Aptamers to Escherichia coli core RNA polymerase that sense its interaction with rifampicin, r-subunit and GreB Andrey Kulbachinskiy1,2, Andrey Feklistov2,3, Igor Krasheninnikov3, Alex G
Trang 1Aptamers to Escherichia coli core RNA polymerase that sense its interaction with rifampicin, r-subunit and GreB
Andrey Kulbachinskiy1,2, Andrey Feklistov2,3, Igor Krasheninnikov3, Alex Goldfarb1and Vadim Nikiforov1,2
1
Public Health Research Institute, Newark, New Jersey, USA;2Institute of Molecular Genetics, Moscow, Russia;
3
Department of Molecular Biology, Moscow State University, Moscow, Russia
Bacterial RNA polymerase (RNAP) is the central enzyme of
gene expression that is responsible for the synthesis of all
types of cellular RNAs The process of transcription is
accompanied by complex structural rearrangements of
RNAP Despite the recent progress in structural studies
of RNAP, detailed mechanisms of conformational changes
of RNAP that occur at different stages of transcription
remain unknown The goal of this work was to obtain novel
ligands to RNAP which would target different epitopes of
the enzyme and serve as specific probes to study the
mech-anism of transcription and conformational flexibility of
RNAP Using in vitro selection methods, we obtained 13
classes of ssDNA aptamers against Escherichia coli core
RNAP The minimal nucleic acid scaffold (an
oligonucleo-tide construct imitating DNA and RNA in elongation
complex), rifampicin and the r70-subunit inhibited binding
of the aptamers to RNAP core but did not affect the disso-ciation rate of preformed RNAP–aptamer complexes We argue that these ligands sterically block access of the aptamers to their binding sites within the main RNAP channel In contrast, transcript cleavage factor GreB increased the rate of dissociation of preformed RNAP– aptamer complexes This suggested that GreB that binds RNAP outside the main channel actively disrupts RNAP– aptamer complexes by inducing conformational changes in the channel We propose that the aptamers obtained in this work will be useful for studying the interactions of RNAP with various ligands and regulatory factors and for investi-gating the conformational flexibility of the enzyme Keywords: aptamers; conformational changes; elongation complex; GreB; RNA polymerase
DNA-directed RNA polymerase (RNAP, EC 2.7.7.6) is a
complex molecular machine undergoing multiple
intra-molecular rearrangements in the process of RNA synthesis
[1–3] During the transcription cycle, RNAP makes specific
and nonspecific contacts with double and single stranded
(ss) DNA, the RNA/DNA hybrid and nascent RNA
Recent advances in structural studies of bacterial and yeast
RNAPs [4–8] made it possible to create three-dimensional
models of the promoter and elongation complexes and to
propose the roles for various RNAP domains in interactions
with DNA and RNA [6,8–11]
The most striking structural feature of RNAP is a deep
cleft (the main channel) formed by the two largest RNAP
subunits (b and b¢ in the bacterial enzyme) that runs along
the full length of the molecule [4,12] In the elongation
complex, the main channel accommodates the RNA/DNA
hybrid, duplex DNA downstream from the hybrid and
RNA behind the hybrid The 8-bp-long DNA/RNA hybrid
is lodged between the catalytic Mg2+ion and a structural element of b¢ called the rudder (Fig 1) [9] The downstream DNA duplex is placed in a trough formed by several domains of b¢ (clamp and jaw) and b (b2 lobe) The b-subunit flexible flap domain closes the main channel from the upstream side leaving a narrow RNA exit channel Rifampicin (Rif), one of the most efficient inhibitors of RNAP, binds the enzyme near the active center at a pocket formed by the b-subunit and sterically blocks RNA synthesis [13] The b¢ F-bridge helix crosses the cleft in the vicinity of the catalytic Mg2+ separating the main and secondary channels (Fig 1B) The secondary channel gives access to the active site for nucleotide substrates [9,14] and for elongation factors GreA and GreB (Fig 1B) [15,16] Despite the great progress of the past few years in structural studies of transcription, many molecular details
of the RNAP–nucleic acid interactions remain unknown Little is also known about the mechanisms of conforma-tional changes of RNAP that occur at different stages of transcription Comparisons of homologous bacterial [17] and yeast RNAP structures [5] suggest significant conform-ational flexibility of RNAP domains that allows for the opening and closing of the main channel The closure of RNAP around the DNA/RNA framework was proposed
to be of crucial importance for the formation of stable elongation complexes [4–6,18] More local conformational changes are thought to occur in the vicinity of the RNAP active center In particular, the movement of the F-bridge helix was hypothesized to accompany the translocation step
Correspondence to A Kulbachinskiy, Laboratory of Molecular
Gen-etics of Microorganisms, Institute of Molecular GenGen-etics, Kurchatov
Sq 2, Moscow 123182, Russia Fax:/Tel.: + 7095 1960015,
E-mail: akulb@img.ras.ru
Abbreviations: RNAP, DNA-directed RNA polymerase; Eco,
Escherichia coli; Taq, Thermus aquaticus; SELEX, systematic
evolu-tion of ligands by exponential enrichment; MS, minimal nucleic acid
scaffold; Rif, rifampicin; ss, single stranded.
