Biochem. J. (2013) 452, 241–248 (Printed in Great Britain)
241
doi:10.1042/BJ20130069
Single-strand promoter traps for bacterial RNA polymerase
Danil PUPOV*1 , Daria ESYUNINA*†1 , Andrey FEKLISTOV*2 and Andrey KULBACHINSKIY*3
*Institute of Molecular Genetics, Russian Academy of Sciences, Moscow 123182, Russia, and †Department of Molecular Biology, Biological Faculty,
Moscow State University, Moscow 119991, Russia
Besides canonical double-strand DNA promoters, multisubunit
RNAPs (RNA polymerases) recognize a number of specific
single-strand DNA and RNA templates, resulting in synthesis
of various types of RNA transcripts. The general recognition
principles and the mechanisms of transcription initiation on these
templates are not fully understood. To investigate further the
molecular mechanisms underlying the transcription of singlestrand templates by bacterial RNAP, we selected high-affinity
single-strand DNA aptamers that are specifically bound by RNAP
holoenzyme, and characterized a novel class of aptamer-based
transcription templates. The aptamer templates have a hairpin
structure that mimics the upstream part of the open promoter
bubble with accordingly placed specific promoter elements.
The affinity of the RNAP holoenzyme to such DNA structures
probably underlies its promoter-melting activity. Depending
on the template structure, the aptamer templates can direct
synthesis of productive RNA transcripts or effectively trap
RNAP in the process of abortive synthesis, involving DNA
scrunching, and competitively inhibit promoter recognition. The
aptamer templates provide a novel tool for structure–function
studies of transcription initiation by bacterial RNAP and its
inhibition.
INTRODUCTION
single-stranded DNA templates recognized by bacterial RNAP
include minus-strand replication origins of filamentous phages
(such as the phage M13 origin, oriM13 [15]) and laggingstrand origins of rolling-circle replication plasmids [16,17], where
RNAP functions as a primase and synthesizes short pRNAs (priming RNAs) for replication initiation [18]. Structure analysis of
phage and plasmid replication origins revealed the presence
of − 35- and − 10-like elements located in partially doublestranded DNA regions, and some reported substitutions in these
elements impaired pRNA synthesis by bacterial RNAP in vitro and
origin function in vivo [16,17,19–21]. At the same time, in vitro
pRNA synthesis on the oriM13 template was observed even in
the absence of specific promoter elements, suggesting that core
RNAP plays a major role in origin recognition [15].
Both bacterial and eukaryotic RNAPs were also shown to utilize
RNA templates of various natures [22,23]. In a striking example
of genetic regulation, bacterial 6S RNA was shown to serve as
a specific inhibitor of σ 70 RNAP holoenzyme during stationary
phase of growth, but to be released from RNAP during recovery
from stationary phase as a result of its transcription by RNAP [24].
Remarkably, the secondary structure of 6S RNA resembles the
structure of the open promoter with unpaired transcription bubble
[24], and the primary determinants for specific 6S RNA binding
by RNAP were shown to reside within regions corresponding to
the − 35 and − 10 promoter elements [25–27]. Similarly to the
oriM13 transcription, transcription of 6S RNA was shown to result
in synthesis of short pRNAs, leading to structural rearrangements
of the pRNA–6S RNA complex and its release from RNAP
[24,28].
Analysis of different single-strand nucleic acid substrates
recognized by RNAP demonstrated that, depending on their
structure and genetic function, they can serve as transcription
templates and direct synthesis of various types of RNA transcripts
and/or specifically inhibit RNAP activity [15,17,24]. However,
Proper expression of genetic information critically depends on
the ability of RNAP (RNA polymerase) to specifically recognize
promoters and initiate RNA synthesis in a highly regulated
manner [1]. In bacteria, transcription initiation is performed by the
RNAP holoenzyme containing a promoter-specificity factor,
the σ subunit. The major σ subunit (σ 70 in Escherichia coli) is
involved in recognition of most cellular promoters which contain
a number of conserved promoter elements, including the − 35
(TTGACA), TG, − 10 (TATAAT) and GGGA (discriminator)
elements recognized by conserved regions 4.2, 3.0, 2.3/2.4 and
1.2 of the σ subunit respectively [1–5] (Figure 1C). Initiation of
transcription on double-stranded DNA requires local melting
of the template around the starting point, promoted by specific
interactions between the σ subunit and the non-template strand
of the − 10 and GGGA elements, as illuminated by recent
structural analysis [6,7]. In addition, core RNAP was proposed
to recognize the melted non-template DNA strand downstream of
the GGGA element, with a particular preference for guanine at
promoter position + 2 [7]. The σ subunit is also implicated in
later steps of transcription initiation, including priming of RNA
synthesis, abortive transcription and promoter escape [8–10]. The
process of abortive synthesis and promoter escape was shown
to be accompanied by significant changes in the conformation
of the DNA template, involving extension of the transcription
bubble and ‘scrunching’ of the melted DNA chains, as a result
of downstream DNA transcription without breaking upstream σ mediated RNAP–promoter contacts [11,12].
