A Novel Indirect Sequence Readout Component in the E. coli Cyclic AMP Receptor Protein Operator

A Novel Indirect Sequence Readout Component in the E. coli CyclicAMP Receptor Protein OperatorSøren 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 CopenhagenN, 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) fromEscherichia coli has been extensively studied for severaldecades. In particular, a detailed characterization of CRPinteraction with DNA has been obtained. The CRP dimerrecognizes a consensus sequence AANTGTGANNNN-NNTCACANTT through direct amino acid nucleobaseinteractions in the major groove of the two operator half-sites. Crystal structure analyses have revealed that theinteraction results in two strong kinks at the TG/CA stepsclosest to the 6-base-pair spacer (N6). This spacer exhibits highsequence variability among the more than 100 natural bindingsites 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 thelacP1 site may be enhanced up to 14-fold or abolished by varying the N6 spacer sequences. Furthermore, on the basis of sequenceanalysis and uranyl (UO2

2+) probing data, we propose that the underlying mechanism relies on N6 deformability.

The cyclic AMP receptor protein (CRP), also known as thecatabolite gene activator protein (CAP), is a global

regulator of gene expression in Gram-negative bacteria and isknown to regulate more than 100 genes in Escherichia coli bymeans of interaction with DNA sites of highly varyingsequence.1,2 CRP was the first transcription activator to haveits structure solved and is one of the best studied proteins interms of DNA recognition. Promoter mutational studies anddetailed analysis of crystal structures of CRP-DNA complexeshave revealed that, upon binding, each CRP monomer interactsdirectly via an α-helix in the DNA major groove, forminghydrogen bonds with the DNA bases at positions 5, 7, and 8w i t h i n e a c h D N A h a l f - s i t e ( i . e . , 5 ′ -A1A2N3T4G5T6G7A8N9N10N11-3′).3−6 The preference for theremaining bases in the consensus sequence is a consequence ofindirect readout (recognition of flexibility and/or DNAstructural elements). Especially, position 6 in the T6G7 base-pair step is not in direct contact with the helix-turn-helix motif ofthe CRP monomer but is nevertheless highly conserved andinvolved in strong kinking at this particular base pair step in thetwo half-sites.3−6 In addition, A/T-rich sequences flanking the 5-bp consensus half-site TGTGA (i.e., A1A2) appear to facilitate theDNA wrapping around the protein accommodated throughelectrostatic interactions between positively charged amino acidson the CRP surface and phosphates in the DNA backbone.5,7,8

Thus, the 5-bp consensus half-sites and the sequences

immediately adjacent to the half-sites have been established asthe 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 anysystematic analysis. However, studies with N6 and N8 spacerregions indicate that the structure of the spacer region mayinfluence the alignment of the two 5-bp half-sites.9−13

Here we have employed an in vitro selection strategy todelineate the relations between the sequence of the N6 spacerregion and CRP affinity. This was accomplished using a DNAlibrary containing a randomized N6 spacer region embedded inthe sequence of the extensively studied CRP binding site LacP1from the E. coli lac promoter.7,14,15

Our results demonstrate that the sequence of the N6 spacer isindeed a significant modulator of CRP binding affinity. Hence,based solely on differences in the sequence of the N6 spacerregion, the affinity of LacP1-derived binding sites could beincreased up to 14-fold relative to the wild type site. In addition,we identified N6 sequence combinations that completely abolishCRP binding. On the basis of sequence analysis of the selectedsites and interpretation of uranyl probing data, we propose thataffinity for the binding sites depends on deformability of the N6region.

Received: November 6, 2013Accepted: January 3, 2014Published: January 3, 2014

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■ RESULTS AND DISCUSSIONCRP Affinity Screening of a Randomized Library.

