Proc. Natl. Acad. Sci. USAVol. 92, pp. 5077-5081, May 1995Biochemistry

A peptide interaction in the major groove of RNA resemblesprotein interactions in the minor groove ofDNALILY CHEN AND ALAN D. FRANKELDepartment of Biochemistry and Biophysics, and Gladstone Institute of Virology and Immunology, University of California, P.O. Box 419100,San Francisco, CA 94141

Communicated by Stephen C. Harrison, Harvard University, Cambridge, MA, February 21, 1995 (received for review November 23, 1994)

ABSTRACT A 17-amino acid arginine-rich peptide fromthe bovine immunodeficiency virus Tat protein has beenshown to bind with high affinity and specificity to bovineimmunodeficiency virus transactivation response element(TAR) RNA, making contacts in the RNA major groove neara bulge. We show that, as in other peptide-RNA complexes,arginine and threonine side chains make important contri-butions to binding but, unexpectedly, that one isoleucine andthree glycine residues also are critical. The isoleucine sidechain may intercalate into a hydrophobic pocket in the RNA.Glycine residues may allow the peptide to bind deeply withinthe RNA major groove and may help determine the confor-mation of the peptide. Similar features have been observed inprotein-DNA and drug-DNA complexes in the DNA minorgroove, including hydrophobic interactions and binding deepwithin the groove, suggesting that the major groove of RNAand minor groove of DNA may share some common recogni-tion features.

A wealth of structural information is now available aboutDNA-protein recognition, largely from crystallographic andNMR studies of DNA-protein complexes. In comparison,relatively little is known about RNA-protein recognition. Themost detailed information is provided by the cocrystal struc-tures of three tRNA synthetase-tRNA complexes (1-5), anR17 coat protein-RNA complex (6), and a UlA ribonucleo-protein domain-RNA complex (7). In the synthetase com-plexes, sequence-specific contacts occur primarily in the minorgroove of tRNAGln (1, 2), in the major groove of tRNAAsP (3,4), and to the phosphate backbone of tRNASer (5). Importantcontacts also are made to anticodon loop nucleotides in theglutaminyl and aspartyl complexes. The overall structures ofthe synthetases are rather different, and aside from RNA con-formational changes observed upon binding, few general fea-tures of recognition have emerged. In the R17 and UlAcomplexes, the most important sequence-specific contacts aremade to bulge and loop nucleotides of RNA hairpins (6, 7).

Recently, several common RNA-binding motifs have beenidentified (for review, see refs. 8 and 9), suggesting thatconserved structural features may be found in some classes ofproteins. One of these motifs, the arginine-rich motif, consistsof a short region of basic amino acids (8-20 residues long)particularly rich in arginine. This motif has been found inbacterial antiterminators, ribosomal proteins, coat proteinsfrom RNA viruses, the human immunodeficiency virus (HIV)Tat and Rev proteins (10), and the bovine immunodeficiencyvirus (BIV) Tat protein (11). Studies with model peptidesderived from the arginine-rich domains of HIV Rev, HIV Tat,and BIV Tat have emphasized the importance of RNA struc-ture in protein recognition and have provided some detailsabout sequence-specific interactions (11-16). In HIV Rev, a17-amino acid peptide specifically recognizes the Rev response

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element (RRE) RNA when the peptide is in an a-helicalconformation and uses 6 amino acids (4 arginines, 1 threonine,and 1 asparagine) for binding (12). NMR studies have shownthat binding induces formation of an internal loop structure inthe RRE containing GG and GA base pairs and two looped-out bases (17, 18). In HIV Tat, a single arginine residue withina 9-amino acid peptide is largely responsible for recognition ofa bulge region in transactivation response element (TAR)RNA (14). The free amino acid arginine binds to TAR by usinga similar set of RNA structural features (19), suggesting thata defined peptide conformation may not be needed for rec-ognition. An NMR model of the arginine-TAR complexsuggests that the guanidinium group of arginine hydrogenbonds to a guanine base in the major groove and to twophosphates on the backbone and that the complex is stabilizedby a base triple interaction between a uracil in the bulge andan A-U base pair above the bulge (15). In both cases, bulgeshelp widen the major grooves of adjacent A-form RNA helices,thereby increasing accessibility to the proteins (13, 20).BIV Tat is closely related to HIV Tat; however, initial

