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Originally published In Press as doi:10.1074/jbc.M702495200 on May 29, 2007

J. Biol. Chem., Vol. 282, Issue 30, 22033-22039, July 27, 2007
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Specific Recognition of the -10 Promoter Element by the Free RNA Polymerase {sigma} Subunit*

Anastasiya Sevostyanova{ddagger}1, Andrey Feklistov{ddagger}2, Nataliya Barinova{ddagger}, Ewa Heyduk§, Irina Bass{ddagger}, Saulius Klimasauskas, Tomasz Heyduk§, and Andrey Kulbachinskiy{ddagger}3

From the {ddagger}Institute of Molecular Genetics, Russian Academy of Sciences, Kurchatov Sq., 2, Moscow 123182, Russia, the §Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, St. Louis, Missouri, 63104, and the Institute of Biotechnology, Vilnius 02241, Lithuania

Received for publication, March 22, 2007 , and in revised form, May 25, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Bacterial RNA polymerase holoenzyme relies on its {sigma} subunit for promoter recognition and opening. In the holoenzyme, regions 2 and 4 of the {sigma} subunit are positioned at an optimal distance to allow specific recognition of the -10 and -35 promoter elements, respectively. In free {sigma}, the promoter binding regions are positioned closer to each other and are masked for interactions with the promoter, with {sigma} region 1 playing a role in the masking. To analyze the DNA-binding properties of the free {sigma}, we selected single-stranded DNA aptamers that are specific to primary {sigma} 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 {sigma} region 2 occurs without major structural rearrangements of {sigma} observed upon the holoenzyme formation and is not inhibited by {sigma} 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 {sigma} subunit and a short aptamer oligonucleotide.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The {sigma} subunit of bacterial RNA polymerase (RNAP)4 holoenzyme plays a key role in promoter recognition and opening. Most promoters of housekeeping genes are recognized by a holoenzyme containing the primary {sigma} subunit, called {sigma}70 in Escherichia coli or {sigma}A in most other bacterial species. Primary {sigma} 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 {sigma} subunit have been implicated in specific recognition of the -10 (consensus sequence TATAAT), the extended -10 (TG), and the -35 (TTGACA) promoter elements, respectively (1). Interactions of {sigma} 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 (1-5). Recognition of the -10 element can be modeled in a complex of holo-RNAP with short oligonucleotides corresponding to the nontemplate promoter strand and containing the -10 sequence (nontemplate oligonucleotides) (6-9). However, despite playing a crucial role in promoter recognition by holo-RNAP, free {sigma} is unable to specifically recognize either promoters or nontemplate oligonucleotides (1, 10-12).

Crystal structures of holo-RNAPs from Thermus aquaticus and Thermus thermophilus revealed that {sigma} contains three domains ({sigma}2, {sigma}3, and {sigma}4, named after corresponding conserved regions) connected by flexible linkers (13, 14). In holoenzyme, these domains are spread on the core enzyme surface, with {sigma}2 and {sigma}4 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 {sigma} is almost identical to their structures in holo-RNAP (13-16). However, the structure of the full-length {sigma} subunit in a free state remains unknown.

Based on indirect biochemical and biophysical data, several mechanisms of inhibition of DNA-binding activity in free {sigma} have been proposed. First, the N-terminal region of {sigma} (region 1.1) was shown to inhibit DNA binding. Deletion of this region allows {sigma}70 to weakly bind double-stranded promoter DNA with some specificity (17, 18) but does not allow the recognition of single-stranded nontemplate oligonucleotides by free {sigma} (15, 19). It was proposed that region 1.1 may inhibit DNA binding by (i) direct masking of {sigma} region 4 (17, 18), (ii) direct interactions with {sigma} region 2 (20, 21), or (iii) by an indirect allosteric mechanism (22).

Second, it was shown that free {sigma} adopts a compact conformation in which DNA-recognition domains {sigma}2 and {sigma}4 are brought closer to each other than in the holoenzyme (23). The sub-optimal positioning of {sigma}2 and {sigma}4 is likely to interfere with simultaneous recognition of the -10 and -35 elements. Furthermore, the close proximity of {sigma}2 and {sigma}4 to each other and to other protein parts may directly mask DNA binding sites in free {sigma} (24-26). Finally, it was proposed that some local differences in the structure of the DNA binding domains in free {sigma} and in holo-RNAP may also be associated with the differences in DNA binding properties of the protein (12, 27). However, because the structure of free {sigma} is unknown, the exact nature of the coreinduced rearrangements and the mechanism of inhibition of DNA binding by free {sigma} are unclear.