Initially, the influence of the N6 spacer sequence in the lacP1site was analyzed by an affinity screening of 15 randomly pickedclones 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 fragmentscontaining the mutant or wild-type lacP1 binding site (180 bpand 75 bp, respectively) compete for a limiting amount of CRP.When separated on a native polyacrylamide gel, four bands arevisible corresponding to the unbound and protein-boundfraction of each of the two DNA fragments. A Krelative value wascalculated corresponding to the ratio of the binding constant forthe mutant relative to the wild-type CRP site.8

Interestingly, this pilot experiment revealed that a large affinityspan exists in the G0 library (Table 1). Among the 15 clones

analyzed, a few (G0.02, G0.04, G0.05, and G0.15) representedbinding sites with CRP affinity slightly higher than or at leastequivalent to that of lacP1, whereas the remaining exhibitedlower affinity. Remarkably, two clones (i.e., G0.03 and G0.14)did not support CRP binding at the experimental conditionsused. Thus, this initial experiment clearly showed that the N6spacer 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 thecentral N6 region, the randomized LacP1 N6 library wassubjected to in vitro selection. After incubation with CRP, gelelectrophoresis was employed to separate CRP-DNA complexfrom free DNA. After 5 rounds of selection, purified DNAfragments (G5) were cloned and sequenced. In total 66individual clones were sequenced representing 47 differentDNA sequences. A significant proportion of the clones contained

mutations in the non-randomized part of the LacP1 DNA site(22 out of the 47 sequences). Specifically, the 5-bp half-site waschanged from TCACT to TCACA in half of the mutants. Thisparticular mutation has previously been shown to significantlyincrease the affinity of lacP1 for CRP.7 Therefore, to have anunbiased data set, all clones containing mutations in the non-randomized part of the LacP1 N6 library DNA template wereomitted, resulting in 25 different N6 sequences (SupportingInformation, Table S1).By closer inspection of the data set we note that certain base

pairs are predominantly found at specific positions resulting inthe presence of a characteristic pyrimidine-purine pattern in theN6 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 thethree bases in the 3′ element (i.e., positions +11 to +9) arefrequently purines (R). The prevalence for purine or pyrimidineat each position is 70−90%, except at position +10, which hasequal frequencies of pyrimidines and purines (C and Gnucleotides, in particular) resulting in the “consensus” Y-Y-Y-R-C/G-R. Consequently, a Y-R base pair step (TA, CG, TG, orCA) 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 naturallyoccurring binding sites likewise reveals a preference for aYYYRRR pattern (Supporting Information table S2). Interest-ingly, a purine-pyrimidine step is not found in the center of any ofthe G5 clones. Rather this base pair step is present in G014, forwhich CRP binding was absent. Pyrimidine-purine steps are themost deformable base pair steps capable of adopting large roll,twist, and slide values, resulting in bending via major groovecompression (i.e., positive roll) and axial kinking.16−21 Inaddition, TC/GA dinucleotides are found at the border to oneof the 5-bp consensus sequences in more than half of the selectedsites giving rise to TGTGATCNNNN or NNNNGATCACTsequences in the binding sites (N6 underlined), therebyindicating a strong selection for a GATC sequence at thejunction 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 relativebinding constants for a subset of the in vitro selected G5 clonesusing the wild-type LacP1 binding site from E. coli as internalstandard.

Quantification of CRP Binding to Selected Sites. Twelveof the in vitro selected G5 clones were chosen for further analysis.As expected, these clones all represent stronger CRP bindingsites compared with wild-type lacP1, exhibiting a 2- to 14-foldincrease in affinity (Table 2). The data show that it is not solelythe presence of a central Y-R base pair step in N6 that determinebinding modulation. For instance, clones G5.01 (5′-TATATG-3′) and G5.59 (5′-TAGATG-3′) exhibit very similar affinity forCRP. Thus a central GA base pair steps in the sequence contextof G5.59 is not unfavorable per se for binding of CRP.Furthermore, the strongest binding sites have three pyrimidinesin the left segment of the N6 region. However, single base pairchange 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 thanCCCGGG (G5.36). Hence, it is not only a combination of threepyrimidines and three purines that determines efficient binding,and as previously noted a strong consensus within the N6sequence is indeed not apparent. However, most strikingly theGATC sequence element overlapping N6 close to the primary