studies with a 17-amino acid peptide suggested that BIV Tatuses a very different set of interactions to recognize BIV TAR(11). The peptide binds to an unusually accessible stem regionadjacent to two single-nucleotide bulges in BIV TAR andrequires an extensive set of determinants in the major groove(Fig. 1) very different from those of HIV TAR. The sequenceof the BIV peptide also is distinct, containing several glycineand proline residues in addition to arginine residues (Fig. 1).In this study, we show that three glycine residues in the peptideare critical for BIV TAR recognition and, most surprisingly,that an isoleucine also is critical. We suggest that the isoleucineintercalates between bases in the major groove of BIV TARand that glycine residues allow the peptide to bind deeply inthe groove, analogous to interactions observed in the minorgroove of DNA.

MATERIALS AND METHODSPlasmids and Chloramphenicol Acetyltransferase (CAT)

Assays. Plasmids encoding BIV Tat peptide mutants wereconstructed by cloning synthetic oligonucleotide cassettes intoa gene encoding a BIV-HIV Tat fusion protein (11). Muta-tions were confirmed by dideoxynucleotide sequencing. Tomeasure transcriptional activation, mutant BIV-HIV Tat plas-mids (50-100 ng) were cotransfected into HeLa cells with anHIV long terminal repeat-CAT reporter plasmid (25-50 ng)in which HIV TAR was replaced with BIV TAR (11), andCAT activity was assayed after 48 hr and quantitated as de-scribed (21).

Peptides and RNAs. BIV Tat peptides used for in vitroRNA-binding experiments were synthesized with C-terminalamides and acetylated at the N termini on an Applied Bio-

Abbreviations: BIV, bovine immunodeficiency virus; HIV, humanimmunodeficiency virus; RRE, Rev response element; CAT, chlor-amphenicol acetyltransferase; Tm, melting temperature; TFE, triflu-oroethanol; TAR, transactivation response element.

5077

5078 Biochemistry: Chen and Frankel

U A // FIG. 1. Secondary structure ofMG 14ell BIVTAR and sequence of the BIVQ G- CTat peptide. Boxed nucleotides in

U A U BIV TAR are required for peptideG C binding and arrows indicate posi-tions of two phosphates whose

F modification interferes with bind-ing (11). The circled U (U10) in-dicates that a bulge nucleotide isneeded but its identity is not im-portant (11). The peptide sequence

SGPRPRGTRGKGRRIRR corresponds to amino acids 65-8165 81 of BIV Tat.

systems model 432A peptide synthesizer as reported (11).Peptides were purified on a C4 reverse-phase HPLC column(Vydac, Hesperia, CA), and peptide identities were confirmedby fast atom bombardment/MS spectrometry (Protein andCarbohydrate Structure Facility, University of Michigan, AnnArbor). The concentration of each peptide was calculated fromA214 values and was confirmed by native gel electrophoresisand Coomassie blue staining with known peptides as stan-dards. Radiolabeled and unlabeled RNAs were transcribed invitro by using T7 RNA polymerase and synthetic oligonucle-otide templates and were purified as described (11). Bindingreaction mixtures (10 ,lI) contained 10 mM Tris-HCl (pH 7.5),70 mM NaCl, 0.2 mM EDTA, 5% (vol/vol) glycerol, and yeasttRNA (25 jig/ml).

