Báo cáo khoa học: Aptamers toEscherichia colicore RNA polymerase that sense its interaction with rifampicin, r-subunit and GreB ppt

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Báo cáo khoa học: Aptamers toEscherichia colicore RNA polymerase that sense its interaction with rifampicin, r-subunit and GreB ppt

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Aptamers to Escherichia coli core RNA polymerase that sense its interaction with rifampicin, r-subunit and GreB Andrey Kulbachinskiy 1,2 , Andrey Feklistov 2,3 , Igor Krasheninnikov 3 , Alex Goldfarb 1 and Vadim Nikiforov 1,2 1 Public Health Research Institute, Newark, New Jersey, USA; 2 Institute 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 mechan isms o f c onformational c han ges of RNAP that occur at different stages of transcription remain unknown. The goal of this work was to obtain novel ligands to RNAP which w ould target d ifferent epitopes o f the enzyme and serve a s specific probes to study the mech- anism of transcription and conformational flexibility of RNAP. Using in vitro selection m ethods, 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 r 70 -subunit inhibited binding of the aptamers t o RNAP core but did not affect the d isso- ciation r ate o f p reformed RNAP–aptamer complexes. We argue t hat 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 R NAP– aptamer complexes by inducing conformational changes in the channel. We propose t hat the aptamers obtained in this work will be useful for s tudying the interactions of RNAP with various ligands and regulatory factors and for investi- gating the conformational flexibility of the enz yme. Keywords: aptamers; conformational changes; elongation complex; Gr eB; R NA polymerase. DNA-directed RNA polymerase ( RNAP, E C 2.7.7.6) is a complex molecular machine undergoing multiple intra- molecular rearrangements in the process of R NA synthe sis [1–3]. D uring the transcription cycle, R NAP 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 c omplexes and to propose the roles for various RNAP domains in interactions with DNA and RNA [6,8–11]. The m ost striking s tructural feature of RNAP is a deep cleft ( the main channel) formed by the t wo largest R NAP 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 a ccommodates t he RNA/DNA hybrid, duplex DNA downstream from the hybrid and RNA behind the hybrid. The 8-bp-long DNA/RNA hybrid is lodged between t he catalytic Mg 2+ ion a nd a s tructural element of b¢ called the rudder (Fig. 1) [9]. The downstream DNA duplex is placed in a ÔtroughÕ 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 th e enzyme near the active center at a pocket formed by the b-subunit and sterically blocks RNA synthesis [13]. The b¢ F-bridge helix cr osses the cleft i n the vicinity of the c atalytic Mg 2+ 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 d etails 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 y east 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 w as proposed to be of crucial importance for the formation of stable elongation complexes [4–6,18]. M ore local conformational changes a re thought to occur i n the vicinity of the RNAP active ce nter. I n p articular, 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 Genetics, 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,systematicevolu- 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 2 5 May 2004, revised 19 October 200 4, accepted 25 October 2004) Eur. J. Biochem. 271, 4921–4931 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04461.x during each c ycle 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 b een 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 m onoclonal antibodies. Not surprisingly, aptamers have drawn significant attention as very promising ligands th at 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 a t 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 v ery effective and highly specific inhibitors of their t argets [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 tRNA Ile [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 m ain c hannel of the enzyme. W e also developed a site-directed SELEX (systematic evolution of ligands by exponential enrichment [ 22]) procedure t hat a llowed 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 m inimal 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, w e showed that the aptamers sensed i nteractions of core R NAP w ith the r 70 -subunit and transcript cleavage factor GreB. The results indicate that