Using Nucleotide Analogs to Probe Protein-RNA Interactions

METHODS 23, 255–263 (2001)doi:10.1006/meth.2000.1136, available online at http://www.idealibrary.com on

Using Nucleotide Analogs to Probe Protein–RNAInteractions

Matthew Elliott, Philip Gottlieb, and Paul Gollnick1

Department of Biological Sciences, State University of New York at Buffalo, Buffalo, New York 14260

Synthetic nucleotide analogs provide the opportunity to evaluate theimportance of individual functional groups on the RNA in protein–RNAcomplexes. The general approach is to incorporate analogs at a definedposition(s) in the RNA target and to evaluate the effect of this substitu-tion on the thermodynamic stability of the protein–RNA complex. Theunderlying assumption is that if the presence of the analog reducesthe stability of the complex, then the functional groups that are alteredin the analog interact with the protein. Here we describe the protocolsfor incorporation of nucleotide analogs either by in vitro transcriptionusing T7 RNA polymerase or by synthetic chemistry. We also describehow we have used this approach to study the interaction of the TRAPprotein from Bacillus subtilis with its cognate RNAs consisting of 11repeats of GAG and/or UAG triplets. By comparing the results of theseanalog studies with the crystal structure of TRAP bound to an RNAcontaining 11 GAG repeats, we are able to see that all the functionalgroups identified by analogs forge direct interactions with the protein.Analog studies also correctly identified residues that do not contactthe protein. Moreover, analogs can have indirect effects on the complexstability by altering the structural properties of the RNA. q 2001

Academic Press

Specific interactions between proteins and RNAmolecules play crucial roles in many important cellu-lar processes (1–3). The availability of synthetic nu-cleotide analogs that contain modifications to func-tional groups on the base, sugar, or phosphatepermits probing the contributions of these individualRNA functional groups to the specificity and stabilityof protein–RNA complexes. The general approachis to use analogs to alter defined RNA residue(s),confining the change to a specific functional group(s)

1 To whom correspondence should be addressed. Fax: (716) 645–2975. E-mail: [email protected].

1046-2023/01 $35.00Copyright q 2001 by Academic PressAll rights of reproduction in any form reserved.

on the base or sugar–phosphate backbone. Then theeffect that this substitution has on the interaction ofthis RNA with the protein of interest is determined.Disruption of the protein–RNA complex, observedas changes to the equilibrium dissociation constant,provides information about the importance of a givenchemical group in complex formation. It is assumedthat the greater the perturbation to the binding con-stant, the more critical that group is for complexformation.

The system that we have been studying with thisapproach is the trp RNA-binding attenuation protein(TRAP) from Bacillus subtilis and other related ba-cilli. TRAP is an RNA-binding protein that nega-tively regulates expression of genes involved in tryp-tophan biosynthesis (4, 5). TRAP consists of 11identical subunits arranged in a symmetrical ring(Fig. 1) (6). The binding of 11 molecules of L-trypto-phan activates TRAP to bind to RNAs containingmultiple (9–11) repeats of GAG and/or UAG (6–9).TRAP binding to a series of 11 G/UAGs in the 58

leader region of the nascent mRNA of the trpEDC-FBA operon promotes formation of a transcriptionterminator (a base-paired stem–loop structure fol-lowed by several U residues). This results in prema-ture termination of transcription and the genes arenot expressed. TRAP also regulates translation ofseveral trp genes, either by directly competing withribosomes for binding to these mRNAs (10, 11) or byaltering the secondary structure of the RNAs so asto sequester the ribosome binding site (12–14).

We recently used a variety of nucleotide analogsto identify functional groups in TRAP-binding RNAs

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ELLIOTT, GOTTLIEB, AND GOLLNICK256

that are essential for complex formation (15). A par-ticular advantage of the TRAP system is the avail-ability of a high-resolution crystal structure of aTRAP–RNA complex (Fig. 1) (16). This structureallows us to visualize the intermolecular contactsbetween the protein and RNA that are presumed tobe required for formation and stability of the com-plex. Comparing the crystal structure and the ther-modynamic data from analog studies shows an excel-lent correlation in most cases. Moreover, theavailability of both types of data has provided newinsights into how the complex achieves specificityand stability, and has also presented new questionsabout this system.

