Articles pubs.acs.org/acschemicalbiology
A Novel Indirect Sequence Readout Component in the E. coli Cyclic AMP Receptor Protein Operator Søren Lindemose,† Peter Eigil Nielsen,† Poul Valentin-Hansen,‡ and Niels Erik Møllegaard*,† †
Department of Cellular and Molecular Medicine, Panum Institute, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark ‡ Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark S Supporting Information *
ABSTRACT: The cyclic AMP receptor protein (CRP) from Escherichia coli has been extensively studied for several decades. In particular, a detailed characterization of CRP interaction with DNA has been obtained. The CRP dimer recognizes a consensus sequence AANTGTGANNNNNNTCACANTT through direct amino acid nucleobase interactions in the major groove of the two operator halfsites. Crystal structure analyses have revealed that the interaction results in two strong kinks at the TG/CA steps closest to the 6-base-pair spacer (N6). This spacer exhibits high sequence variability among the more than 100 natural binding sites in the E. coli genome, but the exact role of the N6 region in CRP interaction has not previously been systematic examined. Here we employ an in vitro selection system based on a randomized N6 spacer region to demonstrate that CRP binding to the lacP1 site may be enhanced up to 14-fold or abolished by varying the N6 spacer sequences. Furthermore, on the basis of sequence analysis and uranyl (UO22+) probing data, we propose that the underlying mechanism relies on N6 deformability.
T
immediately adjacent to the half-sites have been established as the major determinants for binding of CRP.3−8 Despite the extensive studies on the DNA interaction of CRP, the central N6 spacer region has not been subject to any systematic analysis. However, studies with N6 and N8 spacer regions indicate that the structure of the spacer region may influence the alignment of the two 5-bp half-sites.9−13 Here we have employed an in vitro selection strategy to delineate the relations between the sequence of the N6 spacer region and CRP affinity. This was accomplished using a DNA library containing a randomized N6 spacer region embedded in the sequence of the extensively studied CRP binding site LacP1 from the E. coli lac promoter.7,14,15 Our results demonstrate that the sequence of the N6 spacer is indeed a significant modulator of CRP binding affinity. Hence, based solely on differences in the sequence of the N6 spacer region, the affinity of LacP1-derived binding sites could be increased up to 14-fold relative to the wild type site. In addition, we identified N6 sequence combinations that completely abolish CRP binding. On the basis of sequence analysis of the selected sites and interpretation of uranyl probing data, we propose that affinity for the binding sites depends on deformability of the N6 region.
he cyclic AMP receptor protein (CRP), also known as the catabolite gene activator protein (CAP), is a global regulator of gene expression in Gram-negative bacteria and is known to regulate more than 100 genes in Escherichia coli by means of interaction with DNA sites of highly varying sequence.1,2 CRP was the first transcription activator to have its structure solved and is one of the best studied proteins in terms of DNA recognition. Promoter mutational studies and detailed analysis of crystal structures of CRP-DNA complexes have revealed that, upon binding, each CRP monomer interacts directly via an α-helix in the DNA major groove, forming hydrogen bonds with the DNA bases at positions 5, 7, and 8 within each DNA half-site (i.e., 5′A1A2N3T4G5T6G7A8N9N10N11-3′).3−6 The preference for the remaining bases in the consensus sequence is a consequence of indirect readout (recognition of flexibility and/or DNA structural elements). Especially, position 6 in the T6G7 basepair step is not in direct contact with the helix-turn-helix motif of the CRP monomer but is nevertheless highly conserved and involved in strong kinking at this particular base pair step in the two half-sites.3−6 In addition, A/T-rich sequences flanking the 5bp consensus half-site TGTGA (i.e., A1A2) appear to facilitate the DNA wrapping around the protein accommodated through electrostatic interactions between positively charged amino acids on the CRP surface and phosphates in the DNA backbone.5,7,8 Thus, the 5-bp consensus half-sites and the sequences © 2014 American Chemical Society