Besides double-stranded DNA promoters, bacterial and eukaryotic RNAPs were shown to transcribe various types of singlestrand templates, a possible relic of their functions in primordial
world that existed before establishment of the genetic function
of double-stranded DNA [13,14]. The best-studied examples of
Key words: abortive transcription, aptamer, DNA scrunching,
promoter recognition, RNA polymerase, single-strand template.
Abbreviations used: FAM, 6-carboxyfluorescein; hEcap, holoenzyme E. coli aptamer; pRNA, priming RNA; RNAP, RNA polymerase.
1
These authors contributed equally to this work.
Present address: Rockefeller University, 1230 York Avenue, New York, NY 10065, U.S.A.
3
To whom correspondence should be addressed (email [email protected]).
2
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Figure 1
D. Pupov and others
Aptamers to holoenzyme of bacterial RNAP
(A) Sequences of the central stem–loop of the aptamers. Nucleotides from the random and constant library regions are shown in upper- and lower-case letters respectively. Sequence motifs that form
double-stranded stems are shown in grey with underlined complementary nucleotides; the − 10 and TG promoter elements are red and green respectively; pyrimidine-rich motifs at the right part
of the loop region are highlighted in light/dark yellow. The sequence logo of the conserved promoter motif is shown below the aptamer sequences. (B) Proposed secondary structures of shortened
aptamers hEcap1, hEcap5 and hEcap12. (C) The model of the open promoter complex of T. aquaticus RNAP [40]. The active-site magnesium is pink; the promoter DNA is orange; relative positions
of the − 35, TG, − 10 and GGGA promoter elements are indicated. The σ subunit is shown as a tube model, with regions 1.2, 2.3/2.4, 2.5 and 4.2 shown in brown, red, green and blue respectively.
in many cases, the mechanistic details of transcription initiation
on these templates remain poorly understood. To investigate
further possible ways of recognition of single-strand templates
by bacterial RNAP, we obtained high-affinity single-strand DNA
aptamers that are specifically bound by the RNAP holoenzyme,
and characterized a novel class of templates based on the aptamer
sequences. The aptamer templates share important properties
with other single-strand RNAP substrates and can efficiently
inhibit RNAP during the initiation step of transcription, thus
providing a new tool for analysis of transcription initiation and
for development of RNAP inhibitors.
EXPERIMENTAL
Proteins and DNA
E. coli core RNAP and the σ 70 subunit were purified as
described previously [29]. Unmodified and fluorescently labelled
DNA oligonucleotides were purchased from Syntol. The T7A1
promoter fragment was obtained as described in [29].
Aptamer selection
Aptamer selection was performed using a 75-nt-long oligonucleotide library with randomized 32-nt-long central region (Supplementary Figure S1A at http://www.biochemj.org/bj/452/
bj4520241add.htm) essentially as described previously [2,30,31].
In each round of selection, RNAP holoenzyme (10–100 nM) was
mixed with the oligonucleotide pool in binding buffer containing
40 mM Tris/HCl (pH 7.9), 10 mM MgCl2 and increasing salt
concentrations (from 160 mM NaCl/40 mM KCl in the starting
round to 600 mM NaCl/200 mM KCl in final rounds), incubated
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for 30 min at 25 ◦ C, bound to Ni-NTA (Ni2 + -nitrilotriacetate)–
agarose (Qiagen) via a His6 tag present at the C-terminus of
the β’ subunit, and separated from unbound oligonucleotides by
extensive washing. The enriched library was cloned after ten
rounds of selection and 15 randomly chosen clones were sequenced. Sequence logos of the conserved aptamer motif were
created using the WebLogo server (http://weblogo.berkeley.edu/).