Although this approach has been used extensivelyto study protein–DNA interactions, the TRAP–RNAsystem is distinctive because it is one of the fewcharacterized cases where the interaction is entirelywith single-stranded RNA. Nucleotide analogs havealso been used to study ribozymes as well as otherprotein–RNA complexes (17). From a technical pointof view, our results using modified nucleotides in theTRAP–RNA system provide us with an opportunityto assess the general application of analogs and per-haps general limitations in the analysis of protein–RNA interactions.

FIG. 1. Structure of TRAP complexed with an RNA consisting of 11 GAG trinucleotides each separated by an AU dinucleotidespacer. (A) Ribbon diagram of the TRAP 11-mer complexed with RNA. Each subunit is shown in a different shade of gray with the11 bound L-tryptophan molecules shown in van der Waals spheres. The RNA is shown in ball-and-stick models. (B) The interactionof one GAGAU repeat with two adjacent TRAP subunits, one dark and the other lighter gray ribbon. The RNA is labeled G1–U5 andthe critical amino acids that contact the RNA are show in stick models. Dashed lines indicate hydrogen bonds.

OVERVIEW OF THE METHOD

This technique involves incorporation of the nucle-otide analog at a defined position(s) in the RNA poly-mer by either in vitro transcription or chemical syn-thesis. The effect of the analog on the stability of thecomplex is then assessed by a nitrocellulose filterbinding assay. In conjunction with the crystal struc-ture, the relative importance of functional groups onthe RNA was derived and a more complete energeticpicture of the complex was constructed. This methodis but one approach in an arsenal of techniques thatshould be applied to the study of bimolecular com-plexes between proteins and RNA.

In Vitro Transcription Reaction

1. To make DNA templates for runoff transcrip-tion using T7 RNA polymerase, the sequence of inter-est is cloned downstream of a T7 promoter in anappropriate plasmid such as pTZ18U (United StatesBiochemical, Cleveland, OH). Prepare the templateby linearizing the vector with the appropriate restric-tion enzyme. After digestion, extract with phe-nol:chloroform (1:1) followed by chloroform extrac-tion and precipitate the DNA with ethanol. Collect

ANALOGS TO STUDY PROTEIN–RNA INTERACTIONS 257

the nucleic acid by centrifugation and resuspend thetemplate in RNase-free water to a final concentrationof 1 mg/mL.

2. Standard conditions for in vitro transcriptionto make 32P-labeled RNAs are 40 mM Tris–HCl (pH8.0), 8 mM MgCl2, 2 mM spermidine, 25 mM NaCl,1 mM dithiothreitol (DTT), 0.5 mM GTP, UTP, andCTP, 0.025 mM ATP, 100 mCi [a-32P]ATP (3 3 1029

Ci/fmol), 1 mg of linearized DNA template, and 50units of T7 RNA polymerase (Gibco-BRL, Gaithers-burg, MD) (18, 19). The final reaction volume is 0.02mL. Standard incubation conditions are for 1 h at378C; however, transcription of some DNA templatesproduces higher RNA yields when incubated at248C (18).

In Vitro Transcription with Purine Analogs

To obtain high yields of transcripts containing nu-cleotide analogs, the conditions of each transcriptionreaction must be optimized by varying the concentra-tions of DTT, MgCl2, rNTPs, DNA template, and T7RNA polymerase. From the results of these optimiza-tions, we describe below the following modificationsto the standard transcription protocol described instep 2 of the previous section.

1. To prepare RNAs with 7-deazapurine analogs,substitute 0.5 mM ATP or GTP in the standard tran-scription protocol described above with 2 mM C7ATPor with C7GTP, respectively (15, 18). Generally weuse [a-32P]ATP for RNA labeling but when using anadenosine analog such as C7ATP we use 100 mCi of[a-32P]UTP (3 3 1029 Ci/fmol) and 0.025 mM UTPin the transcription reaction.

2. For transcriptions involving inosine, replacethe 0.5 mM GTP from the standard protocol with 6mM ITP (Sigma, St. Louis, MO) and use 3 mg of DNAtemplate. Additionally, incubate the reaction at 308Cinstead of 378C.