Circular Dichroism (CD) and Absorbance Melting Curves.CD spectra were recorded with an Aviv 62DS spectropo-larimeter equipped with a thermoelectric cell holder. Allmeasurements were made at 4°C in solutions containing 10mM potassium phosphate (pH 7.0) and 70 mM KF in 1- to10-mm pathlength cells. Peptide concentrations were 2-50 ,tMand RNA concentrations were 16 ,uM. Data were collected for10 sec at each wavelength and five scans were averaged.Ellipticity was calculated as mean residue ellipticity [0] by usingamino acid residues for the peptide and nucleotide residues forthe RNA and RNA-peptide complex. Melting curves were

A

recorded with an Aviv 14DS spectrophotometer equipped witha thermoelectric cell holder. RNA samples (2-5 ,uM) wererenatured by heating to 85°C for 3 min followed by slow coolingto room temperature in 10 mM potassium phosphate, pH7.5/100 mM KCl. Peptide-RNA complexes were at a 1:1stoichiometry. Melting curves were monitored at 260 nm byusing equilibration times of 1 min/°C, and melting temperature(Tm) values were determined by calculating the first derivativeof each curve. To estimate AG, AH, and AS values fromabsorbance melting curves, concentrations of folded and un-folded molecules were calculated at each temperature, byassuming a two-state unimolecular transition and using base-line absorbance values for the folded and unfolded states (22).Well-defined baselines were observed in the melting profilesand values forKwere determined at each temperature by usingthe equation K = f/(l - f), where f is the fraction of basespaired. Values for AH and AS were calculated from van't Hoffplots (ln K vs. 1/) by using the standard equations d(lnK)/d(1/T) = -AI/R, AG = -RT ln K, and AS = (AH -AG)/T, as described (22). Only points where 0.15 c f ' 0.85were used in the van't Hoff analyses.

RESULTSMutagenesis of the BIV Tat Peptide. To determine which

amino acids in the 17-amino acid BIV Tat peptide are impor-tant for BIV TAR recognition, we individually mutated eachof the 17 residues to alanine (or to lysine if the residue wasarginine) in the context of a hybrid protein composed of theHIV-1 Tat transcriptional activation domain fused to the BIVpeptide. The hybrid protein activates HIV transcription whenHIV TAR is replaced by BIV TAR, and activation in vivocorrelates well with peptide-RNA binding affinities measuredin vitro (11). Mutation of any one of three arginine residues(Arg-70, Arg-73, or Arg-77) dramatically reduced activity (to<3% of wild type) as did mutation of Thr-72, Gly-74, or Ile-79(Fig. 2A). Mutation of Gly-71 or Gly-76 also significantlyreduced activity (to <12% ofwild type). Differences in activitywere not due to differences in the levels of protein expressionor nuclear localization as determined by immunoprecipitationof 35S-labeled mutant proteins (data not shown). To confirm

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FIG. 2. RNA binding by BIV Tat peptide mutants. (A) BIV TAR-dependent CAT activities of BIV peptide mutants fused to the activationdomain of HIV Tat. Activities were assayed on an HIV LTR-CAT reporter containing BIV TAR in place of HIV TAR (11), and activities of themutants were normalized to that of the wild-type fusion protein. Three experiments were averaged for the quantitation shown below. (B)RNA-binding gel shift assays with mutant peptides. A fixed concentration of BIV TAR RNA was titrated with the indicated concentrations (nM)of each peptide and apparent Kd values were estimated as the peptide concentrations required to shift 50% of the unbound RNA into the complex.Three previously described mutant RNAs (UlO bulge deletion, G15-C22, and U15) (11) were used as specificity controls (data not shown).

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that the decreased activities observed in vivo reflected trueeffects on RNA-binding affinity, peptides were synthesizedcorresponding to the most impaired mutants and affinitieswere measured by a gel mobility shift assay. All mutantsshowed a significant reduction in BIV TAR binding affinity(Fig. 2B). In some cases (the Thr-72 mutant in particular), thepeptide-RNA complex migrated significantly faster than thewild-type complex, suggesting that the structure of the boundRNA might be different (see below). Peptides having deletionsof the three N-terminal residues [BIV Tat-(68-81)] or thethree N-terminal and two C-terminal residues [BIV Tat-(68-79)] bound BIV TAR with the same affinity as the wild-typepeptide, further indicating that Ser-65, Gly-66, Pro-67, Arg-80,and Arg-81 are not involved in RNA binding. We cannot ruleout the possibility that Arg-68 and Arg-78 contribute impor-tant electrostatic or hydrogen bonding interactions since theirsubstitution with lysine, which may still form these interactions,had little effect on activity (Fig. 2A).