Analysis of aptamer secondary structures was performed using
The mfold Web Server (http://mfold.rna.albany.edu) in the DNA
folding mode [32]. Equilibrium K d values for the RNAP–
aptamer interactions were determined by the nitrocellulosebinding method [2,31]. 5 -End-labelled aptamers (taken at
0.1 nM) were incubated with a series of dilutions of RNAP
holoenzyme (from 1 nM to 1 μM) for 30 min at room temperature
(25 ◦ C) in 40 μl of the binding buffer containing 400 mM NaCl
and 40 mM KCl; the samples were filtered through 0.45-μmpore-size nitrocellulose filters (HAWP, Millipore), the filters
were washed with 5 ml of the buffer and quantified using the
PhosphorImager (GE Healthcare). The data were fitted to
the hyperbolic equation B = Bmax ×[RNAP]/([RNAP] + K d ),
where B is a percentage of DNA bound, Bmax is the maximum
binding, and K d is the dissociation constant, using GraFit software
(Erithacus Software).
In vitro transcription
Most transcription reactions were performed in transcription
buffer containing 40 mM Tris/HCl (pH 7.9), 10 mM MgCl2 ,
160 mM NaCl and 40 mM KCl at 37 ◦ C. RNAP holoenzyme
(50 nM core RNAP plus 500 nM σ 70 subunit) was incubated with
the aptamer template (10–50 nM) for 5 min at 37 ◦ C followed
by addition of NTP substrates. RNA primers and UTP (with
Aptamer promoter traps for bacterial RNA polymerase
addition of [α-32 P]UTP) were at 25 μM, whereas concentrations
of other NTPs were varied from 30 to 1000 μM each. The
reactions were stopped after 5–10 min by addition of ureacontaining buffer and RNA products were analysed by 15–23 %
PAGE. In transcription inhibition experiments, the aptamers were
mixed with the T7A1 promoter in transcription buffer containing
40 mM Tris/HCl (pH 7.9), 10 mM MgCl2 and 40 mM KCl, then
RNAP holoenzyme was added and transcription reactions were
performed as described above.
KMnO4 footprinting of RNAP–aptamer complexes
KMnO4 probing was performed as described previously [29].
RNAP holoenzyme (100 nM) was mixed with 5 -labelled aptamer
template (10 nM in all reactions) in transcription buffer containing
40 mM Tris/HCl (pH 7.9), 10 mM MgCl2 , 160 mM NaCl
and 40 mM KCl, incubated at 25 ◦ C for 5 min and treated with
2 mM KMnO4 for 30 s. NTPs (500 μM each) were added 2 min
before KMnO4 where indicated. The reactions were processed as
described in [29]. The variations in the total amounts of DNA
in different reactions after sample processing did not exceed
10–15 %. The DNA cleavage products were separated by either
15 or 23 % PAGE to reveal long and short cleavage products
respectively. The KMnO4 experiments were repeated three times
resulting in highly reproducible modification patterns.
Fluorescence measurements
Analysis of aptamer fluorescence was performed in the Modulus
Microplate Multimode Reader (Turner BioSystems) using the
blue filter with the maximum excitation wavelength of 490 nm
and emission wavelength of 510–570 nm. Reaction mixtures
contained 500 nM RNAP holoenzyme and 250 nM hEcap1/51
aptamer derivatives in 60 μl volume of transcription buffer
(40 mM Tris/HCl, pH 7.9, 10 mM MgCl2 and 40 mM KCl) at
25 ◦ C in black-coloured microtitre plates, either in the absence
of NTPs or in the presence of 500 μM ATP, UTP and GTP.
Three measurements were performed in parallel and background
fluorescence values were subtracted. Each experiment was
repeated three to five times, followed by averaging of the relative
fluorescence intensities.
RESULTS
Aptamer-based mimics of the open promoter complex
To explore the DNA-recognition potential of RNAP, we selected
single-strand DNA aptamers that bind the E. coli σ 70 RNAP
holoenzyme with high affinity and specificity (hEcaps, for
holoenzyme E. coli aptamers). Aptamer selection was performed
using a standard protocol from a 75 nt library containing a 32-ntlong random region (Supplementary Figure S1A). Sequencing of
individual clones revealed that all aptamers contained promoterlike motifs corresponding to the TG and − 10 promoter elements
(Figure 1A and Supplementary Figure S1A). In most aptamers
(i.e. aptamers 1–11), these two elements were separated by an
additional nucleotide, reflecting their relative position in doublestranded promoters. In aptamers 12–15, the TG motif immediately
preceded the − 10 element, similarly to previously described
single-stranded DNA aptamers recognized by free σ subunits
from E. coli and Thermus aquaticus [2,31]. Further analysis
is needed to reveal possible differences in the recognition of
the TG motif by RNAP holoenzyme at these two aptamer
positions. Remarkably, the degree of nucleotide conservation at
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all positions of the aptamer promoter motifs (in particular, at the
third, fourth and fifth positions of the − 10 hexamer) was higher
than in the case of natural promoters [33], probably because the
aptamers were specifically selected for the optimal binding to
RNAP.
