amount of the -14tyrD construct resulted in
little to no inhibition of the maxigene reporter ( lane 3). These
findings indicate the URR is involved in formation of stable factor-DNA
complexes.
Figure 4: Sequences preceding tyrD facilitate
stable binding by transcription factors. Template exclusion assays were
carried out in intermediate oocyte extracts for a total of 3 h with
radiolabeled nucleotide. In lanes 2 and 3, 200 ng of
-42tyrD or -14tyrD plasmid DNA were
preincubated in the extract for 1 h to allow factor binding, followed by
addition of 200 ng of a reporter tyrD maxigene, and the reaction
continued for 2 additional h. In lane 1, no DNA was present during
the preincubation period and then the tyrD maxigene was added for
the remaining 2 h of incubation. The RNA products were purified, resolved
in a sequencing gel, and an autoradiograph obtained. The tyrD
maxigene produces a major transcript of greater than 200 bp and smaller
transcripts which probably represent premature termination events. TFIIIC from Late Stage Oocytes Is Unable
to Activate the tyrD Gene Lacking the URR
The factors known to take
part in stable complex formation are TFIIIC and TFIIIB. Previous findings
have shown that the properties of TFIIIC are different in early and late
stage oocytes (19) . Late stage TFIIIC fails to form a
stable, active complex with oocyte-specific tRNA genes, while retaining the
ability to activate the somatic type 5 S RNA gene. The principle binding
site for TFIIIC is the internal B box and A box elements, not the URR
region. However, protein-protein contacts with TFIIIC are thought to be
essential to direct TFIIIB to bind to sequences in or near the URR (14) . Thus, modification to TFIIIC in late stage oocytes could
conceivably alter the interaction with TFIIIB, thereby altering the binding
specificity of TFIIIB. To determine if the change in TFIIIC activity is
mediated through the upstream sequence, these factors were separated by
phosphocellulose chromatography from early and late stage extracts (23) . The resultant fractions are crude, containing many
components in addition to classical TFIIIC and TFIIIB activities. Fraction
TFIIIC from either stage was reconstituted with fraction TFIIIB from late
extracts, which also contains TATA-binding protein and RNA polymerase
III. A template lacking the URR ( -14tyrD) was
transcribed in the presence of early but not late stage TFIIIC (Fig. 5). The template containing the URR (
-42tyrD) was transcribed with either early or late
TFIIIC. In the reverse experiment, with early TFIIIC in all four reactions,
the -14tyrD template was transcribed in the presence of
either early or late TFIIIB (Fig. 5). These results
support a model whereby a change in the properties of TFIIIC, or a
component which copurifies in fraction TFIIIC, can affect transcription by
a mechanism mediated by the upstream
URR.
Figure 5: The tyrD gene lacking the URR is not
transcribed in the presence of TFIIIC from late stage oocytes. In
vitro transcription reactions in the left panel were carried
out in a reconstituted system with partially purified fraction TFIIIB from
late oocytes, and TFIIIC from either late ( lanes 1 and 2)
or early ( lanes 3 and 4) oocytes. In the right
panel, fraction TFIIIC from early oocytes was present along with either
late ( lanes 1 and 2) or early ( lanes 3 and
4) TFIIIB. The templates are the -42TyrD
construct containing the URR, and the -14tyrD construct
lacking the URR. Repositioning the URR Upstream by
One-half Helical Turn Alters the Site of Transcription
Initiation
Yeast studies indicate the TFIIIB initiation complex
contacts DNA sequences upstream and within 45 bp of tRNA genes (14, 32, 33) . Difficulties
in purifying TFIIIB from higher eukaryotes has prevented direct physical
determination of the site of TFIIIB binding; however, there is evidence
from the Bombyx system that the 40-bp region preceding tRNA genes
is involved in TFIIIB binding (24) . To investigate
whether the tyrD URR may be involved in the positioning of the
initiation complex or RNA polymerase III, a 6-base pair sequence was
inserted at position -15, shifting the URR further upstream and to the
opposite side of the DNA helix (Fig. 6). Moving the URR
to the opposite side of the helix could conceivably interfere with
protein-protein contacts between TFIIIB and TFIIIC, which binds to the
internal promoter. Shifting the URR upstream by half a turn was found to
shift the site of initiation. The two original sites of initiation
converged into a single site which was approximately 2 base pairs
upstream. Transcription also decreased by severalfold. It is unlikely that
the 6-bp insertion at position -15 directly affects the choice of
initiation residue by RNA polymerase III, since the insertion is 8-12 bp
away from the usual site of initiation. Instead, this suggests that
shifting the URR further upstream alters the positioning of the initiation
complex, thereby affecting positioning of RNA
polymerase.
Figure 6: Insertion of a 6-bp sequence at position -15
shifts the URR one-half turn upstream and alters the site of
initiation. The diagram shows the site of insertion of the sequence GAATTC
at position -15 preceding the tyrD gene. The arrows
show the sites of initiation previously reported for tyrD (A
residues at -3 and -5) (10) and below is indicated the approximate
site of initiation in the insertion mutant. The -15 insertion
tyrD mutant and the -42tyrD construct were
transcribed in late stage extracts and the transcription products resolved
in sequencing gels as shown below. The -15 insertion construct produced
a slightly larger transcript size.SG requires an
upstream sequence, while a different upstream sequence is required for
transcription of the constitutive tRNA
gene (6, 24) . The sequence preceding the silkgland-specific
tRNA
SG
gene results in a less effective interaction by TFIIIB and polymerase III
(34) , indicating that in higher eukaryotes, as in
yeast, the
40-bp preceding tRNA genes
includes the site of interaction by TFIIIB. In mammalian systems, the
region preceding tRNA genes has also been shown to influence factor binding
and transcriptional activity (4, 35)
. Placement of the Lac repressor binding site at various positions upstream
of a human tRNA gene interfered with formation of transcription complexes,
presumably through steric hindrance, indicating the transcription complex
extends at least 35 bp upstream (35) . The findings
reported here are distinctive from earlier studies in that the tyrD
URR has dramatically different effects on transcription at two stages of
oocyte maturation. In late oocyte extracts, the URR is essential for
transcription, whereas in early oocyte extracts, the URR is
irrelevant. This indicates that the polymerase III machinery changes during
oogenesis with resultant changes in DNA sequence requirements and that the
upstream 40 bp plays a central role in the regulation of these tRNA
genes.
C gene, the
AT-rich character is more important than the actual DNA sequence (24) .
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.