Previously, we characterized ssDNA aptamers that contained the -10 promoter element and were recognized specifically by the free {sigma}A subunit from T. aquaticus (sTaps, for sigma T. aquaticus aptamers) (26). It was shown that the binding of sTaps is accompanied by a conformational change in {sigma}A and leads to an increase in the distance between domains {sigma}2 and {sigma}4. It was concluded that (i) the isolated {sigma}A subunit has all determinants required for specific recognition of the -10 promoter element, and (ii) the increase in the {sigma}2-{sigma}4 distance may be required for the unmasking of the DNA binding sites of {sigma}A. However, it remained unknown whether free {sigma}s 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 {sigma} subunits from various bacterial species. We demonstrate that the aptamers interact with {sigma} region 2 and that this interaction does not require major conformational changes of {sigma}, which has been observed upon the holoenzyme formation (23) and sTap binding (26). The aptamer binding is also not inhibited by {sigma} regions 1 and 4. Thus, specific DNA recognition by {sigma} region 2 can occur without large structural changes of the {sigma} subunit.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Proteins—Wild-type and mutant E. coli {sigma}70, T. aquaticus, and Deinococcus radiodurans {sigma}A were obtained as described (26, 28, 29). The fragment of the rpoD gene encoding for amino acids 1-565 of E. coli {sigma}70 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 (29).

Selection of Aptamers to E. coli {sigma}70—The selection of aptamers was performed essentially as described previously (26). 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 {sigma}70 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 {sigma}70. {sigma}70 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 {sigma}-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 {sigma}-Aptamer Interactions—Determination of equilibrium Kd values for binding of aptamers to the {sigma} subunit was done by the nitrocellulose binding method (30). Oligonucleotides were 5'-end-labeled with [{gamma}-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 {sigma} 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*[{sigma}]/([{sigma}] + Kd), where B is a percentage of DNA bound, Bmax is the maximum binding at infinite concentration of {sigma}, and Kd is the dissociation constant. The fitting was performed by nonlinear regression algorithm using GraFit (Erithacus Software). For each aptamer-{sigma} 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 {sigma}70. 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 beta-mercaptoethanol, and 20% glycerol. The degradation products were separated on 13% SDS-PAGE.

Luminescence Resonance Energy Transfer Distance Measurements—The double-cysteine mutant of {sigma}70 was labeled with europium chelate and Cy5 and purified as described previously (23). Distance measurements were conducted at 25 °C as described (23, 26, 31). {sigma}70 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 {sigma}70 was an sTap1 aptamer described previously (26): 5'-GAGTGTATAATGGGAGCGGTATCGTTCGACATGAG.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Structural Features of Aptamers Recognized by the {sigma}70 Subunit of E. coli RNA Polymerase—As the primary target for aptamer selection, we chose the {sigma}70 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 (1). 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 {sigma}70 (apparent Kd > 10 µM). Control experiments demonstrated that free {sigma}70 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 {sigma}70 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 {sigma}70 (Kd ~ 20 nM) than the full-length variant and was chosen for further studies.


Figure 1
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  		FIGURE 1.
Aptamers against the {sigma}70 subunit of E. coli RNAP. A, representative sequences and the consensus (bottom line) of the variable central region of {sigma}70-specific aptamers are shown. The -10-like element is highlighted dark gray, the TG motif is light gray. B, proposed secondary structure of sEcap1. The conserved aptamer motif is boxed.

 
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 {sigma} -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 {sigma}. 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 (32), we found that the aptamers selected in this work were recognized with equally high efficiencies by {sigma} 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 {sigma}.