Enzymes: DNA-directed RNA polymerase (EC 2.7.7.6).
(Received 25 May 2004, revised 19 October 2004,
accepted 25 October 2004)
Trang 2during each cycle of nucleotide addition [6,8,14] Several
inhibitors of RNAP such as streptolydigin, a-amanitin,
microcin J25 and CBR703 which bind at different sites
near the F-bridge have recently been proposed to act
by restricting the intramolecular mobility of the enzyme
[14,19–21] Thus, the analysis of different ligands that bind
RNAP and stabilize alternative structural states of the
enzyme could open the way for a better understanding of
the conformational flexibility of RNAP
Aptamers are synthetic RNA and ssDNA ligands that
can be obtained to virtually any desired target [22] The
affinities and specificities of aptamers to different protein
targets are comparable to those of monoclonal antibodies
Not surprisingly, aptamers have drawn significant attention
as very promising ligands that can be used in a variety of
biological applications Aptamers to various nucleic acid
binding proteins (including proteins that do not recognize
their substrates sequence specifically) usually bind their
targets at natural RNA or DNA recognition sites [22–26]
Structural analysis of several aptamer–protein complexes
has shown that aptamers mimic the natural nucleic acid
ligand of a protein and bind at the same place even if they
have an unrelated nucleotide sequence and secondary
structure (e.g aptamers to the MS2 phage coat protein
[27], NF-jB [28], reverse transcriptase [24]) As a result,
many aptamers are very effective and highly specific
inhibitors of their targets [29,30]
Aptamers to several enzymes were shown to affect the conformation of the target protein [31–33] For example, ssDNA aptamers to Ile-tRNA synthetase stimulated the editing activity of the enzyme, which is normally induced by tRNAIle [31], while aptamers to hepatitis C virus RNA-dependent RNAP allosterically prevented the entry of an RNA substrate into the enzyme’s active site [32]
Here, we describe the isolation of aptamers to Escheri-chia coli (Eco) core RNAP All selected aptamers are highly potent inhibitors of RNAP and are likely to bind within the main channel of the enzyme We also developed
a site-directed SELEX (systematic evolution of ligands by exponential enrichment [22]) procedure that allowed iden-tification of several aptamers that interact specifically with the Rif-binding pocket of RNAP The RNAP–aptamer complexes were compared with the complex of the core enzyme with the minimal RNA/DNA scaffold (Fig 1) [34], which mimics the natural elongation complex We found that the aptamers and the minimal scaffold bind to overlapping sites on the core enzyme and that the resulting complexes have many similar features Finally, we showed that the aptamers sensed interactions of core RNAP with the r70-subunit and transcript cleavage factor GreB The results indicate that stimulation of the RNAP endonuclease activity by GreB may be accompanied by significant conformational changes of the enzyme We propose that the selected aptamers may be useful in studying the
Fig 1 Structural features of RNAP clasping minimal nucleic acid scaffold (A) Minimal nucleic acid scaffold (MS) used in this study (B) Model of MS in complex with Taq core RNAP [9] MS (ball and stick representation: nontemplate DNA strand, black; template DNA strand, light violet; RNA, red) is placed inside the main RNAP channel Also shown are active site magnesium (light blue), rifampicin (orange) clashing with RNA, b¢ F-bridge helix (green), rudder (red), b¢ coiled-coil r-subunit binding protrusion (dark violet), b flexible flap (blue; region cor-responding to Eco amino acids 885–914 is shown in red), and b¢ elements from the downstream part of the main channel (jaw and
a part of the clamp, light blue; W217His 6
insertion site is red) The b2 (lobe) domain of the b-subunit (amino acids 174–314, corres-ponding to Eco186–433) located above the
MS is outlined by a thick brown line The secondary channel is located just behind the F-bridge The location of the GreB binding site [15] is shown schematically as a yellow oval The ochre contour corresponds to the r-subunit, the position of which was taken from the T thermophilus holoenzyme struc-ture [8] r-Induced conformational changes are not shown The semitransparent area shows the position of r region 3.2.
