Specific Recognition of the -10 Promoter Element by the Free RNA Polymerase σ Subunit
2007; Elsevier BV; Volume: 282; Issue: 30 Linguagem: Inglês
10.1074/jbc.m702495200
ISSN1083-351X
AutoresAnastasiya Sevostyanova, Andrey Feklístov, Nataliya Barinova, Ewa Heyduk, I. A. Bass, Saulius Klimašauskas, Tomasz Heyduk, Andrey Kulbachinskiy,
Tópico(s)Bacterial Genetics and Biotechnology
ResumoBacterial RNA polymerase holoenzyme relies on its σ subunit for promoter recognition and opening. In the holoenzyme, regions 2 and 4 of the σ subunit are positioned at an optimal distance to allow specific recognition of the -10 and -35 promoter elements, respectively. In free σ, the promoter binding regions are positioned closer to each other and are masked for interactions with the promoter, with σ region 1 playing a role in the masking. To analyze the DNA-binding properties of the free σ, we selected single-stranded DNA aptamers that are specific to primary σ subunits from several bacterial species, including Escherichia coli and Thermus aquaticus. The aptamers share a consensus motif, TGTAGAAT, that is similar to the extended -10 promoter. We demonstrate that recognition of this motif by σ region 2 occurs without major structural rearrangements of σ observed upon the holoenzyme formation and is not inhibited by σ regions 1 and 4. Thus, the complex process of the -10 element recognition by RNA polymerase holoenzyme can be reduced to a simple system consisting of an isolated σ subunit and a short aptamer oligonucleotide. Bacterial RNA polymerase holoenzyme relies on its σ subunit for promoter recognition and opening. In the holoenzyme, regions 2 and 4 of the σ subunit are positioned at an optimal distance to allow specific recognition of the -10 and -35 promoter elements, respectively. In free σ, the promoter binding regions are positioned closer to each other and are masked for interactions with the promoter, with σ region 1 playing a role in the masking. To analyze the DNA-binding properties of the free σ, we selected single-stranded DNA aptamers that are specific to primary σ subunits from several bacterial species, including Escherichia coli and Thermus aquaticus. The aptamers share a consensus motif, TGTAGAAT, that is similar to the extended -10 promoter. We demonstrate that recognition of this motif by σ region 2 occurs without major structural rearrangements of σ observed upon the holoenzyme formation and is not inhibited by σ regions 1 and 4. Thus, the complex process of the -10 element recognition by RNA polymerase holoenzyme can be reduced to a simple system consisting of an isolated σ subunit and a short aptamer oligonucleotide. The σ subunit of bacterial RNA polymerase (RNAP) 4The abbreviations used are: RNAP, RNA polymerase; WT, wild-type; ssDNA, single-stranded DNA; sTap, sigma T. aquaticus aptamer; sEcap, sigma E. coli aptamer.4The abbreviations used are: RNAP, RNA polymerase; WT, wild-type; ssDNA, single-stranded DNA; sTap, sigma T. aquaticus aptamer; sEcap, sigma E. coli aptamer. holoenzyme plays a key role in promoter recognition and opening. Most promoters of housekeeping genes are recognized by a holoenzyme containing the primary σ subunit, called σ70 in Escherichia coli or σA in most other bacterial species. Primary σ subunits from different bacteria share four conserved regions each consisting of two or more subregions. Conserved regions 2.4, 3.0, and 4.2 of the σ subunit have been implicated in specific recognition of the -10 (consensus sequence TATAAT), the extended -10 (TG), and the -35 (TTGACA) promoter elements, respectively (1Gross C.A. Chan C. Dombroski A. Gruber T. Sharp M. Tupy J. Young B. Cold Spring Harbor Symp. Quant. Biol. 1998; 63: 141-155Crossref PubMed Scopus (294) Google Scholar). Interactions of σ regions 2.3 and 2.4 with the nontemplate DNA strand at the -10 element have been shown to play a crucial role in RNAP-induced promoter melting and stabilization of the open promoter complex (1Gross C.A. Chan C. Dombroski A. Gruber T. Sharp M. Tupy J. Young B. Cold Spring Harbor Symp. Quant. Biol. 1998; 63: 141-155Crossref PubMed Scopus (294) Google Scholar, 2Juang Y.L. Helmann J.D. J. Mol. Biol. 1994; 235: 1470-1488Crossref PubMed Scopus (143) Google Scholar, 3Fenton M.S. 