Table 1. Relative Binding Constants and Binding Free EnergyCalculations for 15 Randomly Selected G0 Clonesa

clone N6 sequence: 5′−3′ Krelative nΔΔG

(kcal/mol)

LacP1 TGTGA-GTTAGC-TCACTG0.01 TGTGA-TTCGGT-TCACT 0.33 ± 0.01 8 0.65G0.02 TGTGA-CCCTTA-TCACT 1.44 ± 0.05 8 −0.21G0.03 TGTGA-ATTTTT-TCACT 8G0.04 TGTGA-TCACTA-TCACT 1.24 ± 0.06 6 −0.13G0.05 TGTGA-CGGGTG-TCACT 2.28 ± 0.18 5 −0.49G0.06 TGTGA-GCTTCT-TCACT 0.18 ± 0.03 12 1.01G0.07 TGTGA-CCTCCT-TCACT 0.33 ± 0.03 10 0.65G0.08 TGTGA-CGCCCC-TCACT 0.71 ± 0.09 11 0.20G0.09 TGTGA-GTAAAC-TCACT 0.07 ± 0.01 10 1.56G0.10 TGTGA-TCGGCT-TCACT 0.75 ± 0.08 7 0.17G0.11 TGTGA-GAGACA-TCACT 0.63 ± 0.05 6 0.27G0.12 TGTGA-TCTAAT-TCACT 0.65 ± 0.07 6 0.25G0.13 TGTGA-ATAGAA-TCACT 0.05 ± 0.02 6 1.76G0.14 TGTGA-GAATCT-TCACT 8G0.15 TGTGA-TTGGGG-TCACT 1.17 ± 0.19 7 −0.09

aThe sequences of N6 are aligned about the core consensus of the two5-bp half-sites: TGTGA-N6-TCACT. The data were obtained incompetition assays where equimolar amounts lacP1 DNA fragments(reference) and randomly picked binding sites competed for a limitingamount of CRP protein. Krelative and ΔΔG values were calculated asdescribed in the Methods section, and n denotes the number ofexperiments performed. The wild-type LacP1 sequence is shown forcomparison.

ACS Chemical Biology Articles

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kink sites is present in all of the strong binding sites, andsequence changes in this quartet diminish CRP affinity. This isexemplified 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 thesymmetrical ICAP binding site (AAATGTGA-TCTAGA-TCACATTT) containing two GATC quartets with centralsequences that were expected to be detrimental to binding.Indeed, replacement of the ICAP N6 sequence with the G0.14sequence, GAATCT (ICAP“G0.14”) or with an A-tract(ICAP“A‑tracts”) resulted in a strongly reduced affinity (20- and40-fold, respectively) (Supporting Information, Table S3).22 Inaddition, substitution of the ICAP-spacer with N6 sequences ofsome strong lacP1 derived binding sites showed CRP affinitycomparable to that of the intact ICAP site (e.g., ICAP“G5.03”,ICAP“G5.41”, ICAP“G5.44”, and ICAP“G5.54”; Supporting Informa-tion, Table S3). Collectively, the above binding studies establishthat the N6 spacer sequence significantly contributes to bindingaffinity/affects CRP binding.DNA Structural Analysis of the N6 Spacer Region. On