Received: November 6, 2013 Accepted: January 3, 2014 Published: January 3, 2014 752
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Articles
RESULTS AND DISCUSSION CRP Affinity Screening of a Randomized Library. Initially, the influence of the N6 spacer sequence in the lacP1 site was analyzed by an affinity screening of 15 randomly picked clones from an N6 library (G0). The relative binding constants (Krelative) were measured by use of a gel-based competition assay.8 In this assay a mixture of equimolar amounts of DNA fragments containing the mutant or wild-type lacP1 binding site (180 bp and 75 bp, respectively) compete for a limiting amount of CRP. When separated on a native polyacrylamide gel, four bands are visible corresponding to the unbound and protein-bound fraction of each of the two DNA fragments. A Krelative value was calculated corresponding to the ratio of the binding constant for the mutant relative to the wild-type CRP site.8 Interestingly, this pilot experiment revealed that a large affinity span exists in the G0 library (Table 1). Among the 15 clones
mutations in the non-randomized part of the LacP1 DNA site (22 out of the 47 sequences). Specifically, the 5-bp half-site was changed from TCACT to TCACA in half of the mutants. This particular mutation has previously been shown to significantly increase the affinity of lacP1 for CRP.7 Therefore, to have an unbiased data set, all clones containing mutations in the nonrandomized part of the LacP1 N6 library DNA template were omitted, resulting in 25 different N6 sequences (Supporting Information, Table S1). By closer inspection of the data set we note that certain base pairs are predominantly found at specific positions resulting in the presence of a characteristic pyrimidine-purine pattern in the N 6 spacer region (Supporting Information, Table S2). Specifically, the three bases in the 5′ element of N6 (i.e., position −9 to −11) are more frequently pyrimidines (Y), whereas the three bases in the 3′ element (i.e., positions +11 to +9) are frequently purines (R). The prevalence for purine or pyrimidine at each position is 70−90%, except at position +10, which has equal frequencies of pyrimidines and purines (C and G nucleotides, in particular) resulting in the “consensus” Y-Y-YR-C/G-R. Consequently, a Y-R base pair step (TA, CG, TG, or CA) is predominantly found at the center of symmetry (i.e., between positions −11 and +11) (Supporting Information, Tables S1 and S2). It is noted that a comparison of 49 naturally occurring binding sites likewise reveals a preference for a YYYRRR pattern (Supporting Information table S2). Interestingly, a purine-pyrimidine step is not found in the center of any of the G5 clones. Rather this base pair step is present in G014, for which CRP binding was absent. Pyrimidine-purine steps are the most deformable base pair steps capable of adopting large roll, twist, and slide values, resulting in bending via major groove compression (i.e., positive roll) and axial kinking.16−21 In addition, TC/GA dinucleotides are found at the border to one of the 5-bp consensus sequences in more than half of the selected sites giving rise to TGTGATCNNNN or NNNNGATCACT sequences in the binding sites (N6 underlined), thereby indicating a strong selection for a GATC sequence at the junction between N6 and the 5-bp half-sites. In order to decipher the importance of the nucleotide composition in the N6 spacer region, we measured the relative binding constants for a subset of the in vitro selected G5 clones using the wild-type LacP1 binding site from E. coli as internal standard. Quantification of CRP Binding to Selected Sites. Twelve of the in vitro selected G5 clones were chosen for further analysis. As expected, these clones all represent stronger CRP binding sites compared with wild-type lacP1, exhibiting a 2- to 14-fold increase in affinity (Table 2). The data show that it is not solely the presence of a central Y-R base pair step in N6 that determine binding modulation. For instance, clones G5.01 (5′-TATATG3′) and G5.59 (5′-TAGATG-3′) exhibit very similar affinity for CRP. Thus