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Crystal structures of holo-RNAPs from Thermus aquaticus and Thermus thermophilus revealed that σ contains three domains (σ2, σ3, and σ4, named after corresponding conserved regions) connected by flexible linkers (13Murakami K.S. Masuda S. Darst S.A. Science. 2002; 296: 1280-1284Crossref PubMed Scopus (445) Google Scholar, 14Vassylyev D.G. Sekine S. Laptenko O. Lee J. Vassylyeva M.N. Borukhov S. Yokoyama S. Nature. 2002; 417: 712-719Crossref PubMed Scopus (625) Google Scholar). In holoenzyme, these domains are spread on the core enzyme surface, with σ2 and σ4 being positioned at an optimal distance relative to each other to allow interactions with the -10 and -35 promoter elements. The structure of the isolated individual domains of σ is almost identical to their structures in holo-RNAP (13Murakami K.S. Masuda S. Darst S.A. Science. 2002; 296: 1280-1284Crossref PubMed Scopus (445) Google Scholar, 14Vassylyev D.G. Sekine S. Laptenko O. Lee J. Vassylyeva M.N. Borukhov S. Yokoyama S. Nature. 2002; 417: 712-719Crossref PubMed Scopus (625) Google Scholar, 15Severinova E. Severinov K. Fenyo D. Marr M. Brody E.N. Roberts J.W. Chait B.T. Darst S.A. J. Mol. Biol. 1996; 263: 637-647Crossref PubMed Scopus (121) Google Scholar, 16Campbell E.A. Muzzin O. Chlenov M. Sun J.L. Olson C.A. Weinman O. Trester-Zedlitz M.L. Darst S.A. Mol. Cell. 2002; 9: 527-539Abstract Full Text Full Text PDF PubMed Scopus (388) Google Scholar). However, the structure of the full-length σ subunit in a free state remains unknown. Based on indirect biochemical and biophysical data, several mechanisms of inhibition of DNA-binding activity in free σ have been proposed. First, the N-terminal region of σ (region 1.1) was shown to inhibit DNA binding. Deletion of this region allows σ70 to weakly bind double-stranded promoter DNA with some specificity (17Dombroski A.J. Walter W.A. Record Jr., M.T. Siegele D.A. Gross C.A. Cell. 1992; 70: 501-512Abstract Full Text PDF PubMed Scopus (276) Google Scholar, 18Dombroski A.J. Walter W.A. Gross C.A. Genes Dev. 1993; 7: 2446-2455Crossref PubMed Scopus (182) Google Scholar) but does not allow the recognition of single-stranded nontemplate oligonucleotides by free σ (15Severinova E. Severinov K. Fenyo D. Marr M. Brody E.N. Roberts J.W. Chait B.T. Darst S.A. J. Mol. Biol. 1996; 263: 637-647Crossref PubMed Scopus (121) Google Scholar, 19Dombroski A.J. J. Biol. Chem. 1997; 272: 3487-3494Abstract Full Text Full Text PDF PubMed Google Scholar). It was proposed that region 1.1 may inhibit DNA binding by (i) direct masking of σ region 4 (17Dombroski A.J. Walter W.A. Record Jr., M.T. Siegele D.A. Gross C.A. Cell. 1992; 70: 501-512Abstract Full Text PDF PubMed Scopus (276) Google Scholar, 18Dombroski A.J. Walter W.A. Gross C.A. Genes Dev. 1993; 7: 2446-2455Crossref PubMed Scopus (182) Google Scholar), (ii) direct interactions with σ region 2 (20Gopal V. Chatterji D. Eur. J. Biochem. 1997; 244: 613-618Crossref PubMed Scopus (17) Google Scholar, 21Gowrishankar J. Yamamoto K. Subbarayan P.R. Ishihama A. J. Bacteriol. 2003; 185: 2673-2679Crossref PubMed Scopus (20) Google Scholar), or (iii) by an indirect allosteric mechanism (22Camarero J.A. Shekhtman A. Campbell E.A. Chlenov M. Gruber T.M. Bryant D.A. Darst S.A. Cowburn D. Muir T.W. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 8536-8541Crossref PubMed Scopus (104) Google Scholar). Second, it was shown that free σ adopts a compact conformation in which DNA-recognition domains σ2 and σ4 are brought closer to each other than in the holoenzyme (23Callaci S. Heyduk E. Heyduk T. Mol. Cell. 1999; 3: 229-238Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). The sub-optimal positioning of σ2 and σ4 is likely to interfere with simultaneous recognition of the -10 and -35 elements. Furthermore, the close proximity of σ2 and σ4 to each other and to other protein parts may directly mask DNA binding sites in free σ (24Sorenson M.K. Ray S.S. Darst S.A. Mol. Cell. 2004; 14: 127-138Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 25Sorenson M.K. Darst S.A. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 16722-16727Crossref PubMed Scopus (22) Google Scholar, 26Feklistov A. Barinova N. Sevostyanova A. Heyduk E. Bass I. Vvedenskaya I. Kuznedelov K. Merkiene E. Stavrovskaya E. Klimasauskas S. Nikiforov V. Heyduk T. Severinov K. Kulbachinskiy A. Mol. Cell. 2006; 23: 97-107Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Finally, it was proposed that some local differences in the structure of the DNA binding domains in free σ and in holo-RNAP may also be associated with the differences in DNA binding properties of the protein (12Callaci S. Heyduk T. Biochemistry. 1998; 37: 3312-3320Crossref PubMed Scopus (46) Google Scholar, 27Callaci S. Heyduk E. Heyduk T. J. Biol. Chem. 1998; 273: 32995-33001Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). However, because the structure of free σ is unknown, the exact nature of the coreinduced rearrangements and the mechanism of inhibition of DNA binding by free σ are unclear. Previously, we characterized ssDNA aptamers that contained the -10 promoter element and were recognized specifically by the free σA subunit from T. aquaticus (sTaps, for sigma T. aquaticus aptamers) (26Feklistov A. Barinova N. Sevostyanova A. Heyduk E. Bass I. Vvedenskaya I. Kuznedelov K. Merkiene E. Stavrovskaya E. Klimasauskas S. Nikiforov V. Heyduk T. Severinov K. Kulbachinskiy A. Mol. Cell. 2006; 23: 97-107Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). It was shown that the binding of sTaps is accompanied by a conformational change in σA and leads to an increase in the distance between domains σ2 and σ4. It was concluded that (i) the isolated σA subunit has all determinants required for specific recognition of the -10 promoter element, and (ii) the increase in the σ2-σ4 distance may be required for the unmasking of the DNA binding sites of σA. However, it remained unknown whether free σs from other bacterial species can also recognize promoter sequences and whether this requires similar conformational changes in the protein. In this work, we describe a novel class of aptamers that contain motifs similar to the -10 and TG promoter elements and are recognized efficiently by σ subunits from various bacterial species. We demonstrate that the aptamers interact with σ region 2 and that this interaction does not require major conformational changes of σ, which has been observed upon the holoenzyme formation (23Callaci S. Heyduk E. Heyduk T. Mol. Cell. 1999; 3: 229-238Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar) and sTap binding (26Feklistov A. Barinova N. Sevostyanova A. Heyduk E. Bass I. Vvedenskaya I. Kuznedelov K. Merkiene E. Stavrovskaya E. Klimasauskas S. Nikiforov V. Heyduk T. Severinov K. Kulbachinskiy A. Mol. Cell. 2006; 23: 97-107Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). The aptamer binding is also not inhibited by σ regions 1 and 4. Thus, specific DNA recognition by σ region 2 can occur without large structural changes of the σ subunit. Proteins—Wild-type and mutant E. coli σ70, T. aquaticus, and Deinococcus radiodurans σA were obtained as described (26Feklistov A. Barinova N. Sevostyanova A. Heyduk E. Bass I. Vvedenskaya I. Kuznedelov K. Merkiene E. Stavrovskaya E. Klimasauskas S. Nikiforov V. Heyduk T. Severinov K. Kulbachinskiy A. Mol. Cell. 2006; 23: 97-107Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 28Kulbachinskiy A. Bass I. Bogdanova E. Goldfarb A. Nikiforov V. J. Bacteriol. 