Analysis of predicted secondary structures revealed that all
aptamers share a common structural motif containing a short
hairpin formed by self-complementary aptamer regions, with
the TG element located in the upstream double-stranded part
and the − 10 element placed in the loop region (Figure 1B and
Supplementary Figure S1). The aptamer structure is remarkably
similar to the upstream part of the melted DNA bubble in the
open promoter complex (Figure 1C). In most aptamers, the first
nucleotide of the − 10 element was based-paired, corresponding
to the proposed base-pairing in the open complex (Figure 1
and Supplementary Figure S1B) ([6] and references therein). In
some aptamers, the whole − 10 region was located in the loop
region, demonstrating that RNAP holoenzyme can recognize all
six positions of the − 10 hexamer in fully single-strand form.
Similarly, isolated σ subunit was shown previously to recognize
the fully single-stranded − 10 element [2,6,31,34]. All aptamers
also contained a pyrimidine-rich region in the bottom part of
the single-stranded loop opposite the − 10 element (Figure 1A),
suggesting that the RNAP holoenzyme may have preference
for such sequences in this part of the transcription bubble. To
confirm that the aptamers are specifically recognized by RNAP, we
analysed several minimized aptamer variants corresponding to the
proposed secondary-structure motif (Figure 1B). The dissociation
constants (K d values) of holoenzyme–aptamer complexes were
shown to lie in the nanomolar range; in particular, hEcap1
and hEcap5 bound RNAP with K d values of 2.5 and 3.0 nM
respectively, which is comparable with the binding affinities of
natural double-stranded promoters (e.g. [3]).
Aptamer templates guide specific transcription initiation
The structural similarity of the aptamers to the open promoter
bubble suggested that RNAP can use them as transcription
templates. However, the original aptamers lacked the downstream
DNA part that must be placed into the RNAP active-site cleft for
transcription initiation, probably as a result of a limited length
of the DNA library used for aptamer selection. Thus, to obtain
aptamer-based transcription templates, we introduced short DNA
inserts derived from the initially transcribed region of the T7A1
promoter into the downstream loop of hEcap1. To stabilize the
template structure and to increase the strength of RNAP–aptamer
interactions, we introduced additional G/C pairs into the
aptamer stem region, made consensus changes in the − 10
element and put the GGGA element downstream of it (Figures 2A
and 2B).
Several aptamer variants were constructed that differed in the
lengths of the inserted sequences (Figures 2A and 2B). The
aptamer variant without the downstream DNA insert (hEcap1/43)
did not support transcription initiation (Figure 2C, lane 1).
However, aptamer-based templates containing inserts of 5–
12 nt (templates hEcap1/48 to hEcap1/55, Figures 2A and
2B) promoted efficient synthesis of abortive RNA transcripts
(Figure 2C, lanes 2–5). The abortive nature of the observed
RNA products was confirmed in transcription experiments with
immobilized RNAP. The lengths of synthesized RNA transcripts
gradually increased (up to 12 nt) with the increase in the length
of the downstream DNA inserts. However, the downstream loops
present in the aptamer-based templates did not allow RNAP to
proceed to RNA elongation, resulting in its trapping in the process
of abortive synthesis (Figure 2C).
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Figure 2
D. Pupov and others
Transcription properties of aptamer-based templates
(A) Sequences of the hEcap1-based templates. Positions of conserved promoter elements are indicated above the sequences. Sequences that form double-stranded stems are shown in light grey;
the downstream DNA inserts are underlined. (B) The structure of the hEcap1/51 template. The specific promoter elements are indicated; the transcription start point is shown with an arrow. The
downstream DNA insert is boxed; note that in the hEcap1/51–RNAP complex, the downstream hairpin is fully melted (see Figures 3A and 3B). The structures of downstream inserts in other hEcap1
variants are shown. (C) Transcription of hEcap1-based templates containing various downstream DNA inserts. The aptamer lengths are indicated. (D) Localization of the transcription start point in the
hEcap1/51 aptamer template. The experiment was performed with various initiating nucleotides and primers, as indicated. (E) Effects of substitutions in consensus promoter elements on the activity
of the hEcap1/51 template in the presence of all four NTPs (upper panel) or UpA and UTP (lower panel). Positions of characteristic RNA products are indicated (RO, run-off).