3. T7 RNA polymerase initiates transcription inef-ficiently with the purine nucleotide analogs used inour studies. Hence it is crucial to include either 2.5mM GMP or 0.1 mM ApG for transcription initia-tion (15).

Transcriptions to Make Deoxynucleotide-SubstitutedRNA

In vitro transcription reactions to make RNA tran-scripts containing deoxynucleotides, which we callD/RNA chimeras, are performed using a mutant T7

RNA polymerase capable of incorporating both rNTPand dNTPs (20). For these reactions we replace therNTP of interest with 2.5 mM of the correspondingdNTP, and increase the DTT concentration to 50 mM(15). The yield of D/RNA transcript, even under opti-mized reaction conditions, is an average of 15% lowerthan the RNA yield using wild-type polymerase.Therefore it may be necessary to scale up the tran-scription reaction.

Purification of Transcripts

Add 0.5 vol of loading dye containing 95% for-mamide, 20 mM EDTA, 0.05% bromphenol blue, and0.05% xylene cyanol FF to the reaction and heat to708C for 10 min. Load the sample directly onto an 8M urea polyacrylamide gel and run at 30 mA. Visual-ize the RNA bands by exposure to X-ray film or Phos-phorImager screen. Cut out the bands, crush the gelslice to a fine consistency in a 1.5-mL microcentrifugetube using an RNase-free plastic pestle (Kontes),then add 0.4 mL of elution buffer consisting of 0.5M NaCl, 0.1 M Tris–HCl (pH 8.0), and 20 mM EDTA.Elute for 30 min at room temperature, then extractwith phenol:chloroform followed by chloroform. Add10 mg yeast tRNA as a carrier and precipitate thenucleic acid with ethanol. Collect the transcript bycentrifugation and resuspend in a small volume (0.1–0.15 mL) of RNase-free water.

Quantitation

From the specific activity of the labeled nucleotide(Ci/fmol) in the transcription reaction and the num-ber of occurrences of that residue in the transcript,calculate the specific activity of the RNA (dpm/fmol).By counting an aliquot of the purified transcript,determine the concentration of the RNA in fmol/mL.

Chemical Synthesis of Oligonucleotides

The synthesis of 2-aminopurine phosphoramiditehas been described previously (21). All DNA analogswere purchased from Glen Research and dissolvedaccording to the manufacturer’s specifications.Chimeric D/RNA oligonucleotides are synthesizedusing a DNA/RNA automated synthesizer with stan-dard protocols except that longer coupling times arerequired when an RNA phosphoramidite is used. Theanalog is dissolved in dry acetonitrile and placed onthe extra port of the instrument. The coupling times


should be changed depending on the type of phos-phoramidite used (see below).

We initially used riboguanosine phosphoramiditescontaining 28-O-tert-butyldimethylsilyl (tBDMS)groups (Glen Research Corp.) for chemical synthesisof D/RNA chimeras. The disadvantage of these mono-mers is that the coupling reaction rate of the incom-ing 58-hydroxyl is slow (600 s) due to the proximityof the 28-tBDMS group to the 38-phosphine, resultingin decreased product yield. Recent advances in 28-protecting group chemistry have produced RNA mo-nomers with a 28-O-triisopropylsilyloxymethyl(TOM) protecting group (Xeragon AG and Glen Re-search Corp.) with faster coupling times (120 s) andhigher yields. Additionally, a quicker, more reliabledeprotection scheme results from the use of TOMgroups. We describe below two deprotection proto-cols, one for tBDMS groups and one for TOM groups.

Cleavage from the Support and Removal of Base andPhosphate Protecting Groups for 28-tBDMS-ContainingOligonucleotides.

After performing the synthesis, transfer the resinfrom the synthesis column to a tightly sealable vialand add AMA consisting of 1 vol of ammonium hy-droxide and 1 volume of 40% aqueous methylamine.Caution: Methylamine is toxic and should be handledonly while wearing gloves and opened in a fume hood.Use 1 mL AMA for 0.2 mmol synthesis or 2 mL for1 mmol synthesis. Incubate for 10 min at 658C. Coolthe vial on ice and open carefully in a fume hood.Remove the supernatant from the support with asterile pipet and rinse the resin with 1 mL of anethanol:acetonitrile:water (3:1:1) mix. Combine therinse with the decanted solution and evaporate todryness.