2004; 186: 7818-7820Crossref PubMed Scopus (24) Google Scholar, 29Zenkin N. Kulbachinskiy A. Yuzenkova Y. Mustaev A. Bass I. Severinov K. Brodolin K. EMBO J. 2007; 26: 955-964Crossref PubMed Scopus (42) Google Scholar). The fragment of the rpoD gene encoding for amino acids 1-565 of E. coli σ70 was amplified from genomic DNA and cloned between NdeI and EcoRI sites of pET28. The protein was overexpressed in E. coli BL21(DE3) and purified as described (29Zenkin N. Kulbachinskiy A. Yuzenkova Y. Mustaev A. Bass I. Severinov K. Brodolin K. EMBO J. 2007; 26: 955-964Crossref PubMed Scopus (42) Google Scholar). Selection of Aptamers to E. coli σ70—The selection of aptamers was performed essentially as described previously (26Feklistov A. Barinova N. Sevostyanova A. Heyduk E. Bass I. Vvedenskaya I. Kuznedelov K. Merkiene E. Stavrovskaya E. Klimasauskas S. Nikiforov V. Heyduk T. Severinov K. Kulbachinskiy A. Mol. Cell. 2006; 23: 97-107Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). The library used for the selection was 5′-GGGAGCTCAGAATAAACGCTCAA-32N-TTCGACATGAGGCCCGGATC, where N is a random nucleotide. The selection was performed in a buffer containing 20 mm Tris-HCl, pH 7.9, 10 mm MgCl2, 240 mm NaCl, and 60 mm KCl. The amounts of ssDNA and the σ70 subunit were varied from 3 nmol and 500 pmol, respectively, in the first round of selection, to 100 and 10 pmol in subsequent rounds. Selection was performed using N-terminally hexahistidine-tagged σ70. σ70 and DNA were incubated for 30 min in 1 ml of the binding buffer, nickel-nitrilotriacetic acid-agarose (Qiagen) was added, unbound DNA was removed by extensive washing, and σ-DNA complexes were eluted with binding buffer containing 200 mm imidazole. The eluted DNA was amplified using primers corresponding to the fixed regions of the library (5′-GGGAGCTCAGAATAAACGCTCAA and BBB-5′-GATCCGGGCCTCATGTCGAA, where B is a biotin residue), DNA strands were separated by size on 10% denaturing PAGE, and the nonbiotinylated strand was purified and used for the next round of selection. After ten rounds, the enriched library was amplified with primers containing EcoRI and HindIII sites and cloned into the pUC19 plasmid. Individual aptamers were obtained by PCR or purchased from Syntol (Moscow). Analysis of σ-Aptamer Interactions—Determination of equilibrium Kd values for binding of aptamers to the σ subunit was done by the nitrocellulose binding method (30Carey J. Cameron V. de Haseth P.L. Uhlenbeck O.C. Biochemistry. 1983; 22: 2601-2610Crossref PubMed Scopus (203) Google Scholar). Oligonucleotides were 5′-end-labeled with [γ-32P]ATP (6000 Ci/mmol, PerkinElmer Life Sciences) and T4 polynucleotide kinase (New England Biolabs) and purified by PAGE. Each aptamer (0.1 nm) was incubated with a series of dilutions of the σ subunit or its fragments (from 1 nm to 3 μm) in 50 μl of binding buffer for 30 min at room temperature; the samples were filtered through 0.45-μm nitrocellulose filters (HAWP, Millipore), and the filters were washed with 5 ml of the buffer and quantified with PhosphorImager (GE Healthcare). To calculate apparent equilibrium dissociation constants, the data were fit to a hyperbolic equation, B = Bmax*[σ]/([σ] + Kd), where B is a percentage of DNA bound, Bmax is the maximum binding at infinite concentration of σ, and Kd is the dissociation constant. The fitting was performed by nonlinear regression algorithm using GraFit (Erithacus Software). For each aptamer-σ combination, Kd measurements were independently repeated two to three times, and averages were calculated. The experimental variation among replicate measurements usually did not exceed 30% of the average value. Cross-linking Experiments—Cross-linking was performed in 15 μl of the binding buffer. The samples contained 10 nm of 5′-labeled sEcap1 or nontemplate oligonucleotide and 100 nm σ70. The nontemplate oligonucleotide was closely related to the nontemplate strand of a lacUV5 promoter: 5′-ATTGCGTATAATGTGTGGA. The samples were incubated for 15 min at 25 °C, irradiated for 10 min under a 254 nm UV lamp (4 watts, Spectroline), and separated on 5% SDS-PAGE. For the mapping of the cross-linking sites, the DNA-protein complexes were eluted with 400 μl of 0.03% SDS, freeze-dried, and dissolved in 20 μl of 50mm HCl. 10-μl aliquots were withdrawn, and 1 m CNBr was added to the remaining samples to 50 mm. The reaction was stopped after 5 min by adding an equal