the basis of the selection of strong CRP binding sites, it is evidentthat certain sequence elements in or overlapping N6 stronglyinfluence CRP binding, in particular the GATC motif andpyrimidine-purine steps at the center of N6. However, any strongsequence consensus is not apparent and structural features of theDNA helix in the N6 region could therefore play an importantrole. In order to examine for effects of simple isotropic flexibility,we substituted the N6 spacer with nicked, gapped, andmismatched duplexes, which should increase local flexibility ofthe helix (Supporting Information). However, all of thesesubstitutions decreased CRP affinity (Supporting Information,Table S4) indicating that affinity must rely on some structural ordynamic features of the N6 sequences. Furthermore, to studyhelix structure and deformability in the binding, sites weperformed uranyl photocleavage analysis, which probes DNAhelix minor groove width and/or electronegative potential,23,24

as well as DNA helix deformability.25The results are presented inFigure 1A−D and show that the N6 regions of different sequencedisplay uranyl cleavage patterns with distinct similarities.Specifically, increased reactivity is found at positions close tothe primary kink sites within the 5-bp consensus sequence (the

TG/CA steps). Previously, it has been demonstrated that A/Tstretches display uranyl hyper reactivity,25 as also observed in theN6 region of G5.01, G5.40, and G5.28. However, sites ofincreased reactivity are also found inN6 regions that are not A/T-rich (G5.03, G5.32 and G5.44). In general hyper reactivity ispresent at either or both ends of the N6 region. Intriguingly, theGATC quartets present in the eight strongest CRP binding sitesare all more sensitive toward uranyl cleavage, analogously to thereactivity seen for (A/T)n regions.We ascribe this behavior to thehelix plasticity at the GATC site allowing it to take up a uranylinduced fit narrow minor groove structure. Indeed, X-ray crystalstructure and NMR analysis of oligonucleotides containing thisquartet have revealed that the GATC quartet can exist in a varietyof conformations.20,26,27 In particular one crystal structure showsthat this sequence allows the DNA helix to adopt a narrow minorgroove despite the presence of the 2-amino group,26 and NMRstudies provide evidence of possible helix bending.27 Therefore,the helix at the GATC location seems highly deformable, whichmay support an induced fit mechanism in protein−DNAinteraction. A few of the selected sequences do not contain acentral TA step or a GATC quartet. However, one of these(G5.36) contains the CCCGGG sequence that also providesplasticity, e.g., as illustrated by the XmaI and SmaI restrictionenzymes, which upon binding bend the helix in oppositedirection.28 Thus, helix deformability in the N6 regions may be acommon property of the selected high affinity CRP binding sites.

Footprint Analysis of Selected CRP Binding Sites.Crystal structure analyses of CRP DNA complexes (allcontaining nicks in N6) have shown that four phosphatesbetween nucleobase −9/−10 and −10/−11 on the lower strandand +11/+10 and +10/+9 on the upper strand in the N6 regionare contacted by CRP.5,6 Uranyl photoprobing providesinformation about structural deformation as well as phosphatesinvolved in protein interaction.29−32 Therefore, CRP binding tothe 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 cleavageplots (Figure 1E−H). From these data it is evident that 4−6phosphates flanking one of the half-sites are strongly protected.Compared to the natural lac sequence, protection of morephosphates in the flanking regions is observed for the symmetric

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 TGTGA-GTTAGC-TCACTG5.03 TGTGA-CCCAGA-TCACT 14.19 ± 1.54 6 −1.56G5.54 TGTGA-TCTACA-TCACT 12.32 ± 1.12 11 −1.48G5.32 TGTGA-TCTGGG-TCACT 10.40 ± 0.74 6 −1.38G5.28 TGTGA-TCTATA-TCACT 7.06 ± 0.89 6 −1.15G5.41 TGTGA-CCAAGA-TCACT 6.62 ± 0.94 6 −1.11G5.06 TGTGA-TCTAAG-TCACT 6.51 ± 0.45 7 −1.10G5.44 TGTGA-CGCGGA-TCACT 5.74 ± 0.27 6 −1.03G5.46 TGTGA-TCTTGG-TCACT 4.77 ± 0.64 6 −0.92G5.59 TGTGA-TAGATG-TCACT 3.84 ± 0.50 7 −0.79G5.01 TGTGA-TATATG-TCACT 3.35 ± 0.08 6 −0.71G5.40 TGTGA-TTTACA-TCACT 2.91 ± 0.15 6 −0.63G5.36 TGTGA-CCCGGG-TCACT 2.45 ± 0.26 6 −0.53