Isoleucine Mutants. One surprising result of the mutagen-esis was that a hydrophobic side chain (isoleucine) was re-quired. To examine the role of this residue in more detail, wereplaced Ile-79 with 15 other amino acids and measuredRNA-binding activities of these mutants in vivo. While theisoleucine side chain provided the optimal interaction, otherhydrophobic residues of similar size (leucine, tyrosine, orphenylalanine) could substitute reasonably well (20-30% ofwild-type activity; Fig. 3A). Substitution with increasinglysmaller side chains (valine or alanine) showed correspondingdecreases in activity. Substitution with a much larger side chain(tryptophan) abolished activity. Other moderately hydropho-

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indicate the mobilities of unbound RNAs. Experiments were per-Fig.

bic residues (cysteine, methionine, or threonine) showed someactivity (5-20% of wild type), whereas charged or relativelysmall side chains (aspartic acid, lysine, histidine, asparagine,proline, or glycine) showed little to no activity. Peptidescorresponding to moderate (leucine, tyrosine, and methio-nine) and weak (alanine, lysine, and asparagine) bindingmutants were synthesized and RNA-binding affinities weremeasured in vitro (Fig. 3B). Apparent Kd values for the leucine,tyrosine, and methionine mutants were -2 nM (vs. -0.75 nMfor the wild-type peptide) while affinities of the alanine, lysine,and asparagine mutants were slightly lower (Kd = 3 to 10 nM).The relative decreases in affinity were not as dramatic asobserved in vivo, but interestingly, all three weak bindingmutants formed peptide-RNA complexes that migrated mark-edly faster in the gel than the stronger binders. As suggestedfor the Thr-72 mutant, this may indicate that the structure ofthe bound RNAs is altered, which in turn might influencetranscriptional activation in vivo.CD of the Peptide and Peptide-RNA Complexes. CD ex-

periments were used to examine the conformations of thepeptide, RNA, and peptide-RNA complexes. The spectrum ofthe peptide in aqueous buffer indicated an unstructuredconformation (Fig. 4A). Addition of 50% (vol/vol) trifluoro-ethanol (TFE), which often stabilizes a-helical peptide con-formations (23), induced a change in the spectrum consistentwith a mixture of random coil and a small amount of }3-sheetand/or X3-turn. The difference spectrum shows a single mini-mum near 218 nm (Fig. 4B) and is similar to the TFE-inducedspectra of a 13-hairpin fragment of streptococcal protein G (24)and other peptides with l3 tendencies (25). A similar change inthe BIV peptide spectrum was observed in the presence of lowconcentrations of SDS (data not shown), which also has beenproposed to stabilize 13 conformations (25). In contrast, addi-tion of 50% TFE to an HIV Tat peptide (YGRKKRQRRRP)showed a small amount of a-helix formation with characteristicdouble minima at 208 and 222 nm (data not shown), consistentwith the helical tendency observed by NMR (26). It should beemphasized that CD spectra of 13 structures are not as welldefined as spectra of a-helices and the effects of TFE are lesswell understood; however, preliminary NMR experiments areconsistent with a hairpin conformation for the BIV peptide (J.Puglisi, L.C., and A.D.F., unpublished data). The CD spectraof mutant peptides with decreased RNA-binding affinities(70RK, 72TA, 73RK, 77RK, 791A, 791Y, and 79IN; Figs. 2Band 3B) were identical to those of the wild-type peptide inaqueous buffer or in 50% TFE (data not shown), suggestingthat these mutations probably alter direct interactions with theRNA and not the conformational tendency of the peptide. Thespectrum of the 74GA mutant in 50% TFE was slightlydifferent from that of the wild-type peptide (data not shown),suggesting that the glycine may influence the conformationalpreference of the peptide.CD experiments also were used to monitor possible con-