volume of stop buffer containing 2% SDS, 0.5 m Tris-HCl, pH 8.4, 100 mm β-mercaptoethanol, and 20% glycerol. The degradation products were separated on 13% SDS-PAGE. Luminescence Resonance Energy Transfer Distance Measurements—The double-cysteine mutant of σ70 was labeled with europium chelate and Cy5 and purified as described previously (23Callaci S. Heyduk E. Heyduk T. Mol. Cell. 1999; 3: 229-238Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Distance measurements were conducted at 25 °C as described (23Callaci S. Heyduk E. Heyduk T. Mol. Cell. 1999; 3: 229-238Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 26Feklistov A. Barinova N. Sevostyanova A. Heyduk E. Bass I. Vvedenskaya I. Kuznedelov K. Merkiene E. Stavrovskaya E. Klimasauskas S. Nikiforov V. Heyduk T. Severinov K. Kulbachinskiy A. Mol. Cell. 2006; 23: 97-107Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 31Heyduk E. Heyduk T. Biochemistry. 2002; 41: 2876-2883Crossref PubMed Scopus (6) Google Scholar). σ70 was taken at 25 nm. Oligonucleotides and the E. coli core RNAP were added to 100 and 60 nm, when present, respectively. The control oligonucleotide that did not bind σ70 was an sTap1 aptamer described previously (26Feklistov A. Barinova N. Sevostyanova A. Heyduk E. Bass I. Vvedenskaya I. Kuznedelov K. Merkiene E. Stavrovskaya E. Klimasauskas S. Nikiforov V. Heyduk T. Severinov K. Kulbachinskiy A. Mol. Cell. 2006; 23: 97-107Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar): 5′-GAGTGTATAATGGGAGCGGTATCGTTCGACATGAG. Structural Features of Aptamers Recognized by the σ70 Subunit of E. coli RNA Polymerase—As the primary target for aptamer selection, we chose the σ70 subunit from E. coli, which has a well defined promoter specificity and has been used as a model to study the process of promoter recognition for many years (1Gross C.A. Chan C. Dombroski A. Gruber T. Sharp M. Tupy J. Young B. Cold Spring Harbor Symp. Quant. Biol. 1998; 63: 141-155Crossref PubMed Scopus (294) Google Scholar). ssDNA aptamers were selected from a library of 75-nucleotide-long ssDNA containing a 32-nucleotide central region of random sequence surrounded by regions with fixed sequences. The initial ssDNA library showed poor affinity for σ70 (apparent Kd > 10 μm). Control experiments demonstrated that free σ70 also did not bind nontemplate oligonucleotides containing the -10 promoter element (Kd > 10 μm). After 10 rounds of selection, the affinity of the enriched library significantly increased (Kd ∼ 85 nm). The library was cloned, and insert sequences of 30 individual clones were determined. The resulting sequences were named sEcaps (for sigma E. coli aptamer) (Fig. 1A). All the aptamers exhibited high affinity for σ70 and bound it with Kd values ranging from 30 to 100 nm. For several clones, it was possible to minimize the size of the aptamers by removing the whole fixed library regions without decreasing the aptamer affinity. One of the minimal aptamers, sEcap1, had even higher affinity to σ70 (Kd ∼ 20 nm) than the full-length variant and was chosen for further studies. The majority of the aptamers share the octanucleotide sequence TGTAGAAT, which is located at different positions within the central region of the library (Fig. 1A). In this sequence, the underlined motif is similar to the bacterial primary σ -10 promoter element but contains G instead of T at the third position. The dinucleotide TG located to the left of the -10-like sequence matches the TG motif of the extended -10 promoters (see below). In all the aptamers, the conserved 8-nucleotide motif is preceded by G-rich regions, which can potentially form G-quadruplexes. The G-quartet structure is important for aptamer binding, because aptamer variants with substitutions in this region did not interact with σ. The binding of the aptamers also depended on the presence of potassium ions, which are known to stabilize G-quartets (data not shown). The proposed secondary structure of sEcap1 is shown on Fig. 1B. In the structure, the G-quadruplex is followed by a single-stranded region containing the conserved aptamer motif. Although aptamer ligands are usually highly specific to their target proteins (32Gold L. Polisky B. Uhlenbeck O. Yarus M. Annu. Rev. Biochem. 