a central GA base pair steps in the sequence context of G5.59 is not unfavorable per se for binding of CRP. Furthermore, the strongest binding sites have three pyrimidines in the left segment of the N6 region. However, single base pair change from C to T at position 10 (G5.54, TCTACA and G5.40, TTTACA) has a strong negative effect on affinity. Furthermore, CCCAGA (G5.03) provides a much better binding site than CCCGGG (G5.36). Hence, it is not only a combination of three pyrimidines and three purines that determines efficient binding, and as previously noted a strong consensus within the N6 sequence is indeed not apparent. However, most strikingly the GATC sequence element overlapping N6 close to the primary
Table 1. Relative Binding Constants and Binding Free Energy Calculations for 15 Randomly Selected G0 Clonesa clone
N6 sequence: 5′−3′
LacP1 G0.01 G0.02 G0.03 G0.04 G0.05 G0.06 G0.07 G0.08 G0.09 G0.10 G0.11 G0.12 G0.13 G0.14 G0.15
TGTGA-GTTAGC-TCACT TGTGA-TTCGGT-TCACT TGTGA-CCCTTA-TCACT TGTGA-ATTTTT-TCACT TGTGA-TCACTA-TCACT TGTGA-CGGGTG-TCACT TGTGA-GCTTCT-TCACT TGTGA-CCTCCT-TCACT TGTGA-CGCCCC-TCACT TGTGA-GTAAAC-TCACT TGTGA-TCGGCT-TCACT TGTGA-GAGACA-TCACT TGTGA-TCTAAT-TCACT TGTGA-ATAGAA-TCACT TGTGA-GAATCT-TCACT TGTGA-TTGGGG-TCACT
Krelative
n
0.33 ± 0.01 1.44 ± 0.05
8 8 8 6 5 12 10 11 10 7 6 6 6 8 7
1.24 ± 0.06 2.28 ± 0.18 0.18 ± 0.03 0.33 ± 0.03 0.71 ± 0.09 0.07 ± 0.01 0.75 ± 0.08 0.63 ± 0.05 0.65 ± 0.07 0.05 ± 0.02 1.17 ± 0.19
ΔΔG (kcal/mol) 0.65 −0.21 −0.13 −0.49 1.01 0.65 0.20 1.56 0.17 0.27 0.25 1.76 −0.09
a
The sequences of N6 are aligned about the core consensus of the two 5-bp half-sites: TGTGA-N6-TCACT. The data were obtained in competition assays where equimolar amounts lacP1 DNA fragments (reference) and randomly picked binding sites competed for a limiting amount of CRP protein. Krelative and ΔΔG values were calculated as described in the Methods section, and n denotes the number of experiments performed. The wild-type LacP1 sequence is shown for comparison.
analyzed, a few (G0.02, G0.04, G0.05, and G0.15) represented binding sites with CRP affinity slightly higher than or at least equivalent to that of lacP1, whereas the remaining exhibited lower affinity. Remarkably, two clones (i.e., G0.03 and G0.14) did not support CRP binding at the experimental conditions used. Thus, this initial experiment clearly showed that the N6 spacer region plays a pivotal role for CRP binding, conferring >100-fold variation in affinity. In Vitro Selection of CRP Binding Sites. In order to obtain more detailed information about the sequence preference for the central N6 region, the randomized LacP1 N6 library was subjected to in vitro selection. After incubation with CRP, gel electrophoresis was employed to separate CRP-DNA complex from free DNA. After 5 rounds of selection, purified DNA fragments (G5) were cloned and sequenced. In total 66 individual clones were sequenced representing 47 different DNA sequences. A significant proportion of the clones contained 753
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Table 2. Relative Binding Constants and Binding Free Energy Calculations for in Vitro Selected G5 Clonesa clone
N6 sequence: 5′−3′
Krelative
n
ΔΔG (kcal/mol)
LacP1 G5.03 G5.54 G5.32 G5.28 G5.41 G5.06 G5.44 G5.46 G5.59 G5.01 G5.40 G5.36
TGTGA-GTTAGC-TCACT TGTGA-CCCAGA-TCACT TGTGA-TCTACA-TCACT TGTGA-TCTGGG-TCACT TGTGA-TCTATA-TCACT TGTGA-CCAAGA-TCACT TGTGA-TCTAAG-TCACT TGTGA-CGCGGA-TCACT TGTGA-TCTTGG-TCACT TGTGA-TAGATG-TCACT TGTGA-TATATG-TCACT TGTGA-TTTACA-TCACT TGTGA-CCCGGG-TCACT
14.19 ± 1.54 12.32 ± 1.12 10.40 ± 0.74 7.06 ± 0.89 6.62 ± 0.94 6.51 ± 0.45 5.74 ± 0.27 4.77 ± 0.64 3.84 ± 0.50 3.35 ± 0.08 2.91 ± 0.15 2.45 ± 0.26
6 11 6 6 6 7 6 6 7 6 6 6
−1.56 −1.48 −1.38 −1.15 −1.11 −1.10 −1.03 −0.92 −0.79 −0.71 −0.63 −0.53
a
The sequences of N6 are aligned about the core consensus of the two 5-bp half-sites: TGTGA-N6-TCACT. The data were obtained in gel-based competition assays where equimolar amounts of LacP1 DNA (reference) and each of the G5 in vitro selected binding sites competed for a limiting quantity of CRP protein. Krelative and ΔΔG values were calculated as described in the Methods section, and n denotes the number of experiments performed. The wild-type LacP1 sequence is also shown for comparison.