Aptamers Interact with {sigma}70 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 {sigma} 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 {sigma}70 (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 (33). 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 {sigma} (Kd > 1 µM) indicating that the -10 element in sEcaps is recognized by free {sigma}70 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 {sigma} region(s) involved in interactions with the aptamer, we performed cross-linking of {sigma}70 with radioactively labeled sEcap1. Irradiation of the {sigma}70-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 {sigma}70 subunit we used a method of limited chemical cleavage of modified protein at Met residues (34, 35). In a control reaction, we performed cross-linking of {sigma}70 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 {sigma}70 Met residues 413 and 456 (9). The treatment of {sigma}70, 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. 9). 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 {sigma}70, which have been previously implicated in the recognition of the -10 element in promoters.


Figure 2
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  		FIGURE 2.
sEcap1 interacts with region 2 of{sigma}70. A, UV-induced cross-linking of wild-type (WT) and the -11C mutant sEcap1 to {sigma}70. The cross-linked complexes were separated on 7% SDS-PAGE and visualized by autoradiography. B, mapping of the cross-linking site in {sigma}70. The cross-linked complexes of {sigma}70 with the nontemplate oligonucleotide (left panel) or sEcap1 (right panel) were treated with BrCN as described under "Experimental Procedures" and separated on 13% SDS-PAGE followed by autoradiography. Samples in lanes 1 and 4 are controls without BrCN cleavage. Arrows with numbers on the right indicate C-terminal peptides resulting from cleavage at the corresponding Met residues. The shortest labeled peptide corresponds to cleavage at Met-413. The expected positions of the next two C-terminal peptides resulting from cleavage at Met-456 and Met-470 are shown by dashed arrows (see Refs. 29 and 42 for examples of cleavage patterns where these peptides are visible). Continuous lines connect bands corresponding to identical cleavage products in the two panels.

 
{sigma}70 Regions 1, 3, and 4 Are Not Important for Aptamer Recognition—To check whether other parts of {sigma}70, besides region 2, are important for aptamer binding, we analyzed interactions of sEcap1 with three {sigma} fragments, encompassing amino acids 1-565, 1-448, and 102-448 of {sigma}70 (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 {sigma}2, which is conserved in all {sigma}70-family proteins (15, 16) and whose structure has been solved previously (36). We found that the affinities of all three fragments to sEcap1 did not differ significantly from the affinity of the full-length {sigma}70 (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 {sigma}70 is neither required for nor inhibitory to aptamer binding.


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  		TABLE 1
Recognition of sEcap1 and its mutants by E. coli {sigma}70 and T. aquaticus {sigma}A

For each aptamer variant, the sequence of the conserved aptamer region is shown. The Kd values were measured by nitrocellulose filtration method as described under "Experimental Procedures." For each Kd, the averages and standard deviations from two or three independent experiments are presented.

 
The TG Element in Aptamers Is Recognized by {sigma} 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 {sigma}70 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 {sigma} (Kd > 3 µM, Fig. 3A). Therefore, the TG motif is essential for aptamer binding and can be specifically recognized by {sigma}70 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 {sigma} and/or DNA in the {sigma}-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 {sigma}70 (37). Later, region 2.4 was also shown to play a role in the recognition of this motif (38). We therefore used aptamers as model DNA substrates to analyze the roles of these {sigma} regions in the TG recognition. We found that {sigma}70 fragment 1-448 lacking region 3.0 still bound sEcap1 with high affinity but did not bind the TG-less aptamer mutant (Table 1 and Fig. 3B, see also above). The 1-448 fragment also recognized the TGC sEcap1 variant containing C between TG and the -10 element with about the same affinity as full-length {sigma}70 (Table 1 and Fig. 3B). We therefore concluded that {sigma} region 3.0 is not involved in the interactions with the TG motif in the aptamers.

To test the role of region 2.4 in the TG recognition, we analyzed whether mutations at this region could suppress TG mutations in sEcap1. In our experiment, we used the wild-type {sigma}A subunit from T. aquaticus and a {sigma}A mutant with a Gln to His substitution at position 260 of region 2.4 (26). Previously, point mutations of this amino acid in {sigma}A and of the corresponding residue of {sigma}70 (Gln-437) were shown to suppress TG mutations in extended promoters (26, 38). We found that wild-type {sigma}A bound sEcap1 with high affinity (Kd 9.5 nM) but did not bind sEcap1 mutant with the TG to CC substitution (Kd > 3 µM) (Table 1 and Fig. 4). In contrast, the mutant {sigma}A subunit was able to recognize both the wild-type and the mutant aptamers (Kd 20 and 65 nM, respectively) (Table 1 and Fig. 4). However, the mutant {sigma}A did not bind sEcap1 with the -11C mutation at the-10-like element (Kd > 3 µM). Thus, the suppression effect of the Gln-260 -> His substitution is position-specific, and this residue of {sigma}A is likely directly involved in the recognition of the TG element in the aptamers.