Deprotection of 28-tBDMS Groups

1. Resuspend the dried sample in 0.25 mL anhy-drous TEA–THF/NMP solution consisting of 1.5 mLN-methylpyrrolidinone (NMP), 0.75 mL triethyla-mine (TEA), and 1.0 mL triethylamine trihydrofluor-ide (THF) (22). Incubate at 658C for 1.5 h.

2. Precipitate the synthetic product by adding0.025 mL of 3 M sodium acetate (pH 5.2) followedby 1 mL of n-butanol. Store at 2708C for 1 h (canleave overnight). Collect the nucleic acid product bycentrifugation and evaporate to dryness. Resuspendthe pellet in 1:1 formamide:RNase-free water. We

have observed that an insoluble material coprecipi-tates with the nucleic acid product that must be spundown and removed prior to gel purification.

3. Purify the synthetic product by heating the mix-ture to 708C for 10 min and then run it on a 20%polyacrylamide/7 M urea gel. Visualize the nucleicacid using UV shadowing by placing the gel on afluorescent TLC plate and exposing it to short-waveUV light. The RNA appears as a dark shadow in thegel above the fluorescing plate. Cut the band fromthe gel using a razor blade. Extract the nucleic acidfrom the gel slice as described above. Quantitate theproduct by UV absorption.

Cleavage from the Support and Removal of Base andPhosphate Protecting Groups for 28-TOM Group-Containing Oligonucleotides

1. Remove column material and place in a cappedvial. Cleave the D/RNA from the resin by addingammonia hydroxide (30% solution) at room tempera-ture. The vial should be sealed tightly to ensure noleakage of ammonia. After 1 h, cool on ice and trans-fer the ammonia solution to a new vial, seal tightly,and incubate for 2 h at 558C. Cool the solution on ice,open the vial in the fume hood, allow the ammoniato evaporate for 2 h and evaporate the remainingsolution in a Speed-Vac.

2. Some synthesizers have automated cycles thatremove the oligonucleotide from the resin. In thisinstance the ammonia containing the oligonucleotideis heated for an additional 2 h at 558C followed byevaporation of the ammonia in a fume hood and thenremoval of all remaining solution in a Speed-Vac.The 2-h deprotection described above are conditionsused for the more labile protecting groups (such as“Ultrafast” groups from Glen Research). We recom-mend the use of these protecting groups for RNAsynthesis to avoid prolonged exposure of RNA oligo-nucleotide to basic conditions.

Deprotection of 28-TOM Groups

1. Resuspend the dried sample in 1 mL of tetrabu-tylammonium fluoride (TBAF) solution (1 M in THF)(Aldrich Chemical, Milwaukee, WI). Warm the reac-tion solution to 508C with shaking for 10 min. Letcool to 358C and continue shaking for at least 6 h (orovernight). Make sure that the pellets are completelydissolved prior to the 6-h incubation, as TOM-pro-tected oligonucleotides are less soluble than tBDMS-containing nucleic acids.


2. Add 1 mL of 1 M Tris–HCl (pH 7.4) and vortexthoroughly to remove the 28-hemiacetals.

3. Add 1 vol of formamide and purify as describedfor tBDMS-protected oligonucleotides. Note that de-salting and separation of the synthetic nucleic acidfrom TOM by-products becomes especially importantfor RNA oligonucleotides $50 residues in length. Oli-gonucleotides of this length should first be columnpurified using Sephadex G-25 prior to gel purifica-tion (see instructions provided by Glen ResearchCorp.).

Nitrocellulose Filter Binding Assay

One of the quickest and most reliable methods toanalyze the effect of nucleotide analog substitutionon protein binding is the nitrocellulose filter bindingassay. This technique involves incubating the proteinand radiolabeled RNA followed by filtration (andwashing) through a nitrocellulose filter. Nitrocellu-lose binds strongly to proteins but not to RNA. Radio-labeled RNA retained on the filter therefore indicatesthat the nucleic acid is bound to the protein. Datafrom these studies are used to derive a Kd for eachreaction.