TG/CA steps). Previously, it has been demonstrated that A/T stretches display uranyl hyper reactivity,25 as also observed in the N6 region of G5.01, G5.40, and G5.28. However, sites of increased reactivity are also found in N6 regions that are not A/Trich (G5.03, G5.32 and G5.44). In general hyper reactivity is present at either or both ends of the N6 region. Intriguingly, the GATC quartets present in the eight strongest CRP binding sites are all more sensitive toward uranyl cleavage, analogously to the reactivity seen for (A/T)n regions. We ascribe this behavior to the helix plasticity at the GATC site allowing it to take up a uranyl induced fit narrow minor groove structure. Indeed, X-ray crystal structure and NMR analysis of oligonucleotides containing this quartet have revealed that the GATC quartet can exist in a variety of conformations.20,26,27 In particular one crystal structure shows that this sequence allows the DNA helix to adopt a narrow minor groove despite the presence of the 2-amino group,26 and NMR studies provide evidence of possible helix bending.27 Therefore, the helix at the GATC location seems highly deformable, which may support an induced fit mechanism in protein−DNA interaction. A few of the selected sequences do not contain a central TA step or a GATC quartet. However, one of these (G5.36) contains the CCCGGG sequence that also provides plasticity, e.g., as illustrated by the XmaI and SmaI restriction enzymes, which upon binding bend the helix in opposite direction.28 Thus, helix deformability in the N6 regions may be a common property of the selected high affinity CRP binding sites. Footprint Analysis of Selected CRP Binding Sites. Crystal structure analyses of CRP DNA complexes (all containing nicks in N6) have shown that four phosphates between nucleobase −9/−10 and −10/−11 on the lower strand and +11/+10 and +10/+9 on the upper strand in the N6 region are contacted by CRP.5,6 Uranyl photoprobing provides information about structural deformation as well as phosphates involved in protein interaction.29−32 Therefore, CRP binding to the wild type LacP1 site together with five clones (G5.03, G5.32, G5.36, G5.41, and G5.54) and a symmetrical CRP binding site6 were analyzed by uranyl photofootprinting, and unique protein− phosphate contacts were determined from differential cleavage plots (Figure 1E−H). From these data it is evident that 4−6 phosphates flanking one of the half-sites are strongly protected. Compared to the natural lac sequence, protection of more phosphates in the flanking regions is observed for the symmetric
kink sites is present in all of the strong binding sites, and sequence changes in this quartet diminish CRP affinity. This is exemplified by G5.54 (TCTACA) and G5.40 (TTTACA) (Krelative 12.3 versus 2.91). In order to study the effect of N6 sequences on CRP affinity of a very strong CRP site, we substituted the spacer region of the symmetrical ICAP binding site (AAATGTGA-TCTAGATCACATTT) containing two GATC quartets with central sequences that were expected to be detrimental to binding. Indeed, replacement of the ICAP N6 sequence with the G0.14 sequence, GAATCT (ICAP “G0.14” ) or with an A-tract (ICAP“A‑tracts”) resulted in a strongly reduced affinity (20- and 40-fold, respectively) (Supporting Information, Table S3).22 In addition, substitution of the ICAP-spacer with N6 sequences of some strong lacP1 derived binding sites showed CRP affinity comparable to that of the intact ICAP site (e.g., ICAP“G5.03”, ICAP“G5.41”, ICAP“G5.44”, and ICAP“G5.54”; Supporting Information, Table S3). Collectively, the above binding studies establish that the N6 spacer sequence significantly contributes to binding affinity/affects CRP binding. DNA Structural Analysis of the N6 Spacer Region. On the basis of the selection of strong CRP binding sites, it is evident that certain sequence elements in or overlapping N6 strongly influence CRP binding, in particular the GATC motif and pyrimidine-purine steps at the center of N6. However, any strong sequence consensus is not apparent and structural features of the DNA helix in the N6 region could therefore play an important role. In order to examine for effects of simple isotropic flexibility, we substituted the N6 spacer with nicked, gapped, and mismatched duplexes, which should increase local flexibility of the helix (Supporting Information). However, all of these substitutions decreased CRP affinity (Supporting Information, Table S4) indicating that affinity must rely on some structural or dynamic features of the N6 sequences. Furthermore, to study helix structure and deformability in the binding, sites we performed uranyl photocleavage analysis, which probes DNA helix minor groove width and/or electronegative potential,23,24 as well as DNA helix deformability.25The results are presented in Figure 1A−D and show that the N6 regions of different sequence display uranyl cleavage patterns with distinct similarities. Specifically, increased reactivity is found at positions close to the primary kink sites within the 5-bp consensus sequence (the 754
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Figure 1. Uranyl structural probing and footprinting. (A−D) Uranyl structural probing of 12 G5 clones. Three clones are displayed in each panel. The results are displayed as relative uranyl cleavages. (E−H) Differential cleavage plots based on uranyl photofootprinting of CRP binding to six G5 clones, the wildtype lacP1 binding site, and a symmetric CRP binding site (200 nM CRP). 755
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Figure 2. Uranyl, DNase I, and hydroxyl radical footprinting. The symmetric CRP binding site is used for comparison of the three probing methods. The footprinting results are displayed by autoradiographs (A) and by densitometric scans (B). CRP at 200 nM concentration was added in the lanes designated by +. The two lines in the densitometric scans represent uranyl cleavage in the absence of CRP (black line) and in the presence of CRP (blue line). The CRP protections in the uranyl and hydroxyl radical footprints are shown by brackets.