Figure 3
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  		FIGURE 3.
Specific recognition of the TG motif in aptamers by {sigma}70. A, interaction of the wild-type (TG) and mutant (TGC and CC) sEcap1 variants with full-length {sigma}70. B, interaction of the aptamers with the 1-448 {sigma}70 fragment. Shown are representative curves for the binding of radioactively labeled aptamers at increasing protein concentrations as measured by nitrocellulose filtration method (see "Experimental Procedures" for details). The binding is shown in percentages of the maximum binding for wild-type sEcap1.

 


Figure 4
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  		FIGURE 4.
The mutation in the TG motif of sEcap1 is suppressed by the mutation in region 2.4 of the T. aquaticus {sigma}A subunit. Shown is a representative experiment on the binding of the wild-type (WT, open symbols) and the mutant (CC, closed symbols) aptamers to either WT (circles) or Q260H {sigma}A (triangles). The binding was measured by nitrocellulose filtration method. For each aptamer, the binding is shown in percent of maximum binding observed for Q260H {sigma}A.

 
Binding of the Aptamers Does Not Change the Distance between {sigma} Regions 2 and 4—Measurements of distances between different conserved domains of E. coli {sigma}70 using luminescence resonance energy transfer technique revealed large conformational changes in {sigma}70 upon binding to the core and, in particular, a significant increase in the distance between DNA binding domains 2 and 4 (23). We previously showed that the recognition of the sTap aptamers by the T. aquaticus {sigma}A subunit was also accompanied by an increase in the {sigma}2-{sigma}4 distance (26). To test whether these changes are absolutely required for the recognition of DNA by {sigma}, we performed similar experiments with sEcap1. We used a previously described derivative of {sigma}70 (23) that contained a pair of cysteine residues in regions 2 and 4 (at positions 442 and 596). The {sigma} was double-labeled with luminescence donor and acceptor probes and used for luminescence resonance energy transfer measurements. The results are shown in Fig. 5. In agreement with our published data (23), the apparent interdomain distance in free {sigma}70 was short, indicating a compact structure of the protein. The binding of the core enzyme led to a substantial increase in the {sigma}2-{sigma}4 distance. A control oligonucleotide, which did not bind {sigma}70, did not cause any changes in the interdomain distance. Remarkably, the addition of sEcap1 also did not alter the distance between {sigma}70 regions 2 and 4 (Fig. 5). Thus, specific recognition of the -10 element in sEcap1 by {sigma} region 2 does not require separation of {sigma}2 from {sigma}4.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this work, we report that the free primary {sigma} subunit of RNAP is able to specifically recognize the -10 and TG promoter elements in ssDNA aptamers that mimic the nontemplate promoter strand. Because the aptamers contain the -10 promoter element in single-stranded form, their recognition by the {sigma} subunit likely models the interactions of RNAP with nontemplate strand in the open promoter complex after RNAP-induced DNA melting has occurred. Remarkably, {sigma} interacts with the aptamers with high affinity that is comparable to the affinity of holo-RNAP to nontemplate oligonucleotides bearing the -10 promoter element (7, 12, 39). Thus, the aptamers represent a novel type of model promoter substrates that can be efficiently recognized by free {sigma} in the absence of core RNAP.

DNA-protein cross-linking experiments and mutation analysis demonstrated that both the -10 and TG aptamer motifs are recognized by region 2 of {sigma}. The involvement of this region in the recognition of the -10 element does not seem surprising, given its well established role in the recognition of this element in promoters. More interestingly, the TG motif in the aptamers is also recognized by {sigma} region 2.4, whereas region 3.0, which was shown to be involved in its recognition in promoters (37, 38), is not important for aptamer binding. We, therefore, propose that, during promoter recognition, regions 2.4 and 3.0 of {sigma} specifically interact with the TG motif in the nontemplate and template promoter strands, respectively.