stimulation of the R NAP endonuclease activity by GreB may be a ccompanied by significant conformational changes of the e nzyme. We propose that the selected aptamers may be u seful in studying t he Fig. 1. Structural features of RNAP clasping minimal nucleic acid sca ffold. (A) Minimal nucleic acid scaffold (MS) used in th is study. (B) Model of MS in c om plex 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 c hannel. Also shown are active site m agnesium (light blue), rifampicin (orange) clashing w ith RNA, b¢ F-bridge h elix (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 showninred),andb¢ elements from the downstream part of the main channel (j aw and a part of t he clamp, light blue; W217His 6 insertion site is red). The b2(lobe)domainof 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 s hown 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. 4922 A. Kulbachinskiy et al. (Eur. J. Biochem. 271) Ó FEBS 2004 mechanism of transcription and conformational flexibility of RNAP. Materials and methods Proteins Eco core RNAP with a H is 6 tag in t he C terminus of the b¢-subunit and the r 70 -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 Ne w York). Selection of aptamers to Eco core RNAP A ssDNA library (Fig. 2 A) was purchased from Operon Technologies Inc. The amounts of ssDNA and the core enzyme varied f rom 5 nmol and 100 pmol, respectively, in the first ro und 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 -[ 32 P]ATP[cP] (7000 Ci Æmmol )1 ,ICN,CostaMesa,CA,USA)andT4 polynucleotide kinase (New England BioLabs, Beverly, MA, USA), purified by 10% P AGE and added to t he bulk DNA sample to monitor the binding of the library to RNAP. ssDNA was then diluted in 1 mL binding buffer (20 m M Tris/HCl pH 7.9, 10 m M MgCl 2 ,300m M NaCl, 30 m M KCl; in subsequent rounds NaCl and K Cl concen- trations were increased to 400 and 40 m M , respectively), heated for 5 min at 95 °C and cooled rapidly to 0 °C. The DNA solution was passed through a 50-lLNi 2+ –nitrilo- triacetic acid–agarose (Qiagen) microcolumn p re-equili- brated with the binding buffer. The core enzyme w as 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 m in with occasional shaking. The solution containing unbound DNA was r emoved and t he sorbent was washed two to four times with 1 mL of binding buffer (for a total time of 30–60 min). ssDNA–RNAP complexes w ere e luted with 300 lL binding buffer contain- ing 200 m M imidazole. The solution was treated with 300 lL phenol and 300 lL chloroform. DNA was ethanol precipitated, dissolved in water and amplified using V ent DNA polymerase (New England BioLabs) and primers corresponding to fixed regions of the initial library (5¢-GGGAGCTCAGAATAAACGCTCAA-3¢ and BBB- 5¢-GATCCGGGCCTCATGTCGAA-3¢, w here B is a bio- tin r esidue). Two DNA strands were separated b y s ize o n 10% denaturing PAGE, the nonbiotinilated strand was eluted an d 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 f rom the complexes w ith RNAP w as 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 p resence of 2 0 lgÆmL )1 Rif (rifamycin SV, S igma, St Louis, MO, U SA). DNA–protein complexes were adsorbed on Ni 2+ –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 rou nd 11 librarie s (0.1 n M )toEco core RNAP (10 n M )in binding bu ffer containing 440 m M salt. Binding was measured as described i n M ate rials an d methods. One hundred pe r c ent corres- ponds to t he binding in t he absence of Rif. ( C) Sequences of repre- sentative aptamers f rom 13 different c lasses described. Shown are the central 32-nt-lo ng r egio ns of the aptam ers. Ap tam er E 3 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. Ó FEBS 2004 Aptamer probes of RNA polymerase structure (Eur. J. Biochem. 271) 4923 were 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 K d values f or the binding of oligo- nucleotides to RNAP was achieved b y using the nitro- cellulose filtration method as described [41]. All measurements were performed in binding b uffer contain- ing 400 m M NaCl and 4 0 m M KCl unless o therwise indicated. A 5 ¢-end labeled o ligonucleotide ( 0.003 n M ) was incubated with a series of dilutions of core RNAP (from 0.0 1 to 100 n M ) in binding buffer containing 50 lgÆmL )1 BSA for 45–60 min at 22 °Candthen filtered t hrough 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). K i measurements were car ried out at fixed core (1–3 n M ) and aptamer (0.1 n M ) concentrations. Rif, r 70 or GreB were included in the binding reactions 5 m in prior to the addition