In vitro
transcription of a poly(dA)[middot]poly(dT)-containing sequence is inhibited by interaction between the
template and its transcripts
In vitro transcription of a poly(dA)[middot]poly(dT)-containing sequence is inhibited by interaction between the template and its transcripts
Ryoiti
Kiyama*
and
Michio
Oishi
Institute of Molecular and Cellular Biosciences, The University of Tokyo, Yayoi,
Bunkyo-ku,
Tokyo
113,
Japan
Received June 27, 1996;
Revised and Accepted September 20, 1996
ABSTRACT
Transcription of poly(dA)[middot]poly(dT)-containing sequences was investigated
in vitro
using plasmids carrying a (dA)
34
[middot](dT)
34
tract in the coding region of the
lacZ
gene. The efficiency of transcription of the (dT)
34
sequence on the transcribing strand by
Escherichia coli
RNA polymerase was substantially lower (
~
60%) than that of the (dA)
34
sequence or of the control
lacZ
gene. Analysis of the transcription process of the (dT)
34
sequence by T3 RNA polymerase showed that the transcription was frequently
arrested or terminated at the middle as well as immediately proximal of the
(dA)
34
[middot](dT)
34
tract, and it occurred more prominently following accumulation of transcription
products. This inhibition was strongly enhanced by the addition of the oligonucleotide (dT)
34
or poly(U) to the reaction mixture, while (dA)
34
and the duplex (dA)
34
[middot](dT)
34
suppressed the inhibition. A similar transcriptional inhibition was also
observed in transcription mediated by T7 RNA polymerase and eukaryotic RNA
polymerase II. We also demonstrated RNA[middot]DNA complex formation of the (dA)
34
[middot](dT)
34
tract with poly(U), but not with poly(A). These findings strongly suggest that poly(dT)- containing template sequences interact and form a complex with its transcription products, possibly an RNA[middot]DNA triplex, which blocks further transcription. This would explain the instability of the plasmids transcribing
mRNAs with poly(U) but not poly(A) tracts and the underrepresentation of
poly(U) but not poly(A) tracts in mRNAs.
INTRODUCTION
Transcription is generally controlled through interactions between template DNA and RNA polymerases with the help of various types of protein
factors collectively referred to as transcription factors (
1
-
6
). These factors interact with specific DNA sequences in the transcription-regulatory regions. The promoter region located immediately upstream of the genes accommodates binding sites for these
factors, while enhancer and silencer elements modulate the transcriptional
activity from upstream or downstream of the genes. On the other hand, simple
sequences such as (dA)
n
, (dG)
n
, (dA-dC)
n
, which constitute microsatellite DNA are also known to affect the efficiency of
transcription. The presence of A+T-rich
sequences in the promoter region for example, enhances or suppresses transcription in
Escherichia coli
and yeast genes (
7
-
10
), and short poly(dA-dT) sequences can substitute for the function of promoter elements (
9
,
11
,
12
). Sigma factor, an essential component of the transcription machinery in
E.coli
, is released at the poly(dA-dT) tract once the transcription is initiated (
13
). These microsatellite sequences, which are distributed ubiquitously along genomic DNA in most eukaryotes, are likely to be associated with other biological reactions including recombination and replication (
14
-
17
). Although the exact roles played by such sequences remain to be determined, microsatellite DNA sequences could affect
these reactions through formation of unique topological structures or by interactions with specific binding proteins (
12
,
17
-
19
).
Other than specific nucleotide sequences, specific or `unusual' DNA structures
affect the transcription. For example, (dA-dC)
n
sequences affect transcription through Z-DNA formation (
20
,
21
). Bent DNA, formed by short poly(dA) tracts at an interval of 10-11 bp, can alter the transcriptional efficiency (
22
,
23
). Because of low melting temperatures, A+T-rich sequences tend to melt during transcriptional elongation causing transcriptional slippage (
10
,
24
).
Controlling gene expression could be achieved by several strategies. Antisense
RNA or DNA against mRNA is one of the methods to control gene expression at
translation. In contrast, gene expression could be controlled by the formation
of triplex DNA using specific DNA or RNA oligonucleotides which bind to the
regulatory region, to inhibit the initiation of transcription (
25
-
27
). Transcription elongation can also be inhibited by oligonucleotides that form
triplex DNA (
28
,
29
) or by peptide nucleic acids that form a D-loop (
30
).