addressed this question by a competition experiment using other metal ions. Intriguingly, zinc was not only able to decrease the strong cleavage in the center of N6, but also to develop a clear protection (i.e., zinc-footprint) of the position 3′ the hypersensitive site (Figure 3C and D). This result might indicate that the hyperactive position in the center of N6 could be a result of a specific metal ion DNA interaction induced by CRP. The molecular interpretation of this observation needs to be addressed in further studies. Previous work, including DNA binding, gel shift experiments, and structural studies established an important role for DNA distortion in the sequence-specific binding of CRP to DNA sites. It is believed that sequence-dependent deformability of the operator upon CRP binding is achieved largely by two sharp kinks at the conserved pyrimidine-purine step T6-G7, which is known to be highly susceptible to deformation. Furthermore, AT-rich sequences at positions 10 and 11 and GC-rich sequences around position 16 from the dyad axis result in increased bending and increased affinity. Our results provide evidence that the central N6 spacer also plays a significant role in DNA recognition, capable of modulating affinity up to 100-fold despite that this element exhibits high sequence variability among natural sites. On the basis of sequence analysis and uranyl probing data of selected binding sites, we infer that the underlying mechanism relies on N6 deformability. However, we note that no obvious deformation of the N6 sequence was observed in the structures of CRP cocrystallized with DNA. A likely explanation for this discrepancy is that the DNA fragments used in crystallization of CRP-DNA complexes contain nicks in the N6 spacer.4 Consequently, these crystal structures may not accurately reflect protein-induced distortion of the spacer region. In line with this assumption, the
CRP site and for some of the selected G5 variants. Surprisingly, phosphates in the N6 spacer, which by crystallographic studies were identified to interact with CRP, were not protected from uranyl cleavage. On the contrary, positions in the N6 region become hypersensitive to uranyl cleavage upon CRP binding (Figure 1E−H). The strongest hypersensitive site was detected in the N6 sequence of the symmetrical CRP site (Figure 1H). In a previous study using uranyl photocleavage for detecting CRP interaction with DNA binding sites containing inosine and 2,6diamino purine, we likewise showed that CRP binding induces hypersensitive sites in N6 in the binding sites analyzed.29 Hydroxyl radical and DNase I footprinting were performed in order to further investigate interactions in the symmetrical N6 sequence. Interestingly, the exact same positions, which became uranyl hypersensitive were protected from hydroxyl radical cleavage, whereas protection at the A/T-rich sequences outside the 5-bp consensus sequence were identical in the two analyses (Figure 2). The result of DNase I footprinting shows strong protection outside the 5-bp half-sites, whereas protection in N6 is weaker (Figure 2). Finally, it is clearly demonstrated that the appearance of a strong uranyl hypersensitive site in N6 of the symmetric CRP binding site is a result of CRP interaction in the operator since the uranyl cleavage intensity increases with increasing CRP concentration (Figure 3A and B). The very strong uranyl reactivity in the center of N6 could indicate either the presence of a narrow minor groove or deformability.25 However, also the possibility for a protein induced metal ion binding site in the N6 DNA has to be considered, because the divalent uranyl ion has been shown to probe metal ion binding sites in folded DNA and RNA.33,34 Therefore, we asked whether the strong hypersensitive sites could be a strong protein coordinated metal ion binding site. We 756
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Figure 3. (A) Uranyl photofootprinting of CRP interaction with the symmetric binding site. Lane 1: no CRP added; lanes 2−7: 5, 10, 20, 50, 100, and 200 nM CRP added; S: A/G sequence reaction; C: no uranyl reaction. (B) Differential cleavage plot showing the appearance of hypersensitive positions at increasing CRP concentration. The most reactive position within N6 is indicated by an arrow. (C) Uranyl photofootprinting in the presence of Zn2+. Lane 1: no CRP added; lane 2: 200 nM CRP added; lanes 3−6: 200 nM CRP added together with 0.5, 1, 2, and 4 mM ZnCl2. (D) Differential cleavage plot (CRP binding in the presence of Zn2+ (lane 6) relative to CRP binding in the absence of Zn2+ (lane 2).