aThe 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-basedcompetition assays where equimolar amounts of LacP1 DNA (reference) and each of the G5 in vitro selected binding sites competed for a limitingquantity of CRP protein. Krelative and ΔΔG values were calculated as described in the Methods section, and n denotes the number of experimentsperformed. The wild-type LacP1 sequence is also shown for comparison.



Figure 1. Uranyl structural probing and footprinting. (A−D) Uranyl structural probing of 12 G5 clones. Three clones are displayed in each panel. Theresults 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).



CRP site and for some of the selected G5 variants. Surprisingly,phosphates in the N6 spacer, which by crystallographic studieswere identified to interact with CRP, were not protected fromuranyl cleavage. On the contrary, positions in the N6 regionbecome hypersensitive to uranyl cleavage upon CRP binding(Figure 1E−H). The strongest hypersensitive site was detectedin the N6 sequence of the symmetrical CRP site (Figure 1H). In aprevious study using uranyl photocleavage for detecting CRPinteraction with DNA binding sites containing inosine and 2,6-diamino purine, we likewise showed that CRP binding induceshypersensitive sites in N6 in the binding sites analyzed.29

Hydroxyl radical and DNase I footprinting were performed inorder to further investigate interactions in the symmetrical N6

sequence. Interestingly, the exact same positions, which becameuranyl hypersensitive were protected from hydroxyl radicalcleavage, whereas protection at the A/T-rich sequences outsidethe 5-bp consensus sequence were identical in the two analyses(Figure 2). The result of DNase I footprinting shows strongprotection outside the 5-bp half-sites, whereas protection in N6 isweaker (Figure 2). Finally, it is clearly demonstrated that theappearance of a strong uranyl hypersensitive site in N6 of thesymmetric CRP binding site is a result of CRP interaction in theoperator since the uranyl cleavage intensity increases withincreasing 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 ordeformability.25 However, also the possibility for a proteininduced metal ion binding site in the N6 DNA has to beconsidered, because the divalent uranyl ion has been shown toprobe metal ion binding sites in folded DNA and RNA.33,34

Therefore, we asked whether the strong hypersensitive sitescould be a strong protein coordinated metal ion binding site. We

addressed this question by a competition experiment using othermetal ions. Intriguingly, zinc was not only able to decrease thestrong cleavage in the center of N6, but also to develop a clearprotection (i.e., zinc-footprint) of the position 3′ the hyper-sensitive site (Figure 3C and D). This result might indicate thatthe hyperactive position in the center of N6 could be a result of aspecific metal ion DNA interaction induced by CRP. Themolecular interpretation of this observation needs to beaddressed in further studies.Previous work, including DNA binding, gel shift experiments,

and structural studies established an important role for DNAdistortion in the sequence-specific binding of CRP to DNA sites.It is believed that sequence-dependent deformability of theoperator upon CRP binding is achieved largely by two sharpkinks at the conserved pyrimidine-purine step T6-G7, which isknown to be highly susceptible to deformation. Furthermore,AT-rich sequences at positions 10 and 11 and GC-rich sequencesaround position 16 from the dyad axis result in increased bendingand increased affinity.Our results provide evidence that the central N6 spacer also

plays a significant role in DNA recognition, capable ofmodulating affinity up to 100-fold despite that this elementexhibits high sequence variability among natural sites. On thebasis of sequence analysis and uranyl probing data of selectedbinding sites, we infer that the underlying mechanism relies onN6 deformability. However, we note that no obvious deformationof the N6 sequence was observed in the structures of CRP co-crystallized with DNA. A likely explanation for this discrepancy isthat the DNA fragments used in crystallization of CRP-DNAcomplexes contain nicks in the N6 spacer.