of oligonucleotides; the samples were incubated for 1 h a t room temperature and passed through nitrocellulose filters. K d and K i 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 n M ) was preincubated with a labeled aptamer (0.1 n M ) for 60 min, the complex was c hallenged with the corresponding unlabeled aptamer (100 n M ), minimal nucleic acid scaffold (500 n M ), Rif ( 2 lgÆmL )1 ), r 70 - subunit (1 l M )orGreB(3l M ) and aliquots of the sample were filtered a fter increasing time intervals. Control e xperiments 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- tituteMSareshowninFig.1A.MSwaspreparedas described [9]. The RNA oligo (200 p mol, final concentra- tion 10 l M ) was labeled with 10 U T4 polynucleotide kinase and 0.5 mCi [ 32 P]ATP[cP], mixed with template and nontemplate DNA oligonucleotides (final concentrations of the oligonucleotides were 1, 1 and 2 l M , respectively) in the binding buffer, heated to 65 °C and slowly cooled to 20 °C. Determination of K d for the binding of MS to RNAP was performed as described above. In some cases, the binding was measured i n the buffer containing 200 m M salt (20 m M Tris/HCl pH 7.9, 10 m M MgCl 2 , 160 m M NaCl, 40 m M KCl). When s tudying the inh ibitory effect of aptamers on R NAP activity, Eco core enzyme (10 n M ) was added to the mixture of unlabeled MS (10 n M )and aptamers (30 n M ) in binding buffer containing 400 m M NaCl and 40 m M KCl. The samples were incubated for 30 min a t room temperature and supplemented with [ 32 P]UTP[aP] (0.1 l M ,3000CiÆmmol )1 , Perkin Elmer, Wellesley, MA, USA). The reaction was stopped after 10 min by the addition of a formamide-containing stop buffer a nd 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 c ore a nd 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 K d values for some of t hese interactions are in the range of 10 )8 to 10 )10 M [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 rand om sequence to select aptamers that would s pecifically 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 (K d  0.2 n M at 40 m M salt). Such a high level o f 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 (K d > 100 n M at 300 m M salt). Therefore, we performed all selection procedures at elevated monovalent salt concentrations (300–440 m M ). 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 b rief, in each round of the selection the ssDNA library was in cubated with c ore RNAP immobilized on a Ni-affinity sorbent via th e h exahistidine tag p resent a t the C-terminal end of the b¢-subunit. Then un bound DNA was extensively washed out to select se quences that formed stable complexes with RNAP. RNAP–DNA complexes were eluted with imidazole, recovered oligonucleotides were amplified by PCR and used in the next r ound of selection. To avoid s election of nonspecific sequences that bind to the affinity sorbent u sed 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 th at bound specifically to the Rif-bin ding 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. H owever, a l arge number o f 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 o f two consecutive binding reactions. First, we selected sequences that bound to free core RNAP. Seco nd, DNA mole cules that i nteracted with RNAP were incubated w ith core R NAP 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. 4924 A. Kulbachinskiy et al. (Eur. J. Biochem. 271) Ó FEBS 2004 After 11 rounds of selection, the enriched libraries obtained by both protocols bound core polymerase with high affinity (K d  5n M in binding buffer containing 440 m M salt) but exhibited s ubstantially different s ensitivity to Rif addition (Fig. 2B). While the R NAP binding of the ÔconventionallyÕ enriched library was essentially resistant to Rif, binding of the site-specifically selected library was severely inhibitedby 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 E 1–E13 (Fig. 2C; the total number of clones within each class is shown i n Table 1). Each class consisted of several clones w ith identical or closely r elated sequences. Sequences from classes E 1–E4 were found only in the conventionally enriched library, sequences from classes E5–E8 were p resent 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 f old into distinct secondary structures, s uch as h airpins and G-quartets(e.g. aptamers E1, E3, E4, E5) ( data not shown). O ne 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 1 3 classes proved to be high- affinity ligands to Eco core RNAP with apparent K d values ranging