Previously, we reported that
E.coli
plasmid clones containing microsatellite poly(dA)[middot]poly(dT) exhibited marked instability in their maintenance in host cells
(
31
). This observation was further investigated by employing a series of plasmids
containing a (dA)
34
[middot](dT)
34
tract placed downstream of the
lacZ
promoter, and the results suggested that the instability was likely to be
caused by transcription of the poly(dT) strand (
32
). In this report, we show that the
in vitro
transcription of the (dA)
34
[middot](dT)
34
tract was inhibited as a result of the interaction between the poly(U)-containing transcripts and their templates. The possible involvement of an
RNA[middot]DNA triplex structure in the inhibition is discussed.
MATERIALS AND METHODS
Materials
Oligonucleotides were synthesized by a Millipore Cyclone DNA synthesizer and purified with Milligen Oligo-Pak columns. Poly(U) and poly(A) were purchased from Pharmacia.
Plasmid construction
Plasmids pAT19 and pAT18 were constructed from vector pUC19 or pUC18 (
33
) by replacing the
Bam
HI-
Hin
dIII sequence with GGATCC(
Bam
HI)-(A)
32
-AAGCTT(
Hin
dIII) (
32
). pATB3 and pATB12 were constructed by inserting the GGATCC-(A)
32
- AAGCTT sequence into the
Eco
RV site of pBluescript SK(-) (Stratagene, USA) in the direction of the (dT)
34
(pATB3) or (dA)
34
(pATB12) sequence on the transcribing strand for T3 RNA polymerase (therefore,
on the template strand for T7 RNA polymerase). The plasmid pAT[epsilon]3-32 was constructed by inserting the
Eco
RV-
Ssp
I fragment of the human [epsilon]-globin gene (positions -274 to +185 relative to the cap site, thus containing the
whole promoter region) into the
Sma
I site of pATB3 in the direction that the transcription by eukaryotic RNA
polymerase II proceeds to the (dA)
34
[middot](dT)
34
tract. Plasmids used in the transcription assays are summarized in Figure
1
.
In vitro
transcription with
E.coli
RNA polymerase
To avoid read-through transcription, purified plasmid DNA (pUC19, pAT19, pUC18 or pAT18)
was digested with
Pvu
II and
Sca
I, to separate the
bla
and
lacZ
genes. Approximately 1 [mu]g of plasmid DNA was incubated with 1 U of
E.coli
RNA polymerase (Boehringer Mannheim, Germany) in 20 [mu]l of a mixture of 40 mM Tris-HCl (pH 7.5), 6 mM MgCl
2
, 5 mM NaCl, 2 mM spermidine, 10 mM DTT, 0.5 mM each of rNTPs and 1 U RNase
inhibitor (Takara, Kyoto) for 30 min at 37oC, followed by incubation with 70 U DNase I (Takara) for 15 min at 37oC. The reaction was terminated by addition of SDS (0.1%). The reaction
mixture was then spotted on Hybond-N+ (Amersham) nylon membranes. Two identical membranes were made.
Hybridization with
32
P-labeled 24mer sequencing primers #1224 (CGCCAGGGTTTTCCCAGTCACGAC, New
England Biolabs, USA) as the
lacZ
probe, and with
32
P-labeled 20mer oligonucleotides (TATGCGGCGACCGAGTTGCT, positions 2208-2227) as the
bla
probe were performed according to Geliebter
et al.
(
34
). Oligonucleotide probes were labeled with [[gamma]-
32
P]ATP (4500 Ci/mmol, ICN, USA) using T4 polynucleotide kinase (New England
Biolabs) to a specific activity of 1.5 * 10
8
c.p.m./[mu]g. After hybridization, the membranes were washed twice with 3* SSC, 10 mM sodium phosphate buffer (pH 7.2), 10% (v/v) Denhardt
solution, 5% (w/v) SDS for 1 min, once with the same buffer for 1 h and then
with 1* SSC, 1% (w/v) SDS for 1 h. Hybridization and washing membranes was performed at 60oC. The membranes were autoradiographed with a BAS2000 Image Analyzer (Fujix, Japan), and the radioactivities of the RNA-oligonucleotide complexes were quantitated.
In vitro
transcription (run-off assay) with T3 or T7 RNA polymerase, or with nuclear extracts from
HeLa cells
One hundred ng of pATB3 or pATB12 was mixed in 20 [mu]l of the transcription mixture described above except that rCTP was replaced by 5 [mu]Ci of [[alpha]-
32
P]rCTP (650 Ci/mmol), and transcription was initiated by adding 1 [mu]l of T3 (50 U/[mu]l; BRL, USA) or T7 (10 U/[mu]l; Wako, Japan) RNA polymerases. After incubation at 37oC for the indicated lengths of time, the reaction was
terminated by addition of 1 [mu]l of 10% (w/v) SDS. The transcripts were resolved by electrophoresis through a 6% polyacrylamide-7 M urea gel under denaturing conditions. Transcription with the nuclear extract (3 mg/ml proteins) from HeLa cells was carried out with 1 [mu]g of template (
Xho
I digests of pAT[epsilon]3-32 DNA), in 20 [mu]l of buffer containing 12 mM HEPES (pH 7.9), 12% (v/v) glycerol,
0.3 mM DTT, 0.12 mM EDTA, 60 mM KCl, 12 mM MgCl
2
, 0.5 mM rNTPs except for rGTP and 5 [mu]Ci of [[alpha]-
32
P]rGTP (650 Ci/mmol) at 30oC for the indicated lengths of time (
35
). The nuclear extract was prepared according to the method described by Dignam
et al.