4 Consequently, thesecrystal structures may not accurately reflect protein-induceddistortion of the spacer region. In line with this assumption, the

Figure 2.Uranyl, DNase I, and hydroxyl radical footprinting. The symmetric CRP binding site is used for comparison of the three probingmethods. Thefootprinting results are displayed by autoradiographs (A) and by densitometric scans (B). CRP at 200 nM concentration was added in the lanesdesignated by +. The two lines in the densitometric scans represent uranyl cleavage in the absence of CRP (black line) and in the presence of CRP (blueline). The CRP protections in the uranyl and hydroxyl radical footprints are shown by brackets.



footprinting data show that phosphates in the N6 spacer, whichby 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 uponCRP binding (Figure 1E−H). Curiously, we observed that Zn2+specifically prevents the CRP-induced uranyl hyperreactivity at

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, and200 nMCRP added; S: A/G sequence reaction; C: no uranyl reaction. (B) Differential cleavage plot showing the appearance of hypersensitive positionsat increasing CRP concentration. The most reactive position within N6 is indicated by an arrow. (C) Uranyl photofootprinting in the presence of Zn

2+.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 cleavageplot (CRP binding in the presence of Zn2+ (lane 6) relative to CRP binding in the absence of Zn2+ (lane 2).



this position. Thus, it remains possible that CRP-induceddistortion 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-helixbinding proteins.35 Especially, the spacers in DNA sites for the434 and P22 repressors have been thoroughly studied. In bothcases the indirect readout of the spacer sequence influencesprotein affinity.36−41 Specifically, the spacers affect the affinity viathe structure of the unbound DNA and the deformability of thespacer, and for the P22 repressor it has been shown that the easeby which the helix structure in the binding site can be convertedinto a B′ form determines affinity. Thus, helixes such as A-tractswhich already are in a B′ form show the strongest affinity for P22,whereas the presence of G/C base pairs that provide the largestbarrier for the B to B′ transition results in low affinity.41 Thus, thepresent study of CRP-DNA interaction illustrates anotherstriking example of the importance of indirect readout of theDNA helix in which sequence specificity arises through the abilityof certain DNA sequence motifs to accommodate the helixconformation required by the protein for optimal DNAinteraction. We anticipate that as more sequence specific proteinDNA interactions are studied in molecular depth, in terms ofDNA helix conformation, flexibility, and deformability, we shallsee many more examples of how Nature has exploited theseDNA properties for indirect DNA sequence readouts by proteins(and other ligands).

■ METHODSIn Vitro Binding Site Selection. All 32P-labeled DNA fragments

were produced by standard techniques using either T4 polynucleotidekinase or Large Fragment of DNA Polymerase I (Klenow). The in vitroselection assay was modeled after a previous study from ourlaboratory.29 The binding site selection experiments were initiated byuse of 11 ng (∼2.6 × 1011 molecules) of single-stranded N6 in vitroselection template and a double-stranded randomized DNA oligonu-cleotide pool was generated by PCR as described below except only fivePCR cycles were run. To enrich the randomized DNA oligonucleotidepool for CRP-binding sites, the pool was incubated with 50 nMCRP andsubjected to gel electrophoretic mobility shift assay (EMSA). Followingelectrophoresis, the band shifts corresponding to CRP−DNAcomplexes were cut out, and the DNA was purified from the acrylamidegel. Before starting the next round of selection, the obtained DNAfragments were PCR amplified with dNTPs in a volume of 50 μL. Ineach case, the template under study was PCR amplified in a total volumeof 50 μL containing 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mMMgCl2, and 2.5 U of Taq DNA polymerase (Fermentas)) using 50 pmolof each primer and 200 μM dNTPs (Roche). After an initial denaturingstep of 2 min at 94 °C, amplification cycles were performed with eachcycle 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 thelast PCR cycle, the extension segment was continued for 7 min at 72 °Cbefore cooling to RT. The PCR products were gel purified andresuspended in 10 μL of either H2O or CRP binding buffer dependingon the future use of the DNA (PCR or EMSA). PCR for Krelativeexperiments: LacP1 DNA fragments (75 bp and 160 bp) were obtainedby PCR using primer set Lac promoter 1 and 2 and primer set Lacpromoter 3 or 4 using plasmid pUC19 as template. The different in vitroselected G5 clone and mutant DNA fragments (276 bp) were similarlyobtained by PCR using primer set M13F and M13R and the individualpCR2.1-TOPO plasmids containing the appropriate cloned DNAsequences as template.Electrophoretic Mobility Shift Assay (EMSA). 32P-labeled DNA