Trang 3mechanism of transcription and conformational flexibility
of RNAP
Materials and methods
Proteins
Ecocore RNAP with a His6tag in the C terminus of the
b¢-subunit and the r70-subunit were purified as described
[35,36] Eco core RNAP bearing the insertion of six histidine
residues at position 217 of the b¢-subunit was reconstituted
in vitro from individual subunits [37] Mutant Eco core
enzymes with deletions of the b2 (bD186–433) and the
flexible flap (bD885–914) domains were kindly provided by
K Severinov and K Kuznedelov [38,39] Thermus aquaticus
(Taq) core RNAP was purified from Eco cells expressing all
four core subunits from plasmid pET28ABCZ as described
[40] The GreB protein was a generous gift of S Borukhov
(The State University of New York)
Selection of aptamers to Eco core RNAP
A ssDNA library (Fig 2A) was purchased from Operon
Technologies Inc The amounts of ssDNA and the core
enzyme varied from 5 nmol and 100 pmol, respectively, in
the first round of selection to 100 pmol and 10 pmol in
subsequent rounds Prior to each round of selection, a
10-pmol aliquot of ssDNA was labeled with -[32P]ATP[cP]
(7000 CiÆmmol)1, ICN, Costa Mesa, CA, USA) and T4
polynucleotide kinase (New England BioLabs, Beverly,
MA, USA), purified by 10% PAGE and added to the bulk
DNA sample to monitor the binding of the library to
RNAP ssDNA was then diluted in 1 mL binding buffer
(20 mM Tris/HCl pH 7.9, 10 mM MgCl2, 300 mM NaCl,
30 mMKCl; in subsequent rounds NaCl and KCl
concen-trations were increased to 400 and 40 mM, respectively),
heated for 5 min at 95C and cooled rapidly to 0 C The
DNA solution was passed through a 50-lL Ni2+
–nitrilo-triacetic acid–agarose (Qiagen) microcolumn
pre-equili-brated with the binding buffer The core enzyme was then
added to the solution and the mixture was incubated for
15 min at room temperature Thirty microliters
Ni–nitrilo-triacetic acid–agarose was added and the incubation was
continued for a further 20 min with occasional shaking The
solution containing unbound DNA was removed and the
sorbent was washed two to four times with 1 mL of binding
buffer (for a total time of 30–60 min) ssDNA–RNAP
complexes were eluted with 300 lL binding buffer
contain-ing 200 mM imidazole The solution was treated with
300 lL phenol and 300 lL chloroform DNA was ethanol
precipitated, dissolved in water and amplified using Vent
DNA polymerase (New England BioLabs) and primers
corresponding to fixed regions of the initial library
(5¢-GGGAGCTCAGAATAAACGCTCAA-3¢ and
BBB-5¢-GATCCGGGCCTCATGTCGAA-3¢, where B is a
bio-tin residue) Two DNA strands were separated by size on
10% denaturing PAGE, the nonbiotinilated strand was
eluted and used for the next SELEX round In the
Rif-directed SELEX experiment each round of the selection
included two successive partitioning steps The initial
selection of oligonucleotides was carried out as described
above DNA eluted from the complexes with RNAP was
treated with phenol and chloroform and ethanol precipi-tated The resulting enriched library was incubated with the core enzyme (taken in twofold excess relative to the first selection step) in the presence of 20 lgÆmL)1Rif (rifamycin
SV, Sigma, St Louis, MO, USA) DNA–protein complexes were adsorbed on Ni2+–agarose and discarded, while unbound oligonucleotides remaining in the solution were ethanol precipitated, PCR amplified and used in the next SELEX round After the final round of selection, the enriched libraries were amplified with primers containing EcoRI and HindIII sites and cloned into the pUC19 plasmid The sequences of individual aptamers were determined using the standard sequencing protocol Indi-vidual ssDNA aptamers were obtained by PCR with the primers corresponding to aptamer flanks; the DNA strands
Fig 2 Selection of aptamers to Eco core RNAP (A) Random ssDNA library used in selection experiments (B) The effect of Rif on the binding of round 11 libraries (0.1 n M ) to Eco core RNAP (10 n M ) in binding buffer containing 440 m M salt Binding was measured as described in Materials and methods One hundred per cent corres-ponds to the binding in the absence of Rif (C) Sequences of repre-sentative aptamers from 13 different classes described Shown are the central 32-nt-long regions of the aptamers Aptamer E3 contains a
T fi A change at the first position of the right constant region; aptamer E13 contains a single nucleotide deletion at the same site The sequence motif identical in aptamers E9 and E12 is underlined.
Trang 4were separated on denaturing PAGE as described above.