We have used two variations of the filter bindingassay, direct and competition, to study RNA bindingto TRAP (15, 18, 19, 23). In cases where both methodswere used, comparable results were obtained witheither procedure. We use 0.45-mm-pore size nitrocel-lulose (Advantec MFS) for both methods. For directfilter binding assays, filter retention of the radiola-beled RNA is examined as a function of protein con-centration. The advantages of this method are that itis quick and requires small amounts of radiolabelednucleic acid, and analysis of the data is simple (seebelow). The disadvantage of this method is that it isvery difficult to distinguish specific from nonspecificbinding. Therefore this assay works well only forRNAs with a high degree of specificity in their inter-action with the protein of interest.

We have found several instances where D/RNAscontaining nucleotide analogs display significantnonspecific binding to the protein and/or to nitrocel-lulose. In these cases competition filter binding isused to measure the specific binding constant of theD/RNA for TRAP. In this method an unlabeled RNAis examined for its ability to compete with a radiola-beled nucleic acid (which binds to the protein witha high degree of specificity) for binding to the samesite on the protein. Since competition is based solelyon the decrease of radiolabeled nucleic acid on the

filter, nonspecific interactions of the unlabeled com-petitor are not detected and therefore do not obscurespecific binding data. While slightly more labor-intensive and requiring higher concentrations ofanalog-containing oligonucleotides, the competitionassay provides a more accurate measure of specificbinding of nucleic acid to protein than direct filterbinding.

Here we describe the direct and competition filterbinding methods used to analyze the interaction be-tween TRAP and RNA. These approaches are appli-cable to other systems with minor alterations inthe protocol.

Direct Filter Binding Assay

1. Set up twelve 0.1-mL reactions, each containingFBB (250 mM potassium glutamate and 16 mMHepes, pH 8.0), 1 mM L-tryptophan to activate TRAPfor RNA binding, 1 fmol (approximately 1 3 104 dpm)of 32P-labeled RNA, and progressively increasing con-centrations of protein (15, 18, 19). Incubate at 378Cuntil binding equilibrium is achieved. Some TRAP–RNA complexes require incubation for as long as 1h to reach equilibrium, although in most cases 15–20min is adequate. The first tube contains no proteinand is a measure of background RNA retention onthe filter. The range of protein concentrations usedin the remaining reactions varies depending on theaffinity of the RNA for the protein. To accuratelydetermine a binding constant, five tubes should con-tain protein concentrations below the Kd and sixshould have concentrations above the Kd, with bind-ing saturation achieved by the tenth or eleventh reac-tion. A protein range typically used for the TRAP–RNA interaction with a Kd of 3.5 nM is 0.07–500 nM(Fig. 2) (15).

2. We have observed high background retentionin the absence of pretreating the nitrocellulose priorto filtration. Therefore it is necessary to soak theappropriate number of filters in FBB, heat to a weakboil, and cool briefly before filtering. After incubatingthe reactions, filter 0.08 mL of each mixture throughthe nitrocellulose and wash twice with 0.5 mL FBB.Air-dry the filters and measure radioactivity re-tained on each by scintillation counter or Phosphor-Imager analysis.

Data Analysis for Direct Filter Binding

Analyze the data to generate a best-fit bindingcurve (Fig. 2) and to determine the binding constant


(Kd) by using a nonlinear least-squares fitting algo-rithm. We use Prism 3.0 (GraphPad Software Inc.,San Diego, CA) to fit the data to the following equa-tion: Y 5 Bmax X/(Kd 1 X ). This equation describesligand binding to a receptor following the law of massaction. X is the total protein concentration in eachreaction, Bmax is the maximal binding at saturation,and Kd is the ligand concentration necessary for half-maximal binding.