from 0.13 n M for aptamer E1 to 6.3 n M for aptamer E8 at 440 m M salt (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 t ested appreciably bound RNAP at these conditions. All of the a ptamers c ompeted with each other for the binding to core RNAP which indicated that they interacted with overlapping sites o n the RNAP surface (data not shown). We compared the RNAP–aptamer complexes with a complex o f the RNAP core bound to the minimal nucleic acid scaffold (MS) (Fig. 1 A) – a model of the elongation complex [34]. The contacts of MS with Eco core RNAP were mapped previously by nucleic acid–protein crosslink- ing t echniques a nd the results were used to position MS on the t hree-dimensional structure of Taq core RNAP (Fig. 1 B) [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 K d value of  1n M in binding buffer containing 40 m M salt). The complex of MS with Eco core polymerase was transcrip- tionally active at both low (40 m M ) a nd high (440 m M )salt concentrations (data not shown). Remarkably, the a ffinity of MS to RNAP at 440 m M salt (K d ‡ 50 n M )waslower than the affinities of the a ptamers at these conditions. All selected a ptamers competed w ith MS f or binding to core RNAP and efficiently inhibited R NAP activity in t he transcription assay (most probably b y p reventing 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 p recisely we checked the ability of t he aptamers to interact with Eco core RNAP bearing insertion–deletion mutations in several sites o n the periphery of the main channel (Table 1 and Fig. 1B). The mutations were a d eletion of the flexible fl ap domain in the b-subunit (bD885–914), a deletion of the domain b2intheb-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 contain ing 400 m M NaCl and 40 m M KCl. Aptamer SELEX Clones (n) K d (n M ) Binding to mutant RNAPs a Inhibition by III Dflap D186–433 WHis 6 Rif r b GreB c E1 I 10 – 0.13 +/– – + – 8.1 2.5 E2 I 16 – 1.04 + – +/– – 19.9 3.2 E3 I 12 – 1.23 + – – – 28.8 E4 I 6 – 2.23 + – +/– + + 6.3 E5 I + II 2 3 0.72 + +/– +/– + 10.4 3.3 E6 I + II 2 4 1.62 + – +/– + + E7 I + II 2 1 2.21 + + + + + 1.1 E8 I + II 1 4 6.32 + – + + + E9 II – 8 1.43 + – – + 14.2 5.5 E10 II – 4 2.82 + – +/– + + 6.5 E11 II – 5 3.56 +/– – + + + E12 II – 9 4.00 + – +/– + + 5.8 E13 II – 7 4.93 + – + + + 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 l M 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. Ó FEBS 2004 Aptamer probes of RNA polymerase structure (Eur. J. Biochem. 271) 4925 (b¢W217His 6 ). 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 K d values were increased 5.6- and 11.2-fold, respectively). In c ontrast, the binding of mos t of the aptamers was disturbed severely by the b2 domain deletion (for example, K d for E9 increased about 250-fold) and the only a ptamer that bound this mutant with considerable affinity was E7. The most interesting results were obtained with t he b¢W 217His 6 insertion 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 (K d increased  10 0-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 K i of < 0.5 n M (Fig. 4 ). This value is in g ood agreement with earlier r eports on Rif K d for binding to RNAP (0.5–2 n M ) [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 th e same e fficiency as the binding of MS (these aptamers were therefore c alled RifS, for R if- sensitive, aptamers , T able 1 and Fig. 4). In c ontrast, m ost of the a ptamers 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 S ELEX produced mainly RifR aptamers whe reas Rif-directed SELEX succeeded in identifying only RifS sequences. T he high efficiency of the site-directed SELEX protocol used in our work suggests that similar procedures can be used to obtain h igh a ffin ity a ptamers 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 K i  0.5 l M (Fig. 4). At the same time, the mutation did not affect the binding of aptamers. Thus, t he core mutation conferring Rif resistance w eakened Rif binding to RN AP by more than three orders of magnitude while having little or no effect on RNAP–aptamer i nteractions. The r 70 -subunit and GreB suppress the interaction of the core RNAP with aptamers The r 70 -subunit inhibited the binding of all the aptam ers to the core polymerase. Apparent K d s for the binding of different a ptamers t o the holoenzyme of RNAP were increased in the range 8–30 times in comparison with those for the core enzyme (Table 1). When th e binding of the E2 aptamer was measured at fixed core and increasing r 70 - subunit concentrations, r inhibited