In support of this model, a 6-Å resolution structure of a complex of T. aquaticus holo-RNAP with a fork-junction promoter template localizes the TG-like DNA segment between two {alpha}-helixes formed by {sigma} regions 2.4 and 3.0 (Fig. 6) (40). In the structure, amino acids from region 3.0 that have been implicated in the TG recognition (His-455 and Glu-458 in E. coli {sigma}70, corresponding to His-278 and Glu-281 in T. aquaticus {sigma}A, shown in light green in Fig. 6) (37) can potentially contact the template DNA strand. On the other hand, the {alpha}-helix formed by {sigma} region 2.4 likely contacts nucleotides from the nontemplate strand (Fig. 6). We showed that a mutation of {sigma}A Gln260 (Fig. 6, yellow) from this region suppresses substitutions of the TG motif in the aptamers. Previously, substitutions of this and neighboring amino acids in region 2.4 of {sigma}70 were shown to suppress TG mutations in promoters (38). It should be noted that, in the RNAP-DNA model, Gln-260 is located downstream of the TG motif, closer to the first nucleotide of the -10 element (Fig. 6). In agreement with this, mutations of this amino acid in {sigma}70 were previously shown to suppress changes at the first position of the -10 sequence (41). Thus, the conformation and relative positions of promoter DNA and {sigma} may change at different steps of transcription initiation to allow specific interactions between {sigma} region 2.4 and the -10 and TG elements.


Figure 5
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  		FIGURE 5.
Apparent interdomain distances in the {sigma}70 subunit measured by luminescence resonance energy transfer. Apparent distances between two fluorochromes introduced to cysteine residues at positions 442 and 596 of {sigma}70 were measured as described under "Experimental Procedures." Measurements were done with free {sigma}70 or with {sigma}70 in complex with core RNAP or sEcap1. An sTap1 aptamer specific to T. aquaticus {sigma}A was used as a control (C). Averages and standard deviations from three experiments are shown.

 


Figure 6
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  		FIGURE 6.
A structural model for the recognition of the -10 and TG promoter elements by the {sigma} subunit. The model ({alpha}-carbon trace for protein and backbone for DNA is shown) is based on the 6-Å resolution structure of T. aquaticus holo-RNAP in complex with fork-junction DNA template (40). Shown is a {sigma}A fragment encompassing amino acids 100-310 (corresponding to {sigma}70 amino acids 102-487). Conserved region 1.2, nonconserved linker, and region 2.1 are dark gray, region 2.2 is dark green, regions 2.3 and 2.4 are red (Gln-260, yellow), and regions 3.0 and 3.1 are white (Glu-278 and His-281 are light green). The structural domain {sigma}2 capable of specific aptamer binding corresponds to the fragment shown on the figure without regions 3.0 and 3.1. The nontemplate promoter strand (nucleotides from -23 to -7 relative to the transcription start point) is light blue, and the template strand (from -23 to -12) is dark blue. The nucleotides of the TG motif in the two strands are yellow and green, respectively; the nucleotides of the -10 element are orange, the first nucleotide is shown at a larger scale.

 
Previous studies suggested that regions 2 and 4 in free {sigma} are masked for interaction with promoter DNA and that large scale conformational changes of {sigma} induced by core RNAP are required for DNA recognition. In particular, it was proposed that in free {sigma} (i) region 1.1 may inhibit DNA recognition by interacting with region 2, region 4, or both (17-22), and (ii) the close proximity of regions 2 and 4 may mask the DNA binding sites and thus interfere with promoter recognition (24-26) (see the introduction). In this study, we demonstrate that the aptamers containing the promoter-related motif can be efficiently recognized by {sigma} region 2 independently of the presence of regions 1.1 and 4. Furthermore, we show that {sigma}70 in the complex with the aptamers maintains a compact conformation in which domains 2 and 4 are located at the same distance from each other as in free {sigma}. In summary, our data demonstrate that (i) {sigma} regions 1.1 and 4 are unlikely to be directly involved in the masking of DNA binding sites in region 2, and (ii) the increase in the distance between 2 and 4 observed upon holoenzyme formation is not required for the recognition of the -10 element by {sigma} region 2.