Competition Filter Binding Assay

In this assay the radiolabeled nucleic acid usedmust be well characterized for specific binding to theprotein, and have affinity comparable to or higherthan that of the unlabeled competitor. In addition,both the radiolabeled and unlabeled nucleic acidsshould be present in the reaction prior to addition ofprotein to rapidly establish binding equilibrium ofthe system. We use larger amounts of radiolabelednucleic acid than in direct binding so the range ofdetectable RNA binding above baseline is larger,allowing us to more accurately observe small de-creases in binding resulting from competition. Addi-tionally, it is important to ensure that the standarddeviation of counting is #5% of the total counts ofthe reaction without competitor (tube 2, see below).It is essential to add sufficient unlabeled nucleic acidto reach $90% competition, or approximately 900-fold excess of the 32P-labeled nucleic acid dependingon its affinity for the protein. Optimally, five of thetubes contain competitor concentrations resulting in

less than 50% competition with the radiolabeled nu-cleic acid and six result in more than 50% competi-tion, with 90% competition achieved by the tenth oreleventh reaction (Fig. 3). For example, for a TRAP–RNA interaction using 23 pM 32P-labeled D/RNAwith a Kd of 0.5 nM competing against itself, we add0.02–20 nM unlabeled oligonucleotide.

1. Set up twelve 0.1-mL reactions containing FBBand 2.3 fmol (3 3 104 dpm) of a radiolabeled nucleicacid previously characterized for protein binding bydirect filter binding. To tubes 3–12, add increasingamounts of unlabeled competitor nucleic acid. Nextadd TRAP to tubes 2–12 at the concentration equalto the Kd for binding to the 32P-labeled nucleic acid.After 5 min, add 1 mM L-tryptophan to activateTRAP for RNA binding.

2. Incubate, filter, and quantitate each reactionas described in the direct filter binding protocol(see above).

Data Analysis for Competition Filter Binding

Data are analyzed by generating a best-fit curve(Fig. 3) using nonlinear regression using a one-bind-ing-site competition algorithm (GraphPad Prism3.00, GraphPad Software Inc.) with the equation

RO 5 1/2 [KRO 1 (KRO/KRC) Ct 1 Rt 1 Ot

2 !(KRO 1 (KRO/KRC) Ct 1 Rt 1 Ot)2 2 4OtRt] ,

FIG. 2. Effect of inosine substitution examined by direct filterbinding assay. The curves shown are the best fit of the filterbinding data using a nonlinear least-squares fitting algorithm.Background retention in the absence of TRAP was subtractedfrom the binding data. The y-axis values are normalized to thetotal counts retained on the filter at saturation, which was greaterthan 50% of the input counts. Data are the averages of four individ-ual experiments.

FIG. 3. Competition filter binding assay to examine the effectsof deoxyinosine substitution for deoxyadenosine in the second resi-due of each repeat. Both (taGcc)11 and (tiGcc)11 were used to com-pete against 32P-labeled (taGcc)11. Curves shown are the best fitof the filter binding data using a nonlinear regression, one bindingsite competition algorithm. Background retention in the absenceof TRAP was subtracted from the binding data. The y-axis valuesare normalized to the total counts retained on the filter at satura-tion. Data are the averages of four individual experiments.


where Rt, KRO, KRC, Ct, and Ot are the total protein,equilibrium dissociation constant for radiolabelednucleic acid, equilibrium dissociation constant forcompetitor, total competitor nucleic acid, and totalradiolabeled nucleic acid, respectively.

APPLICATIONS AND LIMITATIONS

General Considerations

The interaction of TRAP with RNAs containing 11appropriately spaced GAG or UAG triplets yieldsequilibrium dissociation binding constants of ap-proximately 1 nM (15, 18, 19). However, the numberof unique contacts that are forged to make the specificcomplex are not great. This situation arises due tothe repetitive nature of the RNA and because theprotein consists of 11 identical subunits symmetri-cally arranged in a ring. Hence the effect of an analogsubstitution in each RNA repeat, which alters theinteraction with TRAP, is amplified 11-fold, assum-ing the RNA and the protein interact identically ateach subunit. In this particular case, production ofoligoribonucleotides for the TRAP–RNA studies iseasily achieved either by transcription or by syn-thetic means given the redundancy of sequence. Forother protein–RNA complexes, where the sequenceis less repetitive, the use of synthetic procedures maybe the only approach.