To localize the starting point of transcription on the aptamer
templates, we performed transcription in the presence of different
nucleotide substrate sets. The synthesis of labelled dinucleotide
RNA product was observed from ATP and UTP when either
[γ -32 P]ATP or [α-32 P]UTP was used as a labelled substrate
(Figure 2D, lanes 1 and 2), suggesting that transcription
probably initiates with ATP at position + 1 (indicated on
Figure 2A). Indeed, efficient transcription initiation was observed
in the presence of short primers corresponding to the proposed
transcription start point. In particular, dinucleotide primers UpA
(corresponding to positions − 1/ + 1) and trinucleotide primer
ApUpC (positions + 1/ + 2/ + 3) could be used as initiating substrates (Figure 2D, lanes 3 and 4). Thus the likely starting point of
transcription on the aptamer templates corresponds to the starting
point of transcription in classical double-stranded promoters.
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To reveal how transcription of the aptamer templates depended
on the promoter elements, we analysed several variants
of a representative template, hEcap1/51, containing different
combinations of the elements. The starting template variant
contained TG, − 10 and GGGA consensus elements; in template
hEcap1/51-GGGA, the GGGA motif was replaced with ATAT;
in template hEcap1/51-TG, the TG element was replaced with
AC, preserving the double-stranded stem structure; in template
hEcap1/51- − 10, the − 10 element was replaced with the
TCACCA sequence, and the last two templates also lacked
the GGGA motif (Figures 2A and 2B). The transcription activity
of all four templates was analysed either in the presence of all four
NTPs (Figure 2E, upper panel), or dinucleotide primer UpA and
UTP (Figure 2E, lower panel), allowing for abortive synthesis of a
trinucleotide RNA product. In agreement with the key role of the
Aptamer promoter traps for bacterial RNA polymerase
Figure 3
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DNA scrunching during abortive transcription on aptamer templates
(A) The scheme of the aptamer template showing positions of KMnO4 -modified thymines (numbered starting from the 5 -end) and fluorescent dyes, attached through thymines substituted for
cytosines at indicated positions. The thymine bases that display differential activities in various KMnO4 reactions (Figure 3B) are coloured as follows: grey, bases in the stem region that are
cleaved only at high temperature (reaction 4); blue and red, bases in the RNAP–aptamer complex that are more reactive either in the absence (reaction 2) or in the presence of NTPs (reaction 3)
respectively. (B) KMnO4 footprinting of RNAP–aptamer complexes. The scanned modification profiles are shown on the right. All reactions contained equal total amounts of the DNA template, as
confirmed by phosphorimaging. Reaction 1 (black) contained no RNAP, reaction 2 (blue) contained RNAP–aptamer complex, reaction 3 (red) contained RNAP–aptamer complex in the presence
of NTPs, and reaction 4 (green) contained free aptamer template at 70 ◦ C. The ‘A + G’ is a Maxam–Gilbert sequencing reaction. The colouring of thymine bases corresponds to Figure 4(A).
(C) Relative fluorescence levels of the aptamer–RNAP complexes measured in the absence and in the presence of NTPs (250 μM each). The hEcap1/51 template either contained fluorescein (F) at
position − 12, or was double-labelled with fluorescein and dabsyl (F/Q) at positions − 12 and − 2 respectively. RNAP activity, measured on the control unlabelled hEcap1/51 template (‘C’) or
fluorescently labelled templates in the presence of ATP, UTP and CTP, is shown on the right. (D) Schematics of the aptamer template transcription showing proposed changes in the DNA conformation,
with positions of hyper-reactive thymines and fluorescent dyes indicated. The RNAP active centre is shown by a red dot.