footprinting data show that phosphates in the N6 spacer, which by crystallographic studies were identified to interact with CRP, were not protected from uranyl cleavage. Rather, other positions
in the N6 region become hypersensitive to uranyl cleavage upon CRP binding (Figure 1E−H). Curiously, we observed that Zn2+ specifically prevents the CRP-induced uranyl hyperreactivity at 757
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NP-40, 2 μg mL‑1 calf thymus DNA and 50 μM cAMP) containing 100 μM cAMP for 30 min at 23 °C. After incubation, 9 μL of loading buffer (CRP binding buffer containing 50% glycerol, and 0.1 mg mL‑1 bromophenol blue) was added, and samples were immediately loaded on 6% polyacrylamide gels (55:1). Following electrophoresis, the CRP− DNA complexes were detected by autoradiography or exposure to phosphor imager storage screens. Relative Binding Constants and Binding Free Energy Change. The relative equilibrium binding constants, Krelative, were measured by EMSA as previously described.8 In this assay, a mixture of two different size DNAs (75 and 276 bp, respectively) competes for a limited amount of CRP protein simultaneously. We used 5−20 pM of each DNA fragment, which is well below the Kd for the CRP-LacP1 complex, and all experiments were performed at least in triplicate. Following exposure to phosphor imager storage screens, four different bands were clearly visible, and the amount of radioactivity in each band was quantified using STORM Phosphor Imager scanner and Image Quant software from Molecular Dynamics. The relative equilibrium binding constants were calculated by the formula: Krelative = (Kmutant)/(Kwild‑type) = (Kclone)/ (KLacP1), where Kclone is the ratio of protein-bound G0 or G5 clone DNA divided by free G0 or G5 clone DNA, and KLacP1 is the same ratio for the LacP1 DNA. The binding free energy change, ΔΔG, which is the difference between the binding free energy for CRP−DNAclone complex formation versus the binding free energy for CRP−DNALacP1 complex formation, was calculated from the general assumption: ΔΔG = RT ln(Kd,clone) − RT ln(Kd,LacP1) = −RT ln[(Kd,clone)/(Kd,LacP1)]. This is in our system equivalent to ΔΔG = −RT ln(Krelative) where Krelative is the relative equilibrium binding constant described above, R is the gas constant [8.3145 J/(mol × K)], and T is the temperature in Kelvin. The average Krelative obtained from at least triplicate experiments was used in the expression. Note that positive ΔΔG values indicate a reduction of binding affinity. Uranyl Photocleavage, DNase I and Hydroxyl Radical Footprinting. In order to obtain structural information, uranyl photocleavage was performed as previously described.25,30 Uranyl and DNase footprinting was performed as described in ref 29. Hydroxyl radical footprinting was performed as described42 in the CRP binding buffer (10 mM Tris-HCl, pH 8.0, 50 mM KCl, 2.5 mM MgCl2, 1 mM EDTA, 55 μg mL‑1 bovine serum albumin, 1 mM dithiothreitol, 0.05% NP-40, 2 μg mL‑1 calf thymus DNA, and 100 μM cAMP). A Molecular Dynamics STORM Phosphor Imager was used to collect data from the phosphor storage screens, and baseline corrected scans were obtained by using Image Quant version 5.2 and SAFA software.43 Differential cleavage plots were calculated from the expression ln( fa) − ln( fc) representing the differential cleavage at each bond relative to the control (where fa is the fractional cleavage at any bond in the presence of the protein, and fc is the fractional cleavage of the same bond in the control). Using this expression, positive values indicate enhanced cleavage, whereas negative values indicate cleavage inhibition.