fragments and CRP protein were incubated in 30 μL of CRP bindingbuffer (10 mM Tris-HCl, pH 8.0, 50 mM KCl, 2.5 mM MgCl2, 1 mMEDTA, 55 μg mL‑1 bovine serum albumin, 1 mM dithiothreitol, 0.05%

NP-40, 2 μg mL‑1 calf thymus DNA and 50 μM cAMP) containing 100μM 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 loadedon 6% polyacrylamide gels (55:1). Following electrophoresis, the CRP−DNA complexes were detected by autoradiography or exposure tophosphor imager storage screens.

Relative Binding Constants and Binding Free Energy Change.The relative equilibrium binding constants, Krelative, were measured byEMSA as previously described.8 In this assay, a mixture of two differentsize DNAs (75 and 276 bp, respectively) competes for a limited amountof CRP protein simultaneously. We used 5−20 pM of each DNAfragment, which is well below theKd for the CRP-LacP1 complex, and allexperiments were performed at least in triplicate. Following exposure tophosphor imager storage screens, four different bands were clearlyvisible, and the amount of radioactivity in each bandwas quantified usingSTORM Phosphor Imager scanner and Image Quant software fromMolecular Dynamics. The relative equilibrium binding constants werecalculated by the formula: Krelative = (Kmutant)/(Kwild‑type) = (Kclone)/(KLacP1), where Kclone is the ratio of protein-bound G0 or G5 clone DNAdivided by free G0 or G5 clone DNA, and KLacP1 is the same ratio for theLacP1 DNA. The binding free energy change, ΔΔG, which is thedifference between the binding free energy for CRP−DNAclone complexformation versus the binding free energy for CRP−DNALacP1 complexformation, was calculated from the general assumption: ΔΔG = RTln(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 therelative equilibrium binding constant described above, R is the gasconstant [8.3145 J/(mol × K)], and T is the temperature in Kelvin. Theaverage Krelative obtained from at least triplicate experiments was used inthe expression. Note that positive ΔΔG values indicate a reduction ofbinding affinity.

Uranyl Photocleavage, DNase I and Hydroxyl RadicalFootprinting. In order to obtain structural information, uranylphotocleavage was performed as previously described.25,30 Uranyl andDNase footprinting was performed as described in ref 29. Hydroxylradical footprinting was performed as described42 in the CRP bindingbuffer (10 mM Tris-HCl, pH 8.0, 50 mM KCl, 2.5 mM MgCl2, 1 mMEDTA, 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 MolecularDynamics STORM Phosphor Imager was used to collect data from thephosphor storage screens, and baseline corrected scans were obtained byusing Image Quant version 5.2 and SAFA software.43 Differentialcleavage 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 theprotein, 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.

■ ASSOCIATED CONTENT

*S Supporting InformationThis material is available free of charge via the Internet at http://pubs.acs.org.

■ AUTHOR INFORMATION

Corresponding Author*E-mail: [email protected].

NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTS

We gratefully thank N. J. Harning and N. L. Jansen for technicalassistance



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A Novel Indirect Sequence Readout Component in the E. coli Cyclic AMP Receptor Protein Operator