the interaction w ith an observed K i of  10 n M (Fig. 5 A). This value apparently corresponded to K d for the r 70 –core interaction at these conditions. The r 70 -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 th e 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 add ing [ 32 P]UTP[aP]. T he amount of radio active ly l abele d 9-nt-long RNA product was quantified and normalized to the activity in the a bsence of the i nhib itor. I, Aptamers found o nly in the con - ventional selection experiment; II, aptamers unique to the Rif-directed experiment; I + II, aptamers identifiedinbothselections;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 ofthecoreenzyme,0.1n M oligo- nucleotides and varied amounts of Rif. Monovalent salt concentration in the binding buffer was 440 m M inthecaseofaptamersand200m M in the case o f MS. Bind ing w as measured as described in Materials and methods and n ormalized t o t he binding in the absence of Rif. T he experiment was performed with the wild-type core enzyme (S) a nd Rif- resistant mutant R NAP (S531F, R). 4926 A. Kulbachinskiy et al. (Eur. J. Biochem. 271) Ó FEBS 2004 mutually 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. 1 B) [7,8]. Thus, it is po ssible that r 70 interferes w ith 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 f ound that the b inding of MS to the core polymerase was not affected by GreB. GreB also failed to stimulate the cleavage of the RNA c omponent of MS (data not shown). This, as well as resistance of MS to pyrophosphorolysis (N. Korzheva, personal communica- tion), suggested that MS was captured b y RNAP in a post- translocated state. At the same t ime, GreB suppressed the interaction of Eco RNAP with all the aptamers tested except E7, increasing their apparent K d values three- to sixfold, when present at 1.5 l M (Table 1). The weaker effect of GreB in comparison with the r-subunit is p robably due to its lower affinity to core RNAP. Indeed, t he increase of GreB concentration resulted i n complete inhibition of aptamer binding (Fig. 5B). The apparent K i value for GreB action calculated from the inhibition curve was  100 n M . This value is in goo d agreement with K d reported for the GreB–core interaction [56]. MS, Rif and the r 70 -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, r 70 and GreB on RNAP–aptamer interactions, we measured the dissociation k inetics o f several RNAP–aptamer comple xes in the p resence of these ligands (Fig. 6 ). When the complexes containing radioactively labeled aptamers were Fig. 5. Inhibition of aptamer b in ding by the r 70 -subunit a nd GreB. (A) Inhibition of t he binding of aptamer E2 and MS (0.1 n M )tothecore polymerase (1 and 2 n M , respectively) by increasing concentrations of the r-subunit. Binding buffer cont ained 440 m M salt in the case of the aptamers and 200 m M salt in the case o f MS. (B) Inhibition of t he binding of aptamer E9 (0.03 n M ) to the core enzyme (3 n M )by increasing am ounts o f G reB. Binding was measure d 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 r 70 -subunit or GreB. Aptamer binding was measu red in buffer containing 440 m M salt. The dissociationkineticsisshownforaptamersE4(A),E7(B)andE10(C). Ó FEBS 2004 Aptamer probes of RNA polymerase structure (Eur. J. Biochem. 271) 4927 incubated with an excess of the corresponding unlabeled aptamers, they dissociated with half-life times of more than 1 h . T he dissociation k inetics of t he RNAP–aptamer complexes measured in the presence of MS, Rif (in case of RifS aptamers) o r the r-subunit followed the kinetics observed when the unlabeled aptamer was used as a competitor (Fig. 6). Control e xperiments 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 s tability of several RNAP–aptamer complexes (Fig. 6 and data not shown). In agreement with K d measurements, GreB d id not affect the stability of t he E7–RNAP complex (Fig. 6B). At the same time, when GreB w as 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 w as added b efore the aptamers (Fig. 5 B a nd data not shown). Specific and nonspecific interactions of aptamers with the RNAP main channel The interaction of the a ptamers with RNAP w as found to be highly dependent on the ionic s trength o f t he solution. At elevated ionic strength (440 m M ), 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 m M ), RNAP still bound the aptamers but sequence s pecificity w as apparently lost. Under these conditions all the sequences tested, including the random DNA libr ary, bound the core enzyme with equal affinities (K d  1n M ). MS suppressed the binding of all the oligonucleotides which suggested that the nonspecific bind- ing of s sDNA also oc curred a t RNAP sites involved in the interaction with RNA and DNA in the elongation complex. At elevated ionic strength, Rif a nd r 70 suppre ssed RNAP–aptamer interactions (above). Under low ionic strength conditions, Rif and r 70 hadnoeffectonthe 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 s trength. Discussion The principal result of this work is that the aptamers sense the interaction of RNAP with various ligands, including nucleic acids, an tibiotics and protein f actors. 