Control experiments demonstrated that aptamers did not
bind to the Ni-affinity sorbent and therefore the SELEX
protocol was highly efficient in selecting specific aptamer
sequences
Quantitation of the binding of aptamers to RNAP
Determination of Kd values for the binding of
oligo-nucleotides to RNAP was achieved by using the
nitro-cellulose filtration method as described [41] All
measurements were performed in binding buffer
contain-ing 400 mM NaCl and 40 mM KCl unless otherwise
indicated A 5¢-end labeled oligonucleotide (0.003 nM)
was incubated with a series of dilutions of core RNAP
(from 0.01 to 100 nM) in binding buffer containing
50 lgÆmL)1 BSA for 45–60 min at 22C and then
filtered through 0.45-lm nitrocellulose filters (HAWP,
Millipore) prewetted in the same buffer The filters were
washed with 5 mL buffer and quantified on a
Phosphor-Imager (Molecular Dynamics, Sunnyvale, CA, USA) Ki
measurements were carried out at fixed core (1–3 nM)
and aptamer (0.1 nM) concentrations Rif, r70 or GreB
were included in the binding reactions 5 min prior to the
addition of oligonucleotides; the samples were incubated
for 1 h at room temperature and passed through
nitrocellulose filters Kd and Ki values were calculated
from the binding curves using KALEIDAGRAPH software
(Synergy, Reading, PA, USA) To measure dissociation
kinetics of RNAP–aptamer complexes, the core
polym-erase (3 nM) was preincubated with a labeled aptamer
(0.1 nM) for 60 min, the complex was challenged with the
corresponding unlabeled aptamer (100 nM), minimal
nucleic acid scaffold (500 nM), Rif (2 lgÆmL)1), r70
-subunit (1 lM) or GreB (3 lM) and aliquots of the
sample were filtered after increasing time intervals
Control experiments demonstrated that the level of
RNAP–aptamer binding did not change if the
measure-ments were done in the absence of the inhibitors
Minimal nucleic acid scaffold (MS)
The sequences of DNA and RNA oligos used to
recons-titute MS are shown in Fig 1A MS was prepared as
described [9] The RNA oligo (200 pmol, final
concentra-tion 10 lM) was labeled with 10 U T4 polynucleotide kinase
and 0.5 mCi [32P]ATP[cP], mixed with template and
nontemplate DNA oligonucleotides (final concentrations
of the oligonucleotides were 1, 1 and 2 lM, respectively) in
the binding buffer, heated to 65C and slowly cooled to
20C Determination of Kd for the binding of MS to
RNAP was performed as described above In some cases,
the binding was measured in the buffer containing 200 mM
salt (20 mM Tris/HCl pH 7.9, 10 mM MgCl2, 160 mM
NaCl, 40 mM KCl) When studying the inhibitory effect
of aptamers on RNAP activity, Eco core enzyme (10 nM)
was added to the mixture of unlabeled MS (10 nM) and
aptamers (30 nM) in binding buffer containing 400 mM
NaCl and 40 mM KCl The samples were incubated for
30 min at room temperature and supplemented with
[32P]UTP[aP] (0.1 lM, 3000 CiÆmmol)1, Perkin Elmer,
Wellesley, MA, USA) The reaction was stopped after
10 min by the addition of a formamide-containing stop buffer and applied to 23% urea PAGE The amount of radioactively labeled 9-nt RNA product was quantified by using a PhosphorImager
Results
Selection of aptamers to Eco core RNAP – conventional
vs site-directed SELEX Both the core and holo enzymes of bacterial RNAP bind nucleic acids [42–45] While the holoenzyme is able to recognize specific DNA sequences, the interactions of the core enzyme with DNA and RNA are generally nonspecific There are numerous reports on interactions of the core with total cellular DNA and RNA [43,46], tRNA [47,48], ssDNA [43] and also some individual RNA sequences [49] Repor-ted Kdvalues for some of these interactions are in the range
of 10)8 to 10)10M [43,48,49] and are comparable to the affinities of known aptamers to their protein targets
We used a library of 75-nt long ssDNA containing a 32-nt central region of random sequence to select aptamers that would specifically interact with the RNAP core (Fig 2A)
We found that at low ionic strength, molecules from the unenriched library bound the core enzyme very tightly (Kd 0.2 nMat 40 mMsalt) Such a high level of nonspe-cific affinity of RNAP to nucleic acids could be a serious obstacle for the selection of specific aptamer sequences However, we observed that the nonspecific binding of ssDNA to core RNAP was considerably reduced at increased ionic strength (Kd> 100 nM at 300 mM salt) Therefore, we performed all selection procedures at elevated monovalent salt concentrations (300–440 mM)
We conducted two types of experiments to select aptamers to Eco core RNAP In the first type of experiment (I), the SELEX procedure was performed in a conventional way In brief, in each round of the selection the ssDNA library was incubated with core RNAP immobilized on a Ni-affinity sorbent via the hexahistidine tag present at the C-terminal end of the b¢-subunit Then unbound DNA was extensively washed out to select sequences that formed stable complexes with RNAP RNAP–DNA complexes were eluted with imidazole, recovered oligonucleotides were amplified by PCR and used in the next round of selection
To avoid selection of nonspecific sequences that bind to the affinity sorbent used in the reaction, the library was passed through Ni–agarose column in the absence of RNAP before each SELEX round