In generally, care must be taken when interpretingresults from analog studies because analogs, in addi-tion to perturbing the protein–RNA interface di-rectly, may alter characteristics of the RNA and indi-rectly affect the thermodynamic equilibrium of thecomplex. Examples of both types of effects, direct andindirect, are described below.

DNA versus RNA Analogs

When considering the analog method for analysisfor protein–RNA interaction, a major stumblingblock is the availability of ribonucleotide analogs.Numerous protocols exist for synthesizing base ana-logs but because many synthetic schemes requiremultiple steps to achieve the end product and dueto the necessity for the 28-protecting group, analogsin the ribose series are more difficult to use and areeffectively unavailable. On the other hand, deoxy-nucleotide analogs are abundant and easily pur-chased. For this reason, as well as to explore the roleof 28-hydroxyl groups in the TRAP–RNA complex,

we examined the effects of a series of deoxynucleotidesubstitutions into the RNA (UAGCC)11 using a mu-tant T7 polymerase (15). These studies showed thatthe only requirement for ribose is on the G of eachrepeat. All the other residues can be replaced withthe corresponding 28-deoxynucleotide with no delete-rious effects on binding to TRAP. The crystal struc-ture confirms that the only 28-OH groups that di-rectly interact with the protein are these 11 G’s,which hydrogen bond to the main-chain NH of Phe-32 in each subunit (Fig. 1B). In addition to mappingthis contact, these findings permitted us to use syn-thetic DNA/RNA chimeras to study the TRAP–RNAinteraction (see below). This approach may be ap-plied to other protein–RNA interactions to deter-mine which residues may be replaced with deoxy-nucleotides and their analogs.

Standard versus Competition Binding Analysis

To demonstrate when a standard binding assay isuseful and when a competition assay is required, wedescribe below two examples where sites of interac-tion were detected in the TRAP–RNA complex. Thesedata have been published previously (15) and arepresented here to illustrate the techniques describedin this work.

The RNA (UAGCC)11 contains the sequence 58-UAGCC-38 repeated 11 times. The binding curve gen-erated for this RNA is shown in Fig. 2. From thesedata a Kd of 3.5 6 1.2 nM was obtained (15, 18). Inthe RNA (UAICC)11, the 11 guanosine (G) residueswere replaced with inosine (I), a purine analog lack-ing the 2-amino group of G. This substituted RNAshowed virtually no binding to TRAP (Fig. 2) demon-strating the importance of the exocyclic amine of theguanosine residues in (UAGCC)11. In this example,nonspecific binding is minimal and does not interferewith analysis of the binding curve.

In other cases, significant contributions by nonspe-cific binding of the nucleic acid occur and as a resultthe only means to accurately determine the bindingconstant is by a competition assay. This situationarose when we examined the effect of inosine in thesecond residue of each repeat (normally occupied byadenosine). In this case we used the competitionassay to compare the synthetic D/RNA chimeras(taGcc)11 and (tiGcc)11 binding to TRAP. (Note: upper-case letters represent ribonucleotides while deoxy-nucleotides are in lowercase and i refers to deoxyi-nosine.) The curve for (taGcc)11 competing againstitself is a deep sloping line in which 50% competition


is reached by the fifth data point (Fig. 3). The curvelevels off at the 8th data point where approximately90% competition of the radiolabeled D/RNA isachieved. The apparent Kd of this reaction is 0.5 60.4 nM, which agrees with the value determined forthis D/RNA by direct filter binding (15). No competi-tion by (tiGcc)11 is observed with up to 1 mM competi-tor, resulting in a nearly straight line across the topof the graph. These data suggest that the exocyclicamine and/or the protonated ring nitrogen of theadenosines, which are lacking in inosine, interactwith TRAP on complex formation.

Identification of Direct Protein–RNA Contact Siteswith Nucleotide Analogs

We use a combination of these approaches withseveral base analogs including inosine, 2-aminopu-rine, and nebularine (purine). From these studies weidentified the N6 amine and N1 of A2, and the N1,N2 amine, and O6 of G3 of the G/UAG repeats asbeing essential for binding. Every one of these func-tional groups is seen hydrogen bonding with TRAPin the crystal structure of the TRAP–RNA complex(Fig. 1B).