formational changes in BIV TAR upon peptide binding, as forHIV Tat-TAR (27) and Rev-RRE (28) complexes. We re-corded spectra of the free RNA and a peptide-RNA complex(Fig. 4C) and calculated the difference spectrum (Fig. 4D).The minimum in the difference spectrum observed near 280nm corresponds to ellipticity from the RNA and is consistentwith a change in base stacking. The minimum observed near218 nm is consistent with a 1 conformation of the peptide inthe complex but also is likely to reflect changes in RNAconformation as the free RNA contributes ellipticity at thiswavelength. The minimum observed near 200 nm may alsoreflect contributions from both the RNA and peptide. CDdifference spectra with mutant peptide-RNA complexes wereslightly different from the wild-type complex (data not shown),perhaps suggesting subtle differences in RNA structure andpossibly relating to the differences in gel mobility described

Biochemistry: Chen and Frankel

5080 Biochemistry: Chen and Frankel

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above. The nature of these differences will require moredetailed structural studies.

Thermostability of the Peptide-RNA Complex. Specificpeptide binding was found to significantly enhance the ther-mostability of BIV TAR. In the absence of peptide, the RNAshowed a single cooperative UV melting transition with Tm =

74°C, AH = -73 kcal/mol, and AS = 210 cal per K per mol(Fig. SA; 1 cal = 4.184 J). In the presence of peptide, thestability of the RNA increased substantially, with Tm = 88°C,AlH = -93 kcal/mol, and AS = 260 cal per K per mol. Someof the stabilization may be attributed to the ionic effect of

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FIG. 5. (A) UV absorbance melting curves of BIV TAR RNA andthe peptide-RNA complex. (B) Relationship between specific RNA-binding affinity of mutant peptides (expressed as AG) and increasedthermostability of BIV TAR upon binding. The wild-type peptideincreases the Tm of BIV TAR by 14°C (from 74 to 88°C), whereas themost nonspecific binding mutant (74GA) shows a 5oC increase.

binding a charged peptide, as addition of 1 mM Mg2+ to BIVTAR in the absence of peptide increased the Tm by 5-6°C (datanot shown). The remainder of the stabilization appears toresult from specific complex formation as the increase in Tmmeasured with mutant peptides was proportional to specificRNA-binding affinity (Fig. SB). The 74GA mutant binds BIVTAR with little or no specificity (relative to mutant TARRNAs) and stabilizes the RNA to the same extent (5°C) as theionic stabilization of Mg2e. The unstructured nature of thefree peptide and the increased thermostability of the RNA inthe complex suggest that the peptide probably undergoes adisorder -- order transition that is tightly coupled to specificRNA binding. In the Rev-RRE interaction, the helical con-formation of the peptide is stabilized upon specific RNA bind-ing (28), although some preformed structure is required (12).In many DNA-protein interactions, disordered regions ofproteins also appear to become ordered upon specific binding(29).

DISCUSSIONThe interaction of the BIV Tat peptide with RNA is quitedifferent from the interaction of HIV Tat or Rev arginine-richpeptides with RNA. The specificity of the HIV Tat-TARinteraction is determined largely by the contact of a singlearginine residue, placed in the context of basic amino acids,with a bulge region in TAR (14, 15, 30). In contrast, the HIVRev-RRE interaction requires 6 amino acids (4 arginines, 1threonine, and 1 asparagine), placed in the context of ana-helix, to bind specifically to an internal loop region in theRRE (12). As with Rev, the BIV Tat-TAR interaction re-quires several arginine residues and one threonine but inaddition requires one isoleucine and three glycine residues.The CD data provide preliminary evidence that the BIVpeptide has a tendency to adopt a X3 conformation (althoughwe do not know the conformation of the bound peptide),suggesting that arginine-rich RNA-binding peptides may be