The second type of experiment (II) aimed to identify ligands that bound specifically to the Rif-binding pocket of RNAP Rif is one of the most potent inhibitors of the enzyme and is used as a drug in the therapy of several infectious diseases However, a large number of mutations
in core RNAP conferring resistance to this drug have been described Identification of new ligands that can mimic the effect of Rif is therefore of great importance Each round of site-directed SELEX consisted of two consecutive binding reactions First, we selected sequences that bound to free core RNAP Second, DNA molecules that interacted with RNAP were incubated with core RNAP in the presence of excess Rif DNA molecules that were unable to bind RNAP
in complex with Rif were used in the next round of selection
Trang 5After 11 rounds of selection, the enriched libraries
obtained by both protocols bound core polymerase with
high affinity (Kd 5 nM in binding buffer containing
440 mMsalt) but exhibited substantially different sensitivity
to Rif addition (Fig 2B) While the RNAP binding of the
conventionally enriched library was essentially resistant to
Rif, binding of the site-specifically selected library was
severely inhibited by the antibiotic Both libraries were cloned
and 50 individual clones were sequenced in each case
Analysis of individual clones allowed us to identify 13
different classes of sequences, designated E1–E13 (Fig 2C;
the total number of clones within each class is shown in
Table 1) Each class consisted of several clones with identical
or closely related sequences Sequences from classes E1–E4
were found only in the conventionally enriched library,
sequences from classes E5–E8 were present in both types of
libraries and sequences from classes E9–E13 were unique to
the library obtained by Rif-directed selection All of the
aptamers were predicted to fold into distinct secondary
structures, such as hairpins and G-quartets (e.g aptamers E1,
E3, E4, E5) (data not shown) One aptamer representative
of each class was chosen for further investigation (Fig 2C)
Aptamers bind Eco core RNAP with high affinity and
inhibit the enzyme’s activity
Individual aptamers from all 13 classes proved to be
high-affinity ligands to Eco core RNAP with apparent Kdvalues
ranging from 0.13 nMfor aptamer E1 to 6.3 nMfor aptamer
E8 at 440 mMsalt (Table 1) These affinities are comparable
to the affinity of Rif to Eco core polymerase [50,51] and
greatly exceed those of other small molecule ligands of
RNAP such as streptolydigin [52], microcin J25 [20] and
CBR703 [21] Neither the initial library nor any other
nonspecific oligonucleotide tested appreciably bound
RNAP at these conditions All of the aptamers competed
with each other for the binding to core RNAP which
indicated that they interacted with overlapping sites on the
RNAP surface (data not shown)
We compared the RNAP–aptamer complexes with a complex of the RNAP core bound to the minimal nucleic acid scaffold (MS) (Fig 1A) – a model of the elongation complex [34] The contacts of MS with Eco core RNAP were mapped previously by nucleic acid–protein crosslink-ing techniques and the results were used to position MS on the three-dimensional structure of Taq core RNAP (Fig 1B) [9] The interaction of MS with RNAP was shown to be independent of the MS sequence [9,13] The
MS used in our study consisted of an 18-nt-long down-stream DNA duplex and an 8-nt-long RNA–DNA hetero-duplex separated by two unpaired DNA bases (Fig 1A) Unlike the aptamers, MS bound both Eco and Taq core RNAPs with comparable affinities (with a Kd value of
1 nM in binding buffer containing 40 mM salt) The complex of MS with Eco core polymerase was transcrip-tionally active at both low (40 mM) and high (440 mM) salt concentrations (data not shown) Remarkably, the affinity
of MS to RNAP at 440 mM salt (Kd‡ 50 nM) was lower than the affinities of the aptamers at these conditions All selected aptamers competed with MS for binding to core RNAP and efficiently inhibited RNAP activity in the transcription assay (most probably by preventing the formation of the RNAP–MS complex, see below) (Fig 3) The inhibition of the core polymerase activity by aptamers was specific as much weaker inhibition was observed in the case of the initial random library (Fig 3)
Aptamers interact with distinct sites inside the main channel of core RNAP
In order to locate the aptamer binding sites more precisely
we checked the ability of the aptamers to interact with Eco core RNAP bearing insertion–deletion mutations in several sites on the periphery of the main channel (Table 1 and Fig 1B) The mutations were a deletion of the flexible flap domain in the b-subunit (bD885–914), a deletion of the domain b2 in the b-subunit (bD186–433) and an insertion of six histidine residues at position 217 of the b¢-subunit
Table 1 Properties of the aptamers to Eco core RNAP K d values were measured in the binding buffer containing 400 m M NaCl and 40 m M KCl.
Aptamer SELEX
Clones (n)
K d (n M )
Binding to mutant RNAPs a Inhibition by
a
The increase in K d for aptamer binding to mutant variants of core RNAPs over K d values for the wild-type enzyme: +, 1–5 times; +/ ), 5–20 times; –, more than 20 times b The increase in K d for aptamer binding to the core polymerase in the presence of 0.5 lM r-subunit: +, approximate change in K d is 10–30 times c The increase in K d for aptamer binding in the presence of 1.5 lM GreB Blank cells, no data.