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 r 70 -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 t he aptamers competed with MS for binding to RNAP and inhibited core polymerase activity. Binding of the aptamers w as affected by mutations at several sites in th e main channel t hat were previously implicated in the interactions with nuc leic acids in transcription c omplexes. F urthermore, the binding of 10 out of 13 aptamers was sensitive to Rif. As Rif does not cause any sign ificant 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 t he RNAP–aptamer complexes measured in the presence of MS and Rif followed thesametimecourseasthekineticsmeasuredinthe presence of the unlabeled aptamers. This indicated that these ligands acted by simple trapping of free RNAP and preventing reassociation o f the complexes. Thus, both MS and R if 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]. I n addition, the N-terminal region of r, which is not visible in the holoenzyme structure, was shown to occupy the downstream portion of the m ain channel [57]. The binding of r to the core polymerase causes repositioning of several structural modules of the core, including the c lamp, b1, b2 and flap domains, w hich results in partial c losure of the main channel [ 7]. T hus, 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 t he inhibitory effect of r is that it also directly blocks RNAP sites involved i n a ptamer 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 r 70 ,GreB binds RNAP from the s econdary channel side of t he enzyme, i.e. at the side opposite to the aptamers (see Fig. 1B) [15]. The binding site of the C-terminal do main of GreB near the entrance of the secondary channel is located outside of the enzyme’s catalytic cleft and s eems unlikely t o be involved in aptamer binding. The GreB N-terminal coiled-coil domain protrudes deep i nto the secondary channel, providing two conserved acidic residues which play a k ey role in the RNA cleavage reaction [15,16,58]. Based on these observations, one could s uggest 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 o f the secondary channel and directly interfere w ith GreB b inding. Alternatively, t he aptamers could sense GreB-induced conformational chan- ges inside the RNAP main channel. 4928 A. Kulbachinskiy et al. (Eur. J. Biochem. 271) Ó FEBS 2004 The 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 G reB 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 o f c urrent 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 se ems 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 o f 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 aptam ers could be used to study the conformational changes of RNAP induced by the b inding of these r egulatory factors. The aptamers could also be useful in studies of various RNAP mutations that are thought to change the conformation of th e enzyme. The examples of such mutations i nclude 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-pro tein med iated anti-termination ( pre- sumably by changing the conformation of the interior of the main channel) [63]. It should be noted that such hypothetical conformational c hanges of RNAP are usually very difficult to verify. 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Mol Cell 14, 753–762 62 Nickels, B.E & Hochschild, A (2004) Regulation of RNA polymerase through the secondary channel Cell 118, 281–284 63 Santangelo, T.J., Mooney, R.A., Landick, R & Roberts, J.W (2003) RNA polymerase mutations that impair conversion to a termination-resistant complex by Q antiterminator proteins Genes Dev 17, 1281–1292 . Aptamers to Escherichia coli core RNA polymerase that sense its interaction with rifampicin, r-subunit and GreB Andrey Kulbachinskiy 1,2 , Andrey. 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

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