− 10 element in promoter recognition, substitution of this element
completely abolished transcription initiation (hEcap1/51- − 10,
Figure 2E, lanes 3). At the same time, removal of the GGGA motif
alone only slightly reduced abortive RNA synthesis (compare
templates hEcap1/51 and hEcap1/51-GGGA, Figure 2E, lanes 1
and 4). Notably, however, the hEcap1/51-GGGA template lacking
the GGGA motif produced very small amounts of ∼ 30 nt fulllength RNA corresponding to the transcription to the 5 -end of the
template (Figure 2E, upper panel, lane 4). This RNA product was
completely absent in the case of templates containing the GGGA
motif (Figures 2C and 2E), suggesting that, on these templates,
RNAP is fully trapped in abortive complexes. Substitution of the
TG element (hEcap1/51-TG) moderately decreased trinucleotide
RNA synthesis (Figure 4E, lower panel, lane 2), but greatly
reduced abortive transcription in the presence of all four NTPs
and stimulated full-length RNA synthesis, suggesting that, in this
case, RNAP can disrupt specific contacts with the template and
escape to productive elongation (Figure 4E, upper panel, lane 2).
Thus, depending on the strength of RNAP–DNA interactions, the
single-strand templates can direct synthesis of either abortive or
full-length RNA transcripts.
DNA scrunching during transcription initiation
on aptamer templates
Transcription initiation on double-stranded promoters was shown
to be accompanied by the scrunching of the DNA template within
the RNAP molecule [11,12]. The resulting strain can be relieved
either through release of abortive RNAs or through disruption
of σ -mediated contacts with the promoter and transition to
productive elongation. The trapping of RNAP in the process
of abortive synthesis on the consensus aptamer templates
suggests that, in this case, RNAP retains persistent contacts
with the promoter elements, thus favouring the first scenario
and preventing further RNA elongation. To detect structural
changes of the aptamer template that occur during abortive
RNA synthesis, we performed probing of the RNAP–hEcap1/51
complex with KMnO4 which modifies single-stranded thymine
bases, allowing their further visualization by piperidine cleavage.
Comparison of the modification pattern of the free aptamer
template obtained in the absence of RNAP with the control pattern
observed at high temperature (70 ◦ C, to disrupt the secondary
structure) showed that thymines in the proposed stem region
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D. Pupov and others
change their relative positions and be brought closer to each
other. To detect such changes, we analysed modified hEcap1/51
template containing fluorescent [(FAM (6-carboxyfluorescein)]
and quenching (dabsyl) groups at positions − 12 and − 2
of the template DNA segment respectively (Figure 3A). The
fluorescence was measured in RNAP–template complexes either
in the absence of NTPs or in the presence of a limited NTP set
(ATP, UTP and GTP), allowing for the synthesis of RNAs up to
5 nt in length. Control experiments demonstrated that the modified
template was transcriptionally active under these conditions and
supported abortive RNA synthesis (Figure 3C, right). Remarkably,
the fluorescence intensity was significantly decreased upon NTP
addition (Figure 3C, left), indicative of a decrease in the distance
between the fluorescent dye and the quencher. No fall, but rather
a minor increase, in the fluorescence intensity was observed in
the case of a control template containing a FAM group at position
− 12, but lacking the quencher, demonstrating that the observed
fluorescence changes cannot be explained by changes in the
position of the fluorescent group itself (Figure 3C, left). Together,
these results strongly suggest that the process of abortive initiation
on the aptamer templates is accompanied by the scrunching of the
template DNA segment (Figure 3D).
Transcription inhibition by aptamer templates
Figure 4
Transcription inhibition by aptamer-based promoter traps
The activity of RNAP holoenzyme on DNA template containing the T7A1 promoter and λ tR2
terminator was measured at various NTP concentrations (from 10 to 1000 μM each, as indicated)
in either the absence or the presence of the hEcap1/51 template. Concentrations of RNAP, T7A1
promoter and the aptamer template in the reaction were 30, 100 and 50 nM respectively. The
reaction products were separated on gels of different concentrations to reveal transcripts of
various lengths; tR2, terminated RNA, RO, full-length run-off transcript. The plot shows the
amounts of the T7A1-derived RNAs relative to the maximum activity observed in the absence of
hEcap1/51 at 300 μM NTPs (means +
− S.D. for three experiments).
(positions 5, 7 and 8 from the aptamer 5 -end, shown in grey
in Figure 3A) were protected from cleavage, confirming stem
formation (Figure 3B, compare reactions 1 and 4). Addition
of RNAP increased the reactivities of several thymines in the
loop region (e.g. positions 13, 21, 27 and 31; Figure 3B,
reaction 2), suggesting that this aptamer part was essentially
single-stranded in the RNAP–aptamer complex. Addition of NTP
substrates to the RNAP–aptamer complex resulted in a decrease
in reactivities of thymines at these positions (red compared with
blue intensity profile in Figure 3B). In particular, protection of
thymines at positions 27 and 31 probably reflected formation
of the RNA–DNA hybrid during transcription. Importantly, the
stem aptamer region (thymine bases 5, 7 and 8) remained doublestranded in the presence of NTPs, suggesting that the template
retains its secondary structure during RNA synthesis. Strikingly,
a significant (up to 3-fold) increase in thymine reactivities was
observed for positions 41 and 42, located at the upstream bottom
part of the transcription bubble (shown in red in Figure 3B),
indicative of a strong distortion of the DNA structure in this
region upon RNA synthesis. In the open promoter complex model,
the corresponding segment of the template DNA strand exits
the RNAP main channel to anneal with the complementary nontemplate strand (Figure 1C); this position was proposed to be the
likely place of the template strand scrunching during initiation
[35].