this position. Thus, it remains possible that CRP-induced distortion of the spacer helix might create a (protein assisted) binding site for a metal ion. In prior work spacer sequences have been shown to play a significant role for operator binding of other helix-turn-helix binding proteins.35 Especially, the spacers in DNA sites for the 434 and P22 repressors have been thoroughly studied. In both cases the indirect readout of the spacer sequence influences protein affinity.36−41 Specifically, the spacers affect the affinity via the structure of the unbound DNA and the deformability of the spacer, and for the P22 repressor it has been shown that the ease by which the helix structure in the binding site can be converted into a B′ form determines affinity. Thus, helixes such as A-tracts which already are in a B′ form show the strongest affinity for P22, whereas the presence of G/C base pairs that provide the largest barrier for the B to B′ transition results in low affinity.41 Thus, the present study of CRP-DNA interaction illustrates another striking example of the importance of indirect readout of the DNA helix in which sequence specificity arises through the ability of certain DNA sequence motifs to accommodate the helix conformation required by the protein for optimal DNA interaction. We anticipate that as more sequence specific protein DNA interactions are studied in molecular depth, in terms of DNA helix conformation, flexibility, and deformability, we shall see many more examples of how Nature has exploited these DNA properties for indirect DNA sequence readouts by proteins (and other ligands).
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METHODS
In Vitro Binding Site Selection. All 32P-labeled DNA fragments were produced by standard techniques using either T4 polynucleotide kinase or Large Fragment of DNA Polymerase I (Klenow). The in vitro selection assay was modeled after a previous study from our laboratory.29 The binding site selection experiments were initiated by use of 11 ng (∼2.6 × 1011 molecules) of single-stranded N6 in vitro selection template and a double-stranded randomized DNA oligonucleotide pool was generated by PCR as described below except only five PCR cycles were run. To enrich the randomized DNA oligonucleotide pool for CRP-binding sites, the pool was incubated with 50 nM CRP and subjected to gel electrophoretic mobility shift assay (EMSA). Following electrophoresis, the band shifts corresponding to CRP−DNA complexes were cut out, and the DNA was purified from the acrylamide gel. Before starting the next round of selection, the obtained DNA fragments were PCR amplified with dNTPs in a volume of 50 μL. In each case, the template under study was PCR amplified in a total volume of 50 μL containing 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, and 2.5 U of Taq DNA polymerase (Fermentas)) using 50 pmol of each primer and 200 μM dNTPs (Roche). After an initial denaturing step of 2 min at 94 °C, amplification cycles were performed with each cycle consisting of the following segments: 94 °C for 30 s, 42−48 °C (depending on the primer set used) for 30 s, and 72 °C for 30 s. After the last PCR cycle, the extension segment was continued for 7 min at 72 °C before cooling to RT. The PCR products were gel purified and resuspended in 10 μL of either H2O or CRP binding buffer depending on the future use of the DNA (PCR or EMSA). PCR for Krelative experiments: LacP1 DNA fragments (75 bp and 160 bp) were obtained by PCR using primer set Lac promoter 1 and 2 and primer set Lac promoter 3 or 4 using plasmid pUC19 as template. The different in vitro selected G5 clone and mutant DNA fragments (276 bp) were similarly obtained by PCR using primer set M13F and M13R and the individual pCR2.1-TOPO plasmids containing the appropriate cloned DNA sequences as template. Electrophoretic Mobility Shift Assay (EMSA). 32P-labeled DNA fragments and CRP protein were incubated in 30 μL of CRP binding buffer (10 mM Tris-HCl, pH 8.0, 50 mM KCl, 2.5 mM MgCl2, 1 mM EDTA, 55 μg mL‑1 bovine serum albumin, 1 mM dithiothreitol, 0.05%
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ASSOCIATED CONTENT
S Supporting Information *
This material is available free of charge via the Internet at http:// pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We gratefully thank N. J. Harning and N. L. Jansen for technical assistance 758
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dx.doi.org/10.1021/cb4008309 | ACS Chem. Biol. 2014, 9, 752−760
ACS Chemical Biology
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software for high-throughput quantification of nucleic acid footprinting experiments. RNA 11, 344−354.
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dx.doi.org/10.1021/cb4008309 | ACS Chem. Biol. 2014, 9, 752−760