At the same time, recognition of the -10 promoter element likely requires some local conformational changes in {sigma}. In particular, it was shown that the binding of the core polymerase alters the solvent accessibility and chemical reactivity of amino acids in {sigma} region 2.3 (12, 27). The core region sufficient for the stimulation of DNA binding by region 2 was localized to the coiled-coil motif of the beta' subunit (E. coli amino acids 262-309) that interacts with {sigma} region 2.2 (Fig. 6, dark green) (8). We therefore propose that sEcaps are able to cause similar conformational changes in {sigma} by interacting with the same {sigma} region. Based on the proposed structure of sEcap1 and on the position of the nontemplate promoter strand in the promoter complex model (Fig. 6), one can suggest that {sigma} region 2.2 is likely contacted by the G-quartet portion of the aptamer, which could in this case play a role analogous to that of the beta' coiled-coil region. Thus, structural analysis of aptamer-induced conformational changes of {sigma} would be an important subject of further studies.

Our results define a minimal {sigma} fragment capable of specific DNA binding, which corresponds to structural domain {sigma}2 and contains conserved region 2, a part of region 1.2, and a nonconserved linker between them (Fig. 6). Given the small aptamer size (which roughly corresponds to the size of DNA fragment shown on the figure), the complex of sEcap with this {sigma} fragment represents a very promising minimal system for structural and biochemical studies of the mechanisms of promoter recognition and opening by RNAP.

Recently, we described ssDNA aptamers (sTaps) that were specifically recognized by T. aquaticus {sigma}A and contained the -10 promoter element (26). The aptamers described in this work also contain the -10-like element and are efficiently recognized by {sigma}s from various bacteria, including T. aquaticus. Brief comparison of the properties of the two types of {sigma}-specific aptamers reveals several important differences in the mechanisms of their interaction with {sigma}. First, the -10 element in sTaps and sEcaps is recognized with slightly different specificity, with different preferences for nucleotide at the third position of the -10 element. Second, in the case of sTaps, an additional downstream GGGA motif was required for aptamer binding. In contrast, the recognition of sEcaps does not depend on downstream motifs but requires the presence of the TG motif and a G-quartet structure upstream of the -10-like element. Remarkably, {sigma} is able to recognize the TG motif located at different positions relative to the -10 element in sEcaps. Third, the recognition of sEcaps does not involve major conformational changes in {sigma} that were observed in the case of sTaps (26). This difference may result from the different structure of the two types of aptamers, with sTaps bearing a hairpin structure downstream and sEcaps containing a G-quartet structure upstream of the -10 element. Thus, the {sigma} subunit shows a remarkable plasticity in the recognition of the promoter elements in model DNA substrates. This property may underlie the ability of RNAP to recognize a large diversity of promoters that can differ in the sequences of the conserved elements and distances between them. Analysis of different types of {sigma}-specific DNA ligands, including the aptamers described in this work, opens a way for better understanding of delicate mechanisms of promoter recognition by RNAP.


   FOOTNOTES
 
* This work was supported in part by the Russian Foundation for Basic Research (Grants 05-04-49405 and 07-04-00247), by National Institutes of Health Grant GM50514 (to T. H.), by the President of the Russian Federation (Grant MK-952.2005.4 to A. K.), and by an international research fellowship grant from the Howard Hughes Medical Institute (to S. K.). Colloberation between the Institute of Biotechnology in Vilnius was supported by a travel grant from the U.S. National Academy of Sciences with partial support from the Howard Hughes Medical Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Present address: Dept. of Microbiology, The Ohio State University, Columbus, OH 43210. Back

2 Present address: The Rockefeller University, New York, NY 10021. Back

1 To whom correspondence should be addressed: Institute of Molecular Genetics, Russian Academy of Sciences, 2 Kurchatov sq., Moscow 123182, Russia. Tel./Fax: 7(499)1960015; E-mail: akulb{at}img.ras.ru.

4 The abbreviations used are: RNAP, RNA polymerase; WT, wild-type; ssDNA, single-stranded DNA; sTap, sigma T. aquaticus aptamer; sEcap, sigma E. coli aptamer. Back


   ACKNOWLEDGMENTS
 
We thank V. Nikiforov, K. Severinov, and I. Artsimovitch for helpful discussions. A. K. is thankful to all members of the S. Klimasauskas laboratory for their help during the work.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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