Using Analogs to Identify Nonessential FunctionalGroups

In addition to identifying functional groups in theRNA that interact with the protein in the complex,nucleotide analogs can be used to identify residuesthat do not contact the protein. Our most extremeexample involves using an abasic analog (dspacer)from Glen Research that contains a deoxyribosesugar lacking a base. Replacing the spacer residuesbetween the GAG or UAG repeats with these abasicanalogs had no effect on the affinity of the result-ing chimera for TRAP, strongly indicating thatTRAP does not interact with the bases from theseresidues. This conclusion was confirmed by the crys-tal structure.

Indirect Effects on Binding by Nucleotide Analogs

In addition to identifying direct interactions be-tween the RNA and protein, we have observed indi-rect effects of nucleotide analogs on complex forma-tion. When 7-deazapurine analogs were introducedinto TRAP-binding RNAs, the affinity of these RNAsfor TRAP increased significantly (18). This effect was

most pronounced for RNAs with CC dinucleotidespacers between the GAG or UAG repeats and corre-lated with the ability of these RNAs to form stablesecondary structure. Indeed, introduction of the 7-deaza analogs significantly lowered the Tm of theseRNAs as seen in UV melting curves. From thesestudies, as well as by studying TRAP binding to theseRNAs as a function of temperature, we were able todeduce that the protein does not interact directlywith the N7 of the purines. Rather the presence of7-deaza analogs lowers the energy required to unfoldthe RNA, allowing it wrap around the protein at alower cost of energy. These observations were sup-ported by the crystal structure, which shows no inter-actions between the N7’s of the purines and the pro-tein (Fig. 1B). One of the important take-homelessons from these studies that we would like to em-phasize is the necessity of using multiple biophysicalmethods when attempting to define any molecularassociation process.

Thermodynamic Measurements versus CrystalStructure

All of the cases described above showed an excel-lent correlation between energetic contributions tothe complex when specific functional groups are al-tered and the X-ray crystal structure. In some in-stances, however, chemical groups predicted to beinvolved in binding by X-ray crystallography do notresult in significant perturbations to the complexwhen an appropriate analog is used. For example,in the crystal structure of TRAP complexed to anRNA with 11 GAG repeats (16), a hydrogen bondis predicted between the 2-amino group of the firstguanosine of each triplet and Asp-39 of TRAP (Fig.1B). However, substituting these residues with ino-sine, which lacks this NH2 group, does not signifi-cantly change TRAP binding affinity (15). Further-more, substituting Asp-39 with Ala does not resultin decreased RNA binding by TRAP (24). These datasuggest that while Asp-39 and the exocyclic amineof this guanosine are in appropriate proximity forhydrogen bonding, no energetic contribution to com-plex stability is made from this putative interaction.

Limits to Interpretation of Data

When making nucleotide analog substitutions andassaying their affect on protein–nucleic acid interac-tions, one assumes that the bimolecular complex in-teracts in a global manner that is identical to the

ANALOGS TO STUDY PROT

wild-type complex. The goal of the analog approachis to minimize perturbations to the general complexbut to introduce local changes to the complex to accu-rately assess the interactive process at the interfaceof the two molecules. Generally this condition is metwhen a given functional group is removed. Whenmultiple substitutions are introduced or more radicalchanges are made, the level of interference betweenthe two molecules can reach significant levels. At this
level of disruption, the protein–nucleic acid complex may assemble or adapt to an alternative interactionto compensate for the large positive change in freeenergy. Consequently it is difficult to assign individ-ual energetic contributions to a predicted hydrogenbond. For this reason, when we introduce nucleotideanalogs in multiple sites along the RNA and a signifi-cant level of disruption is observed, we cannot pre-cisely assign the source of the free energy change without the aid of additional techniques such as crystallographic analysis. Even in such circumstancescaution should be used.
ACKNOWLEDGMENTS

We acknowledge Dr. Fred Antson from the University of York,
England, who performed the crystallographic studies of the TRAP-RNA interaction and who prepared the figures shown in Fig. 1.This work was supported by Grant MCB 9603594 from the Na-tional Science Foundation.
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Using Nucleotide Analogs to Probe Protein-RNA Interactions