1995; 64: 763-797Crossref PubMed Scopus (742) Google Scholar), we found that the aptamers selected in this work were recognized with equally high efficiencies by σ subunits from various bacterial species, including T. aquaticus and D. radiodurans (Kd values for sEcap1 were 9.5 and 6.4 nm, respectively). This indicated that the aptamers interact with a highly conserved epitope of σ. Aptamers Interact with σ70 Region 2—The presence of the conserved -10-like element in sEcaps suggested that it is essential for aptamer binding and is likely recognized by σ conserved region 2, similarly to the -10 element in promoters. Indeed, a mutant variant of sEcap1 bearing an ATCTCG sequence instead of the -10-like motif did not bind σ70 (data not shown). Adenine at the second position of the -10 element (-11A) is the most conserved promoter base and was previously shown to be critical for promoter recognition and opening (33Lim H.M. Lee H.J. Roy S. Adhya S. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14849-14852Crossref PubMed Scopus (58) Google Scholar). We found that a substitution of C for A at this position of the -10-like motif in sEcap1 completely abolished aptamer binding (Kd > 3 μm). Interestingly, an sEcap1 mutant bearing the perfect -10 element consensus (TATAAT) also exhibited poor binding to σ (Kd > 1 μm) indicating that the -10 element in sEcaps is recognized by free σ70 with a somewhat different specificity than the -10 promoter element by holo-RNAP. At the same time, changes at other positions of the TAGAAT motif had less dramatic effect on aptamer recognition (Kd for aptamer variants with point nucleotide changes at the first, fourth, and fifth positions of this motif ranged from 20 to 80 nm). To localize the σ region(s) involved in interactions with the aptamer, we performed cross-linking of σ70 with radioactively labeled sEcap1. Irradiation of the σ70-sEcap1 complex with UV light at 254 nm resulted in the appearance of a cross-linked DNA-protein complex that had lower mobility than free DNA on a denaturing gel (Fig. 2A, lane 1). The cross-link was highly specific, because its efficiency was dramatically reduced in the case of the sEcap1 variant that contained the -11A to C mutation (Fig. 2A, lane 2). For mapping of the cross-linking sites in the σ70 subunit we used a method of limited chemical cleavage of modified protein at Met residues (34Grachev M.A. Lukhtanov E.A. Mustaev A.A. Zaychikov E.F. Abdukayumov M.N. Rabinov I.V. Richter V.I. Skoblov Y.S. Chistyakov P.G. Eur. J. Biochem. 1989; 180: 577-585Crossref PubMed Scopus (80) Google Scholar, 35Mustaev A. Zaychikov E. Grachev M. Kozlov M. Severinov K. Epshtein V. Korzheva N. Bereshchenko O. Markovtsov V. Lukhtanov E. Tsarev I. Maximova T. Kashlev M. Bass I. Nikiforov V. Goldfarb A. Methods Enzymol. 2003; 371: 191-206Crossref PubMed Scopus (9) Google Scholar). In a control reaction, we performed cross-linking of σ70 in a complex of E. coli holo-RNAP with a single-stranded nontemplate oligonucleotide containing the -10 element (Fig. 2B). The cross-linking site of this oligonucleotide was previously mapped between σ70 Met residues 413 and 456 (9Kulbachinskiy A. Mustaev A. Goldfarb A. Nikiforov V. FEBS Lett. 1999; 454: 71-74Crossref PubMed Scopus (16) Google Scholar). The treatment of σ70, radioactively labeled with sEcap1, with BrCN in single-hit conditions resulted in the formation of a characteristic pattern of radiolabeled peptides corresponding to the C terminus of the protein (Fig. 2B, lane 3). This pattern was identical to that observed in the case of the control nontemplate oligonucleotide (lane 2). The shortest labeled peptide corresponded to cleavage at Met-413. Shorter peptides resulting from cleavage at and C-terminal to Met-456 were not detected on the autograph and, therefore, did not contain the site of modification (Fig. 2B, see also Ref. 