Trang 6(b¢W217His6) The aptamers differed in their affinity to the
mutants (Table 1) The flap deletion had the least
pro-nounced effect on the interactions of the aptamers with
RNAP, significantly affecting the binding of only two of
them, E1 and E11 (their Kdvalues were increased 5.6- and
11.2-fold, respectively) In contrast, the binding of most of
the aptamers was disturbed severely by the b2 domain
deletion (for example, Kdfor E9 increased about 250-fold)
and the only aptamer that bound this mutant with
considerable affinity was E7 The most interesting results
were obtained with the b¢W217His6insertion mutant While
some of the aptamers (E13, E8) interacted with the mutant
with unchanged affinity, binding of the others was
weak-ened to different degrees (Table 1) The strongest effect was
for aptamer E3 (Kd increased 100-fold) The simplest
interpretation of the observed effects is that the regions of
RNAP changed by the mutations are parts of the aptamers’
binding sites
Effect of rifampicin on the binding of aptamers
Rif binds near the RNAP active center at the so-called
Rif-binding pocket of the b-subunit and sterically prevents the
synthesis of RNAs longer than a dinucleotide (Fig 1B) Rif
also prevents the binding of MS to the core enzyme [13] We
confirmed this result and found that Rif inhibited MS
binding with an apparent Ki of < 0.5 nM (Fig 4) This
value is in good agreement with earlier reports on Rif Kd
for binding to RNAP (0.5–2 nM) [50,51]
Rif exhibited different effects on the interaction of various
aptamers with RNAP (Table 1) The binding of all the
aptamers obtained through Rif-directed selection (E5–E13)
was inhibited by Rif with the same efficiency as the binding
of MS (these aptamers were therefore called RifS, for
Rif-sensitive, aptamers, Table 1 and Fig 4) In contrast, most
of the aptamers unique to the conventional selection procedure (E1–E3) were insensitive to Rif (RifR, for Rif-resistant, aptamers, Table 1 and Fig 4) and only one of them (E4) was found to be RifS RifS sequences from classes E5–E8 which were identified in both selection experiments comprised only a small fraction of all sequences in the first SELEX population (Table 1) Thus, conventional SELEX produced mainly RifR aptamers whereas Rif-directed SELEX succeeded in identifying only RifS sequences The high efficiency of the site-directed SELEX protocol used in our work suggests that similar procedures can be used to obtain high affinity aptamers to antibiotic-binding sites of many proteins of interest
We repeated the binding assay using Rif-resistant core RNAP carrying an S531F substitution in the b-subunit
In this case, the effect of Rif was much weaker with
Ki 0.5 lM(Fig 4) At the same time, the mutation did not affect the binding of aptamers Thus, the core mutation conferring Rif resistance weakened Rif binding to RNAP
by more than three orders of magnitude while having little
or no effect on RNAP–aptamer interactions
The r70-subunit and GreB suppress the interaction
of the core RNAP with aptamers The r70-subunit inhibited the binding of all the aptamers to the core polymerase Apparent Kds for the binding of different aptamers to the holoenzyme of RNAP were increased in the range 8–30 times in comparison with those for the core enzyme (Table 1) When the binding of the E2 aptamer was measured at fixed core and increasing r70 -subunit concentrations, r inhibited the interaction with an observed Kiof 10 nM (Fig 5A) This value apparently corresponded to Kd for the r70–core interaction at these conditions The r70-subunit also suppressed the interaction
of the core enzyme with MS (Fig 5A) This result is in agreement with previous studies which demonstrated that the binding of r and RNA in the elongation complex was
Fig 3 Inhibition of the Eco core polymerase activity by aptamers.
RNAP activity was measured as described in Materials and methods.
The core enzyme was added to the mixture of MS and aptamers in
binding buffer containing 440 m M salt and transcription was initiated
by adding [ 32 P]UTP[aP] The amount of radioactively labeled
9-nt-long RNA product was quantified and normalized to the activity
in the absence of the inhibitor I, Aptamers found only in the
con-ventional selection experiment; II, aptamers unique to the Rif-directed
experiment; I + II, aptamers identified in both selections; N, the
initial library.
Fig 4 Effect of Rif on the binding of aptamers and MS to core RNAP Binding reactions contained 10 n M of the core enzyme, 0.1 n M oligo-nucleotides and varied amounts of Rif Monovalent salt concentration
in the binding buffer was 440 m M in the case of aptamers and 200 m M
in the case of MS Binding was measured as described in Materials and methods and normalized to the binding in the absence of Rif The experiment was performed with the wild-type core enzyme (S) and Rif-resistant mutant RNAP (S531F, R).