(
36
) and detailed previously (
37
).
Detection of RNA[middot]DNA complex
RNA[middot]DNA complex formation was assayed first by incubating 400 [mu]M (nucleotide-equivalent) of
32
P-labeled third strand, poly(A) or poly(U) and 20 ng/[mu]l of
Xmn
I-digested plasmid DNA in 25 [mu]l of 10 mM Tris-HCl (pH 7.5), 10 mM MgCl
2
, 50 mM NaCl (TMN buffer) for 30 min at 37oC, followed by electrophoresis on an agarose gel (0.7%) in 50 mM Tris-borate (pH 8.3) and 10 mM MgCl
2
overnight at 4oC (
37
). The gel was then soaked in 10% (w/v) trichloroacetic acid, dried under paper towels and autoradiographed. Poly(A) and poly(U) were labeled with T4 polynucleotide kinase and [[gamma]-
32
P]ATP (4500 Ci/mmol).
RESULTS
Inhibition of transcription by
E.coli
RNA polymerase
The efficiency of transcription of the (dA)
34
[middot](dT)
34
tract was first examined
in vitro
with
E.coli
RNA polymerase using several template plasmids (pUC19, pAT19, pUC18 and pAT18) producing either poly(U)-containing (pAT19) or poly(A)-containing (pAT18) transcripts, or
lacZ
(control) transcripts (pUC19 and pUC18). These plasmids were used for the
analysis of plasmid instability (
29
), and among them, only pAT19 exhibited a marked instability. This instability was shown to be closely related with the transcription of
lacZ
gene (
29
). Full-length run-off transcripts were detected by hybridization with
lacZ
probe (#1224 oligonucleotide) located just downstream of the (dA)
34
[middot](dT)
34
tract (Table
1
). By this assay, the transcripts containing the poly(U) or poly(A) tract were
quantitated. We found that the level of
lacZ
transcripts with pAT19 was significantly lower than the others; ~60% of those with the other plasmids. Meanwhile, the level of transcription
of the control (
bla
) gene was the same among all the plasmids (data not shown) and was used to
normalize the
lacZ
transcripts. This result suggested that transcription was inhibited when a (dT)
34
sequence was present on the transcribing strand. The inhibition by the presence
of (dT)
34
sequence apparently occurred only when it was located in a
cis
position because transcription of (dT)
34
did not affect
bla
gene transcription in the same reaction mixture. Since a number of strong and
weak transcription initiation sites exist in the pUC plasmids for
E.coli
RNA polymerase (data not shown) which may complicate analysis of the results,
we examined transcription by T3 RNA polymerase.
In vitro
transcription of poly(dA)[middot]poly(dT)-containing sequences by
E.coli
RNA polymerase
Relative rate of transcription
a
Plasmid
pUC19
pAT19
pUC18
pAT18
Experiment
1
1.00
0.65
1.08
0.90
2
1.00
0.53
1.09
1.04
Average
1.00
0.59
1.09
0.97
a
Transcripts of
lacZ
gene were quantitated by dot hybridization with
32
P-labeled #1224 oligonucleotides as described in Materials and Methods. The
radioactivity was counted and the ratios of the counts of the
lacZ
probe to those of the
bla
probe were calculated and normalized by the ratio for pUC19 as 1.00.
Inhibition of transcription by T3 RNA polymerase
In vitro
transcription of the (dA)
34
[middot](dT)
34
tract was further examined with plasmid pBluescript and its derivatives (pATB3
and pATB12) where the tract was placed between the T3 and T7 promoters (Fig.
2
). When the plasmid DNA linearized with
Kpn
I was used as a template for run-off assay, T3 RNA polymerase produced 166 nt (U)
34
-containing (for pATB3) or (A)
34
-containing (for pATB12) transcripts, while control pBluescript vector produced 117 nt transcripts (Fig.
1
). The results with pATB3 indicated a strong termination site in the middle as
well as at the immediate proximal region of the (dT)
34
sequence on the transcribing strand (lower bracketed regions in Fig.