9Kulbachinskiy A. Mustaev A. Goldfarb A. Nikiforov V. FEBS Lett. 1999; 454: 71-74Crossref PubMed Scopus (16) Google Scholar). This indicated that the site of cross-linking is located between Met-413 and Met-456. This region contains conserved regions 2.3 and 2.4 of σ70, which have been previously implicated in the recognition of the -10 element in promoters. σ70 Regions 1, 3, and 4 Are Not Important for Aptamer Recognition—To check whether other parts of σ70, besides region 2, are important for aptamer binding, we analyzed interactions of sEcap1 with three σ fragments, encompassing amino acids 1-565, 1-448, and 102-448 of σ70 (Table 1). The first and the second of the fragments lacked conserved region 4.2 and regions 3 and 4, respectively. The third fragment lacked regions 3 and 4 and the N-terminal segment, including region 1.1 (amino acids 1-93) and the first 8 amino acids from region 1.2, and corresponded to structural domain σ2, which is conserved in all σ70-family proteins (15Severinova E. Severinov K. Fenyo D. Marr M. Brody E.N. Roberts J.W. Chait B.T. Darst S.A. J. Mol. Biol. 1996; 263: 637-647Crossref PubMed Scopus (121) Google Scholar, 16Campbell E.A. Muzzin O. Chlenov M. Sun J.L. Olson C.A. Weinman O. Trester-Zedlitz M.L. Darst S.A. Mol. Cell. 2002; 9: 527-539Abstract Full Text Full Text PDF PubMed Scopus (388) Google Scholar) and whose structure has been solved previously (36Malhotra A. Severinova E. Darst S.A. Cell. 1996; 87: 127-136Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar). We found that the affinities of all three fragments to sEcap1 did not differ significantly from the affinity of the full-length σ70 (Table 1). All fragments recognized sEcap1 specifically, because the -11C mutation abolished aptamer binding (Kd > 3 μm). This indicates that the presence of regions 1, 3, and 4 in full-length σ70 is neither required for nor inhibitory to aptamer binding.TABLE 1Recognition of sEcap1 and its mutants by E. coli σ70 and T. aquaticus σAσ SubunitAptamer KdTGTAGAATTGCTAGAATCCTAGAATnmσ701-613 (WT)aWT, full-length wild type σ subunit.18.7 ± 5.669.5 ± 15.3>30001-56529.3 ± 6.7NDbND, not determined.>30001-44822.7 ± 7.389.0 ± 20.7>3000102-44817.6 ± 6.5ND>3000σAWT9.5 ± 3.1ND>3000Q260H20.0 ± 5.3ND65.9 ± 21.3a WT, full-length wild type σ subunit.b ND, not determined. Open table in a new tab The TG Element in Aptamers Is Recognized by σ Region 2.4—The TG motif present in all the aptamers resembles the TG element of the extended -10 promoters but, in contrast to the promoters, is located immediately upstream of the -10-like sequence. To check whether the position of TG is essential for aptamer recognition, we studied an sEcap1 mutant, which contained an additional cytosine nucleotide inserted between the TG motif and the -10-like element ("TGC sEcap1"). This aptamer was recognized by σ70 with almost as high affinity as wild-type sEcap1 (Kd ∼ 70 nm, Table 1 and Fig. 3A). At the same time, an aptamer mutant in which the TG motif was substituted by CC ("CC sEcap1") did not bind σ (Kd > 3 μm, Fig. 3A). Therefore, the TG motif is essential for aptamer binding and can be specifically recognized by σ70 when located at the same position as in the extended -10 promoters. The observed difference in the TG position in the aptamers and promoters likely reflects some differences in conformations of σ and/or DNA in the σ-sEcap complex and in the holoenzyme RNAP-promoter complex during transcription initiation. Suppression genetics studies initially suggested that TG in the extended -10 promoters is recognized by region 3.0 of σ70 (37Barne K.A. Bown J.A. Busby S.J. Minchin S.D. EMBO J. 1997; 16: 4034-4040Crossref PubMed Scopus (231) Google Scholar). Later, region 2.4 was also shown to play a role in the recognition of this motif (38Sanderson A. Mitchell J.E. Minchin S.D. Busby S.J. FEBS Lett. 2003; 544: 199-205Crossref PubMed Scopus (37) Google Scholar). We therefore used aptamers as model DNA substrates to analyze the roles of these σ regions in
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