During DNA scrunching, nucleotides located at opposite
edges of the single-stranded template DNA segment should
c The Authors Journal compilation c 2013 Biochemical Society
Natural single-strand templates were shown to play regulatory
roles in transcription, as exemplified by 6S RNA that inhibits
promoter recognition by bacterial RNAP holoenzyme [24].
Since holoenzyme-specific aptamers mimic natural promoter
substrates, we expected them to also inhibit promoter-dependent
transcription.
To reveal the effects of the aptamer templates on promoterdependent transcription we analysed T7A1-dependent RNAP
activity in the presence of hEcap1/51 at different NTP
concentrations. The aptamer template strongly inhibited synthesis
of full-length T7A1 RNAs and switched transcription to the
synthesis of short abortive RNAs, even in the presence of a 2fold excess of the promoter (Figure 4). Remarkably, efficient
inhibition was observed even at high NTP concentrations (up to
1 mM), suggesting that the transcription of the aptamer template
does not relieve its inhibitory effect on promoter recognition. This
contrasts with 6S RNA that was shown to dissociate from RNAP
upon pRNA synthesis [24,28]. Thus, unlike 6S RNA, the aptamers
probably do not undergo RNA-dependent secondary-structure
rearrangements and remain bound to RNAP during transcription.
DISCUSSION
Transcription of single-strand DNA and RNA templates by
cellular RNAPs plays important biological functions in both
bacteria and eukaryotes [13,16–19,23,24]. Analysis of singlestrand aptamer templates specifically recognized by bacterial
RNAP, performed in the present study, suggests general principles
in the recognition and function of various types of singlestrand templates and double-strand promoters. The aptamer
structure reveals a striking similarity to the structure of the
open promoter bubble containing the unpaired − 10 promoter
element and double-stranded TG motif upstream of it. Thus
the in vitro selection experiment demonstrated that bacterial
RNAP holoenzyme has a strong preference for such DNA
templates over other structural and sequence DNA motifs. This
preference probably underlies RNAP DNA melting activity
during formation of the open complex on double-stranded
promoters. Previous studies have identified additional elements
Aptamer promoter traps for bacterial RNA polymerase
in the melted promoter bubble that are specifically recognized by
RNAP holoenzyme, including the GGGA/discriminator element
immediately downstream of the − 10 element [2–5] and the ‘corerecognition element’ at promoter positions − 4/ + 2 [7]. However,
these elements were not revealed in the holoenzyme-specific
aptamers, which may be explained by strong RNAP–aptamer
interactions that are formed even in the absence of these motifs
or by limited aptamer size that might preclude their selection.
Subtle modification of the structure of aptamers converted
them into efficient transcription templates, specifically recognized
by RNAP holoenzyme. Depending on the downstream template
structure and the presence of consensus promoter elements, we
were able to obtain template variants capable of synthesis of RNA
transcripts of various lengths and types. We therefore propose
that the specific aptamer part can be considered as a recognition
module for specific initiation of RNA synthesis on single-stranded
DNA templates of various structures and origins, which may have
various practical applications.
Comparison of the aptamer templates with other single-strand
RNAP substrates characterized reveals common features in their
recognition by RNAP. Similarly to the aptamer templates, singlestrand phage and plasmid replication origins recognized by
bacterial RNAP contain promoter-like motifs, including − 10
and − 35-like elements, that were shown to be important for
their function in vivo [16,17,19–21]. Furthermore, similarly to the
aptamers, all natural single-strand DNA and RNA substrates of
RNAP fold into partially double-stranded structures containing
hairpins and single-stranded bulges that are important for their
binding to RNAP [16,19,20,23,24,36].