2
A). On the other hand, transcription of pATB12 with T3 RNA polymerase showed
little inhibition at these regions. The kinetics of the appearance of radioactivity in the major bands (corresponding to 166 and 117 nt full-length transcripts) (Fig.
2
B) indicated that incorporation for pATB3 reached a plateau earlier than for pATB12 or the control vector. As
shown in Figure
2
C, while the ratios of the full-length to the premature transcripts for pATB12 increased up to 60 min of
incubation, the ratio for pATB3 started to decline after 10 min of incubation,
reaching 50% within the next 90 min. These results may be best explained by
inhibition of transcription of the full-length RNA midway when the (dT)
34
was present in the transcribing strand, and the effect became more prominent when the transcripts were accumulated at the later stage of incubation. Although there were several other minor bands corresponding to the premature
transcripts including the ~130 nt transcripts with pATB12 (Fig.
2
A), none showed this type of kinetics, indicating that they are not inhibited by
the full-length transcripts but represent the products of premature termination.
Involvement of complex formation between transcripts and their templates in the
inhibition
To investigate further the mechanism of the observed transcriptional inhibition, we analyzed the transcription process in the presence of oligodeoxyribonucloetides, (dT)
34
or (dA)
34
, or an oligoribonucleotide, poly(U) (~40-50 nt). As shown in Figure
3
A, when at least 10 nM of (dT)
34
was added in advance to the transcription mixture, the level of the premature
transcripts increased. On the other hand, addition of at least 10 nM (dA)
34
decreased the level of inhibition. Poly(U) caused an effect similar to that seen with (dT)
34
(Fig.
3
A). Note that the concentration of poly(U) (2 [mu]M), which was nucleotide-equivalent, was roughly equivalent to 40-50 nM in the number of poly(U) molecules. When the transcription was compared as a function of time among the reactions with 1 [mu]M (dT)
34
or (dA)
34
(Fig.
3
B) or without oligonucleotides (Fig.
2
A), the ratio of full-length to premature transcripts for the transcription with (dT)
34
decreased without a lag (summarized in Fig.
3
C). In contrast, the ratio for the transcription with (dA)
34
first increased to 2.4-fold between 1 and 10 min, but decreased later to the original level.
These results indicate that transcription was also inhibited in the presence of
(dT)
34
, although the inhibition occurred without a delay. We found, however, that when
(dA)
34
was present in the reaction mixture, the degree of inhibition was apparently reduced. This could be explained by the oligonucleotide forming a duplex with the (U)
34
sequence in the transcripts and, as a result, effectively removing the free transcripts. If this was the case,
(U)
34
-containing transcripts interacted with the (dA)
34
[middot](dT)
34
tract, which would result in inhibition of transcription.
We also examined the transcription in the presence of the 44 bp duplex DNA containing a (dA)
34
[middot](dT)
34
tract in the middle (Fig.
3
D). While there was no effect of this DNA at 0.01 and 0.1 [mu]M (data not shown), the suppression of inhibition was observed at 1 and 4 [mu]M (Fig.
3
D, left or right panel, respectively). Furthermore, an apparent reduction of
some of the premature transcripts (shown by arrowheads) was observed between
them.
Inhibition of transcription by T7 RNA polymerase and RNA polymerase II
Inhibition of transcription by the (dA)
34
[middot](dT)
34
tract was also examined with other RNA polymerases. Figure
4
shows transcription with T7 RNA polymerase and eukaryotic RNA polymerase II using pATB12 and pAT[epsilon]3-32, containing the promoter from the human [epsilon]-globin gene, respectively. In both cases,
transcription was inhibited in the middle and/or in front of the (dT)
34
tract. However, the degrees and the positions of the inhibition varied among
the polymerases.
RNA[middot]DNA complex formation
To examine the strand specificity of the interaction between the plasmid DNA and
transcripts containing a poly(U) sequence, the plasmid (pUC19, pAT19, pUC18 or
pAT18) digested with
Xmn
I was mixed with
32
P-labeled poly(A) or poly(U) and subjected to electrophoresis in a buffer
containing Mg
2+
(Fig.
5
). Complex formation was observed only in the combination of pAT19 or pAT18 and
poly(U) (Fig.
5
, lanes 8 and 10), indicating that RNA transcripts containing a poly(U)
sequence, but not poly(A), can interact or form an RNA[middot]DNA complex with the template plasmid DNA at the (dA)
34
[middot](dT)
34
tract. pAT18 has the ability to form a complex if poly(U) is supplied. Mg
2+
was required for this complex formation (data not shown).
DISCUSSION
ACKNOWLEDGEMENTS
This work was supported by Grants-in-Aid from the Ministry of Education of Japan. We thank Yuko Wada-Kiyama for information regarding the eukaryotic
in vitro
transcription system.
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