Trang 7mutually exclusive [53,54] In the three-dimensional
struc-ture of the holoenzyme polymerase, region 3.2 of r seems to
clash with the 5¢ end of growing RNA during initiation
(Fig 1B) [7,8] Thus, it is possible that r70interferes with
MS binding by competing with its RNA component for the
same site on core RNAP
GreB exerts its effect on the elongation complex in a
backtracked state stimulating the nuclease activity of the
RNAP active center [55] We found that the binding of MS
to the core polymerase was not affected by GreB GreB also
failed to stimulate the cleavage of the RNA component of
MS (data not shown) This, as well as resistance of MS to
pyrophosphorolysis (N Korzheva, personal
communica-tion), suggested that MS was captured by RNAP in a
post-translocated state At the same time, GreB suppressed the
interaction of Eco RNAP with all the aptamers tested except
E7, increasing their apparent Kd values three- to sixfold,
when present at 1.5 lM (Table 1) The weaker effect of
GreB in comparison with the r-subunit is probably due to
its lower affinity to core RNAP Indeed, the increase of
GreB concentration resulted in complete inhibition of aptamer binding (Fig 5B) The apparent Kivalue for GreB action calculated from the inhibition curve was 100 nM This value is in good agreement with Kdreported for the GreB–core interaction [56]
MS, Rif and the r70-subunit do not affect the stability
of RNAP–aptamer complexes while GreB promotes their rapid dissociation
To investigate the nature of the effects of MS, Rif, r70and GreB on RNAP–aptamer interactions, we measured the dissociation kinetics of several RNAP–aptamer complexes
in the presence of these ligands (Fig 6) When the complexes containing radioactively labeled aptamers were
Fig 5 Inhibition of aptamer binding by the r 70 -subunit and GreB (A)
Inhibition of the binding of aptamer E2 and MS (0.1 n M ) to the core
polymerase (1 and 2 n M , respectively) by increasing concentrations of
the r-subunit Binding buffer contained 440 m M salt in the case of the
aptamers and 200 m M salt in the case of MS (B) Inhibition of the
binding of aptamer E9 (0.03 n M ) to the core enzyme (3 n M ) by
increasing amounts of GreB Binding was measured in buffer
con-taining 440 m M salt. Fig 6 Dissociation kinetics of RNAP–aptamer complexes in the pres-ence of various competitors The core enzyme was preincubated with a
labeled aptamer and the complex was challenged with the corres-ponding unlabeled aptamer, MS, Rif, the r70-subunit or GreB Aptamer binding was measured in buffer containing 440 m M salt The dissociation kinetics is shown for aptamers E4 (A), E7 (B) and E10 (C).
Trang 8incubated with an excess of the corresponding unlabeled
aptamers, they dissociated with half-life times of more than
1 h The dissociation kinetics of the RNAP–aptamer
complexes measured in the presence of MS, Rif (in case of
RifS aptamers) or the r-subunit followed the kinetics
observed when the unlabeled aptamer was used as a
competitor (Fig 6) Control experiments demonstrated
that when these ligands were added to RNAP before the
aptamers, they completely suppressed complex formation
(data not shown)
In contrast, GreB greatly reduced the stability of several
RNAP–aptamer complexes (Fig 6 and data not shown) In
agreement with Kdmeasurements, GreB did not affect the
stability of the E7–RNAP complex (Fig 6B) At the same
time, when GreB was added to the preformed complexes of
RNAP with E4 and E10 aptamers, it caused their rapid
dissociation; half-life times of the complexes were reduced
by more than 10 times (5 min in comparison with > 1 h
when the kinetics was measured without GreB) (Fig 6A
and C) The residual binding of aptamers measured at large
time intervals corresponded to the maximum inhibition
observed when GreB was added before the aptamers
(Fig 5B and data not shown)
Specific and nonspecific interactions of aptamers
with the RNAP main channel
The interaction of the aptamers with RNAP was found to
be highly dependent on the ionic strength of the solution
At elevated ionic strength (440 mM), the binding of the
aptamers was very sequence specific as even point mutations
of aptamers’ sequences disrupted their interaction with
RNAP The aptamers were also specific to Eco core RNAP
and neither of them bound Taq RNAP (data not shown)
At lower ionic strength (< 200 mM), RNAP still bound
the aptamers but sequence specificity was apparently lost
Under these conditions all the sequences tested, including
the random DNA library, bound the core enzyme with equal
affinities (Kd 1 nM) MS suppressed the binding of all the
oligonucleotides which suggested that the nonspecific
bind-ing of ssDNA also occurred at RNAP sites involved in the
interaction with RNA and DNA in the elongation complex
At elevated ionic strength, Rif and r70 suppressed
RNAP–aptamer interactions (above) Under low ionic
strength conditions, Rif and r70 had no effect on the
binding of RifS aptamers to core RNAP (data not shown)
Therefore, the structure of nonspecific complexes of RNAP
with the aptamers differs from the structure of the
complexes formed at high ionic strength
Discussion
The principal result of this work is that the aptamers sense
the interaction of RNAP with various ligands, including
nucleic acids, antibiotics and protein factors Based on the
mechanism of the inhibition of aptamer binding, these
ligands can be divided into two groups The minimal
nucleic acid scaffold, Rif and the r70-subunit seem to
inhibit RNAP–aptamer interactions by steric blocking of
the aptamer binding sites on the RNAP molecule, while
GreB is likely to affect aptamer binding in an allosteric
manner
Several facts indicated that the aptamers interact with the main channel of RNAP where nucleic acids in natural transcription complexes are held All of the aptamers competed with MS for binding to RNAP and inhibited core polymerase activity Binding of the aptamers was affected