Transcription initiation on double-stranded promoters was
shown to involve scrunching of the DNA strands in the melted
region within the transcription complex, a process that is believed
to facilitate promoter escape by RNAP [11,12,35]. Similarly,
the DNA scrunching was proposed to occur during σ -dependent
pausing in elongation complexes [37,38]. Our data suggest that
transcription initiation on single-strand templates proceeds via a
similar mechanism, involving scrunching of the template DNA
segment (Figure 3D). Using the aptamer templates, we directly
detected changes in the conformation of the template DNA
strand during initiation. It should be noted that, owing to the
specific hairpin structure of the template, the ‘non-template’
DNA segment is unlikely to be scrunched, but should rather
enter the RNAP active centre for transcription, which makes
RNA extension dependent on the size of the downstream hairpin.
Indeed, we observed that the aptamer templates promoted highly
efficient abortive RNA synthesis that depended on the downstream
DNA length. In the case of fully consensus aptamer templates, the
σ -mediated contacts of RNAP with specific promoter elements
cannot be broken and the resulting stress is relieved through
dissociation of abortive RNA transcripts (Figure 3D). As a
result, the templates act as suicidal promoter substrates for
RNAP that sequester the enzyme in the process of abortive
initiation and inhibit its interactions with promoters. At the
same time, we propose that adjusting the strength, respective
position of the promoter elements and the sequence of the initially
transcribed region (in particular, its G/C-content) may allow
efficient transition to elongation on the aptamer templates and
may help to obtain single-strand promoters for in vitro
and in vivo RNA production.
In conclusion, the holoenzyme-specific aptamers and aptamerbased templates described in the present paper provide a tool
for structural analysis of different steps of promoter recognition
and transcription initiation by bacterial RNAP, including the
processes of DNA scrunching and abortive cycling, and for
analysis of RNAP inhibition. We also propose that an aptamer-
247
based approach can be useful to probe sequence specificity of the
plethora of alternative σ factors in various bacteria for which only
a very limited information on promoter specificity is currently
available [39]. Detailed understanding of how these σ factors
recognize their promoter elements will provide important insights
into the mechanisms of transcription regulation and will allow
rational design of novel σ –promoter pairs for controlled gene
expression in synthetic biology applications.
AUTHOR CONTRIBUTION
Andrey Kulbachinskiy and Danil Pupov designed the research. Danil Pupov, Daria Esyunina
and Andrey Feklistov performed the research. All authors analysed the data. Andrey
Kulbachinskiy wrote the paper with the help of all other authors.
ACKNOWLEDGEMENTS
We thank I. Artsimovitch for plasmids, N. Zenkin for helpful discussions and S.A. Darst
for the co-ordinates of the open promoter complex model.
FUNDING
This work was supported in part by the Russian Academy of Sciences Presidium Program in
Molecular and Cellular Biology (grant to A.K.), the Russian Foundation for Basic Research
[grant numbers 12-04-33187 and 12-04-32042], and by the Ministry of Education and
Science of Russia [projects P335 and 8106].
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doi:10.1042/BJ20130069
SUPPLEMENTARY ONLINE DATA
Single-strand promoter traps for bacterial RNA polymerase
Danil PUPOV*1 , Daria ESYUNINA*†1 , Andrey FEKLISTOV*2 and Andrey KULBACHINSKIY*3
*Institute of Molecular Genetics, Russian Academy of Sciences, Moscow 123182, Russia, and †Department of Molecular Biology, Biological Faculty,
Moscow State University, Moscow 119991, Russia
Supplementary Figure S1 is on the following page.
1
2
3
These authors contributed equally to this work.
Present address: Rockefeller University, 1230 York Avenue, New York, NY 10065, U.S.A.
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2013 Biochemical Society
D. Pupov and others
Figure S1
Sequences and proposed secondary structures of aptamers to E. coli holoenzyme RNAP
(A) Full-length aptamer sequences are shown. The colour code corresponds to Figure 1(A) of the main text. The − 10 and TG promoter elements are shown in red and green respectively. (B) The
secondary aptamer structure was predicted using the mfold Web Server (http://mfold.rna.albany.edu) in the DNA folding mode [1]. The predicted wobble G:T base pairs are indicated with dotted
lines. The aptamers are grouped on the basis of similarities of their sequences and proposed secondary structures.
REFERENCE
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Nucleic Acids Res. 31, 3406–3415
Received 11 January 2013/7 March 2013; accepted 22 March 2013
Published as BJ Immediate Publication 22 March 2013, doi:10.1042/BJ20130069
c The Authors Journal compilation c 2013 Biochemical Society