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Proc. NatL Acad ScL USA 92 (1995)

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Proc. Nati Acad Sci. USA 92 (1995) 5081

capable of adopting a wide range of structures. It is possiblethat glycine residues help provide conformational flexibilityand may help favor formation of turn structures. Because theBIV peptide is likely to become ordered only upon specificbinding to BIV TAR, the conformational tendencies of thepeptide may be important in allowing the RNA to "mold" thepeptide into the appropriate shape.The finding that glycine residues and a hydrophobic side

chain are used to recognize the major groove of BIV TARsuggests parallels to minor-groove DNA recognition and apossible resemblance between the RNA major groove andDNA minor groove. It is known that the major groove of anA-form RNA helix is relatively deep and narrow, roughlysimilar in shape to the minor groove of a B-form DNA helix(31). Proteins and drugs that bind to the DNA minor grooveoften use the shape of the groove as a recognition feature. Forexample, a peptide from the HMG-I/Y protein (PRGRP)binds to the minor groove of an AT-rich sequence and requiresthe small glycine side chain to allow the peptide to sit deeplywithin the groove (32). We speculate that one or more glycineresidues in the BIV peptide may be needed to allow bindingdeep within the RNA major groove. DNA binding by theHMG-I/Y peptide and a related arginine/glycine-rich peptidefrom the DAT1 protein (33) appear to mimic the shape-selective binding of netropsin and distamycin to AT-rich minorgrooves (32), which, interestingly, also bind selectively to themajor groove of a helix in tRNAPhe (34). Other RNA-bindingdomains rich in arginine and glycine, such as the RGG motif(9), may show related modes of binding. One RGG-containingprotein, known as heterogeneous nuclear ribonucleoprotein Uor SAF-A, binds to both RNA and AT-rich scaffold-attachedregion DNA (35).

Striking hydrophobic interactions have been observed in theDNA minor groove, including stacking of phenylalanine res-idues in TATA-binding protein-TATA box complexes (36,37),intercalation of an isoleucine in an SRY-DNA complex (38),and intercalation of a leucine in a PurR-DNA complex (39).Mutation of the isoleucine in SRY (an HMG-box protein)reduces DNA-binding affinity and is associated with pheno-typic sex reversal (38), and the leucine in PurR is highlyconserved among related DNA-binding repressors (39). Be-cause isoleucine in the BIV peptide can be replaced withtyrosine, phenylalanine, or leucine, it seems reasonable thatthis hydrophobic side chain may intercaiate between bases inthe RNA major groove. A single nucleotide bulge (U10) inBIV TAR is required for specific peptide binding (Fig. 1 andref. 11), and it is possible that the isoleucine interacts in themajor groove near the bulge, which is expected to be wide andaccessible to a protein (13, 20). The structure of the UlAribonucleoprotein domain-RNA complex shows several stack-ing interactions between aromatic protein side chains andRNA bases in a hairpin loop (7). A recent in vitro selectionexperiment has identified RNAs that selectively bind L-valine(40), and an isoleucine mutation in the putative RNA-bindingdomain of the FMR1 protein has been associated with fragileX chromosome syndrome and causes decreased nonspecificRNA-binding affinity (41), suggesting that hydrophobic con-tacts will make important contributions in other RNA-proteininteractions.

We thank Rene Tipton and Jim Cullem for technical assistance,Jody Puglisi for many helpful discussions, and Carl Pabo, Jody Puglisi,Raul Andino, Mark Feinberg, John Young, and members of thelaboratory for comments on the manuscript. This work was supportedby National Institutes of Health (NIH) Grant A129135 (A.D.F.) andby NIH Postdoctoral Fellowship AI08591 (L.C.).

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Biochemistry: Chen and Frankel