by mutations at several sites in the main channel that were previously implicated in the interactions with nucleic acids
in transcription complexes Furthermore, the binding of 10 out of 13 aptamers was sensitive to Rif As Rif does not cause any significant conformational changes of the core polymerase [13], its effect must result from direct compe-tition with aptamers for the Rif pocket of the b-subunit Finally, the dissociation kinetics of the RNAP–aptamer complexes measured in the presence of MS and Rif followed the same time course as the kinetics measured in the presence of the unlabeled aptamers This indicated that these ligands acted by simple trapping of free RNAP and preventing reassociation of the complexes Thus, both MS and Rif are likely to compete with the aptamers for the binding sites in the main channel
The r-subunit also binds within the main channel of RNAP The main docking sites of r on the core polymerase include the clamp domain of b¢ and the flexible flap domain
of the b-subunit (Fig 1B) [7,8] In addition, the N-terminal region of r, which is not visible in the holoenzyme structure, was shown to occupy the downstream portion of the main channel [57] The binding of r to the core polymerase causes repositioning of several structural modules of the core, including the clamp, b1, b2 and flap domains, which results
in partial closure of the main channel [7] Thus, the inhibition of aptamer binding by r could occur by both steric and allosteric mechanisms We found that, similarly to
MS and Rif, r did not affect the dissociation rate of RNAP–aptamer complexes Thus, the most likely inter-pretation of the inhibitory effect of r is that it also directly blocks RNAP sites involved in aptamer binding The steric competition between aptamers and r is not surprising, when taking into account the extensive interaction interface between r and the core polymerase Hopefully, further studies of mutant variants of r as well as testing various alternative r-subunits will help to establish the regions of r which are responsible for the inhibition of aptamer binding
In contrast to MS, Rif and the r-subunit, GreB dramatically increased the dissociation rate of RNAP– aptamer complexes and therefore actively disrupted RNAP–aptamer interactions As opposed to r70, GreB binds RNAP from the secondary channel side of the enzyme, i.e at the side opposite to the aptamers (see Fig 1B) [15] The binding site of the C-terminal domain of GreB near the entrance of the secondary channel is located outside of the enzyme’s catalytic cleft and seems unlikely to
be involved in aptamer binding The GreB N-terminal coiled-coil domain protrudes deep into the secondary channel, providing two conserved acidic residues which play a key role in the RNA cleavage reaction [15,16,58] Based on these observations, one could suggest two mechanisms of GreB action on the binding of the aptamers One possibility is that the aptamers bound in the main channel might occupy the mouth of the secondary channel and directly interfere with GreB binding Alternatively, the aptamers could sense GreB-induced conformational chan-ges inside the RNAP main channel
Trang 9The strong stimulatory effect of GreB on the
disso-ciation of RNAP–aptamer complexes provides serious
evidence in support of the allosteric mechanism of GreB
action Sensing of GreB binding by several aptamers,
each interacting with RNAP in a different way, as well
as different strengths of GreB effect on various aptamers
(Table 1) is also consistent with the allosteric mechanism
Our data thus give evidence that the interaction of GreB
with RNAP may result in structural changes of the core
polymerase The resolution of current structural data
does not allow us to verify such changes [15] However,
conformational rearrangements in the main channel were
observed in the complex of yeast RNAPII with
elonga-tion factor TFIIS, which also protrudes into the
secon-dary channel and seems to utilize very similar
mechanisms to stimulate RNA cleavage [59]
GreB-induced conformational changes of RNAP detected with
the aptamers may be essential for the stimulation of the
endonuclease activity of the enzyme
Recent studies demonstrated that other protein factors
(e.g DksA) and antibiotics (microcin) also bind RNAP
within the secondary channel and seem to affect RNAP
conformation [60–62] We propose that the aptamers
could be used to study the conformational changes of
RNAP induced by the binding of these regulatory
factors The aptamers could also be useful in studies of
various RNAP mutations that are thought to change the
conformation of the enzyme The examples of such
mutations include the substitution at position 934 near
the F-bridge helix in the b¢-subunit that was proposed to
shift the conformation of the F-bridge toward the bent
form [14] and mutations on the surface of the b-subunit
that impair Q-protein mediated anti-termination
(pre-sumably by changing the conformation of the interior of
the main channel) [63] It should be noted that such
hypothetical conformational changes of RNAP are
usually very difficult to verify The aptamers thus
represent a very useful tool to probe RNAP structure
in many experimental systems
Acknowledgements
We thank K Severinov for protein and plasmid samples and for
reading the manuscript, K Kuznedelov and S Borukhov for materials,
A Stolyarenko for reading the manuscript A.K is especially grateful
to N Korzheva, V Epshtein and A Mustaev for help in doing some
experiments This work was supported by the NIH grant GM30717 to
A.G and by the Russian Foundation for Basic Research grant
02-04-48525.
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